Human infections with avian H7N9 subtype influenza viruses are a major public health concern and vaccines against H7N9 are urgently needed for pandemic preparedness. In early 2013, novel H7N9 influenza viruses emerged in China that caused about 1600 human cases of infection with a high associated case fatality rate. In this study, two H7N9 split virion vaccines with or without AS03 adjuvant were tested in the naive ferret model. Serological analyses demonstrated that homologous hemagglutination inhibition and microneutralization antibody titers were detectable in the ferrets after the first immunization with the AS03-adjuvanted vaccines that were further boosted by the second immunization. In addition, heterologous antibody titers against older H7 subtype viruses of the North American lineage  and newer H7 subtype viruses of the Eurasian lineage (H7N9) were detected in the animals receiving the AS03-adjuvanted vaccines. Animals receiving two immunizations of the AS03-adjuvanted vaccines were protected from weight loss and fever in the homologous challenge study and had no detectable virus in throat or lung samples. In addition, microscopic examination post-challenge showed animals immunized with the AS03-adjuvanted vaccines had the least signs of lung injury and inflammation, consistent with the greater relative efficacy of the adjuvanted vaccines. In conclusion, this study demonstrated that the AS03-adjuvanted H7N9 vaccines elicited high levels of homologous and heterologous antibodies and protected against H7N9 virus damage post-challenge. In addition to seasonally occurring human infections, zoonotic infections caused by avian influenza A viruses are a major public health concern and pose a pandemic threat. In 2013, an avian H7N9 virus strain emerged in China that caused hundreds of human infections. From 2013 to 2017, the H7N9 virus led to annual epidemics. During these five waves of epidemics ~1600 laboratory-confirmed cases have been reported, coupled with a case fatality rate of almost 40%2. Human infections with H7N9 viruses occurred each year and the viruses gained virulence markers that potentially enhance the risk for humans and may have increased their spread into the human population, making this virus a notable pandemic threat4. During the fifth wave of H7N9 epidemics the virus split into two phylogenetically distinct lineages, the Yangtze River Delta and Pearl River Delta clades5. Ferret antisera raised against existing candidate vaccine viruses (CVVs) showed reduced cross-reactivity against these novel H7N9 viruses6. In addition, highly pathogenic avian influenza (HPAI) H7N9 viruses emerged that featured a polybasic cleavage site in the hemagglutinin (HA) and were lethal for poultry8. However, no community transmission has been reported and infections occurred almost exclusively after exposure to infected poultry. Nevertheless, the large number of human cases increased the likelihood of genetic reassortment of H7N9 viruses with human seasonal influenza viruses, which could lead to sustained human-to-human transmission9. In China, cases of people co-infected with both H7N9 and seasonal influenza virus strains have been reported during the period of overlapping seasonal and H7N9 epidemics10. In addition, an avian H7N2 virus caused an outbreak in cats in an animal shelter in New York that led to one human case11.Influenza viruses infect millions of people worldwide and result in ~290,000–650,000 influenza-related deaths each year12. Currently, there is no licensed H7N9 vaccine available and people infected with H7N9 viruses are only treated therapeutically with neuraminidase inhibitors. However, H7N9 is quickly acquiring resistance to neuraminidase inhibitors13 which is leading to an unreliable public health strategy to combat this virus. To provide more effective protection, vaccines are urgently needed. A phase I/II trial investigating a pre-pandemic H7N9 vaccine, that is also being evaluated in the present study, showed promising results in eliciting robust immune responses in healthy adults, but it is unknown if this vaccine can induce cross-reactive antibody responses against recent fifth wave H7N9 viruses and elicit protective efficacy upon live virus challenge14. In this study, H7N9 split virion vaccines, produced at two different GlaxoSmithKline (GSK) production sites (Quebec (Q) and Dresden (D)), are being evaluated. In context of pandemic preparedness, testing of vaccines produced in different regions of world is of importance and contributes to global preparedness efforts.Humans are immunologically naive to H7 subtype viruses and possess little to no pre-existing, humoral immunity16. Previous preclinical studies in the ferret model have shown that protective immune responses against homologous and heterologous infection with H5N1 viruses can be induced with low doses of H5N1 inactivated split virion vaccines adjuvanted with AS03 . The use of AS03 adjuvant reduced the required dose of HA antigen and resulted in higher and cross-protective immune responses17. An AS03-adjuvanted H5N1 split virus vaccine (produced at the GSK Quebec site) has been licensed by FDA for human use, however, there is no FDA approved H7N9 vaccine available yet.Ferrets are highly susceptible to influenza virus infection, show clinical symptoms similar to humans and are the preferred animal model to provide evidence of efficacy of influenza virus vaccines especially against highly pathogenic avian influenza virusesThe present study was designed to evaluate the potential of AS03-adjuvanted H7N9 A/Shanghai/2/2013 vaccines to induce humoral immunity to a panel of H7 viruses and protection against vaccine-homologous challenge with wild-type H7N9 A/Anhui/1/2013 virus in the naive ferret model.n = 8 per group) were immunized with non-adjuvanted standard-dose H7N9 vaccines or AS03-adjuvanted half-dose H7N9 vaccines twice 21 days apart. Blood for serological testing was taken before the first immunization and after the first (prime) and second (booster) immunizations. The experimental overview, vaccination groups, and virus strains used for vaccination, serological testing, and virus challenge are shown in Fig. Female ferrets  against homologous H7N9 A/Shanghai/2/2013 and A/Anhui/1/2013 virus. Notably, six animals  seroconverted against heterologous H7N3 virus after a single immunization, whereas only five ferrets (1 in Q-H7N9 and 4 in D-H7N9) had a rise in HI antibodies against H7N7 virus. These titers were strongly boosted after the second immunization, reaching high reciprocal GMTs of 666 (Q-H7N9) and 788 (D-H7N9) against A/Shanghai/2/2013 and 629 (Q-H7N9) and 635 (D-H7N9) against A/Anhui/1/2013 H7N9 virus at day 49 post-second immunization Fig. .Next, virus-neutralizing (VN) responses against the same viruses were measured Fig. . A primiThese results support and highlight the beneficial effects of adding the AS03 adjuvant to the H7N9 vaccines in order to increase the immunogenicity of an H7 HA vaccine antigen, to increase the breadth of the functional antibody response, and to enable an antigen-sparing vaccine approach.11. These viruses circulated more recently than the virus strain used in the vaccine and are antigenically distinct from the vaccine strain virus  Fig. . For theTo test if immunization with a Eurasian lineage H7N9 virus vaccine can also induce antibodies cross-reactive toward a North American lineage H7 virus, we tested a feline H7N2 virus in HI and VN assays Fig. c, f. ImmTo investigate cross-reactive antibody responses in more detail, IgG binding antibodies were measured by ELISA in the animals immunized with AS03-adjuvanted half-dose H7N9 split virion vaccines, which showed robust binding against all tested HAs from the panel of H7 viruses  virus on day 49. The body temperatures were monitored continuously, and nose and throat swabs were collected daily. Body weights were recorded at challenge and on the day of scheduled euthanasia . The lungs and nasal turbinates were also harvested on the day of scheduled euthanasia Fig. . The aniThe animals receiving the AS03-adjuvanted half-dose H7N9 vaccines were protected against morbidity in terms of little to no body weight loss post-challenge with a mean body weight loss of 1% (Q-H7N9) or 3% (D-H7N9). Significantly higher body weight loss was observed in ferrets receiving non-adjuvanted standard-dose vaccines  or PBS placebo (13% mean body weight loss) Fig. .To investigate viral replication in the upper respiratory tract, nose and throat swabs were collected daily post-challenge and levels of virus shedding measured by PCR and/or virus titration on MDCK cells. The ferrets immunized with non-adjuvanted standard-dose H7N9 vaccines or injected with PBS placebo showed high levels of viral RNA in the throat after challenge, whereas immunization with the AS03-adjuvanted half-dose H7N9 vaccines reduced the amount of detectable viral RNA in the throat region by at least 100-fold Fig. . Samples50/g lung tissue and all animals (8/8) in the non-adjuvanted standard-dose D-H7N9 group had detectable virus with a GM of 972 TCID50/g  than animals receiving the standard-dose vaccines  in the non-adjuvanted standard-dose Q-H7N9 group had replication-competent virus in lung tissues with a geometric mean of 126 TCIDnes Fig. . Virus tThese data taken together demonstrate that immunization with AS03-adjuvanted half-dose H7N9 split virion vaccines confer H7 cross-reactive antibody responses and protective immunity against wild-type H7N9 virus infection in the ferret model.The effect of live virus challenge on the immunized animals was investigated in more detail by performing gross and histopathological evaluation on the trachea and lungs. There was no evidence from the post-mortem examination that the H7N9 influenza virus challenge was confounded by any other infecting microorganisms or underlying disease that was present at the start of the study or acquired during the course of the study.The intratracheal challenge of ferrets with H7N9 influenza virus led to rhinitis across the treatment groups, although approximately one-third of the animals in the study had no rhinitis at all. On average, animals from one of the AS03-adjuvanted half-dose vaccine groups (D-H7N9) and animals from the PBS placebo group were most severely affected Table . TherefoThe intratracheal challenge of ferrets with H7N9 influenza virus resulted in alveolitis/alveolar damage, alveolar edema, and hemorrhage that ranged from mild to severe. The animals that received the AS03-adjuvanted half-dose vaccines were least affected in this location Table . Convers18. In addition, the animals that received the AS03-adjuvanted half-dose vaccines  seen in the current study are consistent with those reported previously in the setup of this preclinical model/2013 see19. In 2013, an avian influenza H7N9 virus emerged in China that caused five consecutive, annual waves of infections20. The virus caused infections in humans that had close contact with infected animals, but it did not gain the ability for sustained human-to-human transmission. Nevertheless, vaccines against the H7N9 virus are urgently needed as countermeasures against possible outbreaks and for pandemic preparedness3. Humans have no pre-existing, humoral immunity to H7 subtype viruses and it has become clear in earlier studies that an effective adjuvant and/or two-dose vaccination are necessary to achieve protective antibody levels23.Avian influenza A viruses are a major public health threat. Spillover events of animal viruses into the human population have the potential to cause epidemics or, in the worst case, pandemics17.In the present study, AS03-adjuvanted and non-adjuvanted H7N9 inactivated split virion vaccines administered twice (second injection administered 3 weeks after first injection) were evaluated in the naive ferret model and the protective efficacy tested in a non-lethal challenge study. The present study demonstrated that a half-dose of H7N9 inactivated split virion vaccines induced a potent humoral immune response in ferrets when formulated with an effective adjuvant such as AS03. A standard dose of non-adjuvanted vaccines was unable to induce a high percentage of HI seroconversion, highlighting the benefit of the adjuvant. These findings are consistent with results reported in preclinical studies testing an AS03-adjuvanted H5N1 split virion vaccine in ferrets. The addition of the AS03 adjuvant allowed for dose-sparing and elicited robust antibody responses24. During the fifth epidemic wave, H7N9 viruses showed more genetic diversity, but recent structural studies report that fifth wave H7 HAs have similar overall structures as compared to a first wave H7 HA. However, based on several amino acid changes in antigenic site A of LPAI H7 HA and the occurrence of HPAI viruses with changes in HA antigenic sites A, C, and D, new candidate vaccine viruses have been proposed to cover these changes25.Furthermore, two administrations of AS03-adjuvanted half-dose H7N9 vaccines induced cross-reactive antibodies that inhibit hemagglutination by Eurasian lineage H7 viruses  and neutralized the same viruses in vitro. In addition, vaccination with the AS03-adjuvanted half-dose vaccines elicited HI and neutralizing antibodies that reacted against fifth wave H7N9 viruses as well as a feline H7N2 virus of the North American lineage. These results indicate that an AS03-adjuvanted HA vaccine antigen derived from a first wave virus can induce cross-reactive antibodies reacting to more recent fifth wave viruses as well as to other H7 viruses. This cross-reactivity is likely based on conserved shared epitopes, either on the H7 HA head or HA stalk domain, that are not subjected to immune pressure18. The viral challenge experiment demonstrated that the adjuvanted vaccines can suppress fever, minimize body weight loss, and reduce the viral load in throat swabs and lungs In conclusion, the present study demonstrated that H7N9 split virion vaccines adjuvanted with AS03 induced broad and potent antibody responses in naive ferrets and protected against homologous viral challenge with low pathogenic H7N9 virus. These preclinical results together with previously reported phase I/II results support the advanced clinical development of the AS03-adjuvanted H7N9 split virion vaccines.14. Both vaccines were used for the ferret immunizations as final bulk material manufactured under good manufacturing practice (GMP). AS0335 was manufactured under good manufacturing practice (GMP) by GSK. AS03 is an Adjuvant System containing α-tocopherol and squalene in an oil-in-water emulsion (11.86 mg α-tocopherol). The AS03 used for the study described herein is the equivalent of the adult human dose. The detergent-split virion vaccines were ad-mixed with the AS03 by gentle inversion preceding each immunization.The investigational monovalent H7N9 egg-derived, inactivated, detergent-split virion vaccines used in this study were manufactured from A/Shanghai/2/2013 (H7N9)-PR8-IDCDC-RG32A reassortant vaccine virus  using reverse genetics; the virus contains the HA and NA genes of low pathogenic avian A/Shanghai/2/2013 (H7N9) virus plus six genes from A/Puerto Rico/8/1934 (H1N1) virus. The sequence is based on a virus initially isolated from a patient with fatal disease. The vaccine was manufactured at two different GSK production sites, Quebec (Q-H7N9) and Dresden (D-H7N9). The Q-H7N9 vaccine was manufactured at GSK, Sainte-Foy, Québec, Canada, using the FluLaval Quadrivalent seasonal influenza vaccine process, whereas the D-H7N9 vaccine was manufactured at GSK, Dresden, Germany, using the Fluarix Quadrivalent seasonal influenza vaccine process slightly modified. The Q-H7N9 vaccine was further developed and tested in a human clinical trialThe standard-dose non-adjuvanted vaccine formulation consisted of a target dose of 15 µg HA, whereas the half-dose adjuvanted formulations consisted of a target dose of 7.5 µg HA mixed at a 1:1 ratio with AS03 adjuvant containing 47.44 mg/ml α-tocopherol. Standard and half-dose formulations refer to standard or half doses of split virion influenza vaccines typically given to human adults. The HA content for formulation was measured by single radial immunodiffusion (SRID) assay. The antigen dose for the Q-H7N9 vaccines were less than the initially intended concentrations of 15 and 7.5 µg HA because the SRID assay used to measure the antigen concentration overestimated the antigen concentration in relation to later available reagents provided by the Center for Biologics Evaluation and Research (CBER). The adjusted vaccine doses for the Q-H7N9 vaccines based on revised SRID values were 10.5 µg HA in the non-adjuvanted standard-dose vaccine and 5.25 µg HA in the half-dose adjuvanted vaccine. Both vaccine formulations contained 0.001% thimerosal. Sterile phosphate-buffered saline  was given to the placebo group. The vaccine formulations were prepared on the day of administration (day 0 and day 21).The animal study was carried out in the central animal facilities of The Netherlands Vaccine Institute . The NVI is approved for animal work under local legislation; Dutch Animal Experimentation Act, 1977 , which is the Dutch equivalent to the European Council Directive 86/609 EEG. The protocol was licensed under protocol number 201300081 by the animal ethics committee. The study was conducted in accordance with the GSK Policy on the Care, Welfare, and Treatment of Laboratory Animals and it was reviewed by the Institutional Animal Care and Use Committee at the NVI.The ferrets were serologically tested for Aleutian disease using counterimmunoelectrophoresis (CIEP) and were confirmed seronegative for circulating H1N1 and H3N2 virus as well as H7N9 (A/Anhui/1/2013) virus in hemagglutination inhibition assays. Only animals that were found seronegative for Aleutian disease and influenza viruses were used for the current study. The animals were housed in normal cages in groups of eight animals during the acclimation phase and immunization phase, and they were transferred to glovebox isolator cages on the day of the challenge. The animals had free access to food and water. For all experimental manipulations, the ferrets were anesthetized by intramuscular injection with ketamine or a mixture of ketamine and medetomidine.g for 10 min at room temperature, serum was collected, and stored at −80 °C until used for serology. The immunization scheme and overview of the study groups can be seen in Fig. 5.5 tissue culture infectious dose 50 (TCID50) wild-type H7N9 (A/Anhui/1/2013) virus under biosafety level 3 containment as previously established18. Throat and nose swabs were taken on days 49 through 54, inclusive. Body temperatures were monitored continuously with a 10 min interval by means of an implanted recorder . The recorder was implanted into the peritoneal cavity 36 days prior the first immunization. The animals were euthanized on day 54 and lung tissue and nasal turbinates collected for viral load determination and histopathology.Eight female ferrets were used per vaccine or PBS placebo group. Animals were immunized on days 0 and 21 with non-adjuvanted standard-dose H7N9 split virion Q-H7N9 or D-H7N9 vaccine, AS03-adjuvanted half-dose H7N9 split virion Q-H7N9 or D-H7N9, or PBS placebo. On days 0, 21, and 49, 2.5 mL of blood was collected from the jugular vein in clot tubes from anesthetized animals. The tubes were centrifuged at 2000 × Madin Darby canine kidney (MDCK) and human embryonic kidney (293T) cells were maintained in Dulbecco’s modified Eagle’s medium  supplemented with penicillin-streptomycin antibiotic mix  and fetal bovine serum . BTI-TN5B1-4 (Trichoplusia ni) cells were grown in serum-free SFX medium (HyClone), containing antibiotics .37. The polybasic cleavage site of the highly pathogenic avian isolate A/Guangdong/17SF003/2016 was removed, resulting in a low pathogenic avian influenza H7N9 cleavage site and increased protein stability.The recombinant proteins, including H7 from A/Anhui/1/2013, H7 from A/Guangdong/17SF003/2016, H7 from A/Hunan/02285/2017, H7 from A/Hong Kong/2014/2017, and H7 from A/feline/New York/16-040082/2016 were produced in the baculovirus expression vector system as previously described38. Briefly, the H7 HA segment of A/Hong Kong/2014/2017 and A/Hunan/02285/2017 were combined, respectively, with the seven other genomic influenza A segments of PR8, resulting in 7:1 reassortants. The H7 HA and N2 NA segment of A/feline/New York/16-040082/2016 were combined with the six internal segments of PR8, resulting in 6:2 virus variants. The HA and NA cDNA was synthetically produced (Thermo Fisher), and all virus reassortants were sequenced to confirm the genotype. The viruses were grown in 8–10-day-old embryonated chicken eggs (Charles River Laboratories) at 37 °C for 48 h and the allantoic fluid was harvested.The H7 low pathogenic virus variants were rescued in a A/Puerto Rico/8/1934 (PR8) background by reverse genetics techniques as previously described18.The wild-type influenza H7N9 A/Anhui/1/2013 virus isolate used for the challenge phase was passaged three times in embryonated chicken eggs and once in MDCK cells. The virus is based on an isolate from a fatal human case in China and was kindly provided by the Pandemic Influenza Preparedness FrameworkViruses and proteins used in serological assays in this study are indicated in the phylogenetic tree in Fig. Microtiter plates  were coated with recombinant H7 HA proteins at a concentration of 2 µg/mL diluted in coating buffer (SeraCare) at 4 °C overnight. The following day, the plates were washed three times with PBS (Gibco) supplemented with 0.1% Tween 20 (PBS-T) and blocked with 220 µL of blocking solution (PBS-T supplemented with 3% goat serum (Life Technologies) and 0.5% milk powder (American Bio) for 1 h at room temperature. Ferret serum samples were diluted to an initial starting concentration of 1:100, serially diluted 1:2 in blocking solution, and incubated for 2 h at room temperature. The plates were washed three times with PBS-T and 50 µL anti-ferret IgG (goat anti-ferret IgG (gamma chain-specific) horseradish peroxidase (HRP) conjugate, Alpha Diagnostics, #70530) per well was added. The 96-well plates were incubated for 1 h at room temperature, washed four times with PBS-T, and developed with 100 µL/well of SigmaFast o-phenylenediamine dihydrochloride . To stop the reaction, 50 µL/well 3 M hydrochloric acid (Thermo Fisher) was added and the plates were read at 490 nm with a microtiter plate reader (BioTek). The data were analyzed in Microsoft Excel and GraphPad Prism Version 8. The cutoff value was defined as the average of all blank wells plus three times the standard deviation of the blanks. The area under the curve (AUC) was calculated based on the cutoff value.39. In brief, after treatment with cholera filtrate  and heat-inactivation at 56 °C, the sera were tested for the presence of anti-HA antibodies. For this purpose, 1% turkey erythrocytes were used and four HA-units of the different H7 influenza strains. Titers of 1:5 were assigned to HI-negative samples to facilitate data analysis and data representation.Hemagglutination inhibition (HI) antibody titers against H7N9 A/Shanghai/2/2013, H7N9 A/Anhui/1/2013, H7N7 A/Netherlands/219/2003, and H7N3 A/mallard/Netherlands/12/2000 were determined at Viroclinics as described previoulsySerum antibody titers against the fifth wave viruses  and H7N2 (A/feline/New York/16-040082/2016) were measured in a slightly modified HI assay at the Icahn School of Medicine at Mount Sinai. The ferret sera were treated with RDE (Denka Seiken) to remove nonspecific inhibition of hemagglutination. In brief, for 25 µL sera 75 µL RDE was added and incubated overnight at 37 °C. The reaction was stopped by the addition of 75 µL sodium citrate (2.5%) and incubation at 56 °C for 1 h. The sera were diluted with 75 µL PBS, resulting in an overall dilution of 1:10 (limit of detection of the assay). The pre-diluted sera were serially diluted 1:2 in v-bottom 96-well plates (Thermo Fisher). The viruses  were diluted to 8 hemagglutination units (HAU)/50 µL in PBS and 25 µL/well of virus was added to the serially diluted sera in the v-bottom plates (Sigma). The plates were incubated for 30 min at room temperature on a shaker. Chicken red blood cells  were diluted to a concentration of 0.5% in PBS and 50 µL/well were added to the sera/virus mixture. The plates were incubated at 4 °C for 1 h, the results recorded in Microsoft Excel and analyzed in GraphPad Prism Version 8. The HI titer was defined as the last dilution in which hemagglutination does not occur. Negative samples  were assigned a value of 5  for GMT calculation and data representation.39. In brief, sera were tested after heat-inactivation at 56 °C for 30 min for the presence of VN antibodies using a microneutralization assay with ~100× TCID50 of the different H7 influenza strains. Neutralization titers were calculated as the dilution giving 50% neutralization and expressed as the reciprocal of the dilution. Samples below the limit of detection were assigned a value of 8 for GMT calculations and data representation.Virus neutralization (VN) titers against H7N9 (A/Shanghai/2/2013 and A/Anhui/1/2013), H7N7, and H7N3 were measured at Viroclinics as described previously5 cells/mL complete DMEM in 96-well tissue culture plates (Sigma) and incubated overnight at 37 °C. Ferret sera were RDE-treated as described above, resulting in an initial dilution of 1:10 (limit of detection). The pre-diluted sera were serially diluted 1:2 in UltraMDCK media (Lonza), containing tosyl phenylalanyl chloromethyl ketone (TPCK)-treated trypsin  at a concentration of 1 μg/mL, in 96-well culture plates. The viruses were diluted to a concentration of 100-TCID50, added to the serially diluted sera, and incubated for 1 h at room temperature on a shaker. The MDCK cells were washed with 220 µL PBS/well and 100 µL/well of the virus/sera mixture was added to the cells. The plates were incubated for 72 h at 33 °C. Chicken RBCs were diluted to a concentration of 0.5% in PBS and 50 µL of cell supernatant was transferred to v-bottom plates (Sigma). RBCs (50 µL/well) were added to the v-bottom plates and the plates were incubated for 1 h at 4 °C. The neutralization titer was defined as the last dilution in which hemagglutination does not occur. Negative samples were assigned a value of 5  for GMT calculation and data representation. The results were recorded in Microsoft Excel and analyzed in GraphPad Prism Version 8.The more recently circulating H7 viruses were tested in a modified microneutralization assay at the Icahn School of Medicine at Mount Sinai. MDCK cells (100 µL/well) were seeded at a concentration of 1.6 × 1050/mL, respectively. CDU were determined from a standard curve produced from a stock virus with a known titer which was serially diluted in a 10-fold dilution series, with each dilution undergoing nucleic acid extraction and Taqman PCR amplification in the same manner as test samples. The undiluted virus (virus stock) had a titer of 8.5 log10 TCID50/mL, was diluted from 100 to 10−8 (in 10-fold dilution steps) and tested in triplicate in the PCR resulting in a slope of the standard curve of 3.49 and intercept of 40.84. On the basis of the standard curve, the lower limit of quantification (LLOQ) of PCR was determined to be 0.8 log10 CDU/mL. Samples below the LLOQ are shown as <0.8 log10 CDU/mL. All samples that gave a Ct value were analyzed by virus titration. Virus titration was performed on a newly thawed aliquot as described elsewhere40. The LLOQ of virus titration was 0.8 log10 TCID50/mL. Samples below this cutoff value were scored as <0.8 log10 TCID50/mL. Virus RNA load and infectious virus titer in tissues were expressed as log10 CDU/gram of tissue and log10 TCID50/g of tissue, respectively. All tissue samples were analyzed by TaqMan PCR and virus titration. The LLOQ of each lung and turbinate sample varied according to the individual tissue weight.Levels of viral RNA (from infectious and non-infectious virus) were determined by means of influenza virus A matrix gene-specific TaqMan polymerase chain reaction (PCR) and levels of infectious (replication-competent) virus were determined by virus titration on MDCK cells. Virus RNA load and infectious virus titer in the upper respiratory tract (URT) were expressed as Control Dilution Units (CDU)/mL and TCIDAt the time of scheduled euthanasia on day 54, a complete macroscopic gross-pathological assessment of all organ systems was performed, and all abnormalities were noted. All trachea and lung lobes were visually inspected and weighed, and any abnormal lesions were noted and described. The nasal turbinates and left lung with trachea of each animal were collected and these tissues were fixed with 10% neutral-buffered formalin for histopathological examination. Following fixation, tissue sections were embedded in paraffin blocks for processing, micro-sectioned, and stained with hematoxylin and eosin for histopathological examination by light microscopy. The following histopathological abnormalities were noted when present in the nasal turbinates or the lungs: severity of inflammatory cell infiltration, congestion, damage, edema, hemorrhage, hyperplasia/inflammation. All personnel performing the analyses in which interpretation of data was required were unaware of the study treatment assignments, and specimens were masked by allocating a unique sample number to each sample collected.Further information on research design is available in the Supplementary MaterialReporting Summary


The seminal description of the cellular restriction factor APOBEC3G and its antagonism by HIV-1 Vif has underpinned two decades of research on the host-virus interaction. We recently reported that HIV-1 Vif is also able to degrade the PPP2R5 family of regulatory subunits of key cellular phosphatase PP2A . We now identify amino acid polymorphisms at positions 31 and 128 of HIV-1 Vif which selectively regulate the degradation of PPP2R5 family proteins. These residues covary across HIV-1 viruses in vivo, favouring depletion of PPP2R5A-E. Through analysis of point mutants and naturally occurring Vif variants, we further show that degradation of PPP2R5 family subunits is both necessary and sufficient for Vif-dependent G2/M cell cycle arrest. Antagonism of PP2A by HIV-1 Vif is therefore independent of APOBEC3 family proteins, and regulates cell cycle progression in HIV-infected cells. The canonical function of HIV-1 Vif is to recruit the cellular restriction factor APOBEC3G for CUL5 E3 ligase and ubiquitin-proteasome-dependent degradation in infected cells, preventing APOBEC3G encapsidation and enhancing virion infectivity . This inThe other cell biological phenotype associated with Vif in multiple studies is the induction of G2/M cell cycle arrest . Vif-depWe have recently discovered that, in addition to APOBEC3 family proteins, Vif is also able to degrade the B56 family of regulatory subunits of the ubiquitous heterotrimeric serine-threonine phosphatase PP2A (PPP2R5A-E) in HIV-1-infected CEM-T4 T cells  and primThe ability of Vif to degrade PPP2R5 subunits  are also able to restrict HIV replication , and VifFirst, we tested the ability of Vif mutants lacking the ability to deplete PPP2R5B to deplete HA-tagged APOBEC3F in 293Ts, similar to our initial flow cytometry screen . As prevTo avoid the possibility of over-expression artefacts, we next focussed on endogenous APOBEC3 and PPP2R5 family proteins expressed in CEM-T4 T cells (CEM-T4s). These cells were transduced with the panel of I31, I128 and R33/K34 Vif mutants found in our flow cytometry screens to be specifically defective for PPP2R5B depletion, together with a Y44 mutant  to serve as a control.Levels of an indicative PPP2R5 subunit for which a reliable antibody is available -based functional proteomic approach . CEM-T4 In total, we identified 8781 proteins , includiAs expected, all Vif mutants tested were defective for PPP2R5B depletion, and the Y44A mutant was also defective for APOBEC3G depletion . In addiWe previously showed that expression of Vif results in extensive remodelling of the phosphoproteome in HIV-infected cells, including activation of the aurora kinases AURKA and AURKB, effects we attributed to PP2A antagonism . As expeTogether with APOBEC3 family antagonism, it has been known for >10 years that certain Vif variants (including NL4-3 Vif) are also able to induce G2/M cell cycle arrest, and that this is dependent on CUL5 E3 ligase recruitment and the ubiquitin-proteasome system . The VifSince both PP2A-B56  and aurora kinases are required to coordinate mitotic progression , we hypoTo confirm this result and formally evaluate cell cycle progression, we measured DNA content of CEM-T4s 48 hr after transduction with WT or mutant Vif variants. Again, WT Vif, but not Vif mutants lacking the ability to deplete PPP2R5 subunits, caused G2/M arrest . As a coIn addition to APOBEC3 and PPP2R5 family proteins, we recently showed that NL4-3 Vif is also able to target FMR1 and DPH7 . Both I1Consistent with PPP2R5A-E depletion by Vif, inhibition of PP2A with okadaic acid causes G2/M cell cycle arrest in CEM-T4s . HoweverSince Vif-dependent cell cycle arrest is abrogated by point mutations which rescue quite distinct PPP2R5 subunits compare , some fuFirst, we confirmed that, as in CEM-T4s, expression of WT NL4-3 Vif in HeLas causes cell cycle arrest . LikewisTo confirm that knockdown of all PPP2R5 subunits is necessary for cell cycle arrest, we repeated the experiment using pools of siRNA targeting 4 out of 5 PPP2R5 subunits (‘minus one’). Again, and with the exception of PPP2R5B, cell cycle arrest was only observed when all subunits were knocked down together . InteresThat depletion of PPP2R5B is neither sufficient  nor requFinally, knockdown of FMR1 and DPH7  did not The ability to cause G2/M cell cycle arrest is known to vary between naturally occurring HIV-1 Vif variants from clade B viruses (such as NL4-3 and HXB2), as well as viruses from other clades . We therInterestingly, residues 31 (I or V), 33  and 128  all showed obvious polymorphism, with NL4-3 Vif encoding the commonest amino acids at positions 31 (I) and 128 (I), and the second commonest amino acid at position 33 (R). We therefore evaluated each of the common polymorphisms as single point mutations on a background of NL4-3 Vif using our flow cytometric screen. Conservative substitutions in positions 31 and 128 partially impaired the ability of NL4-3 Vif to deplete PPP2R5B , whereas I128R resulted in more marked impairment . Likewis31V, 33G and 128I), and the macrophage-tropic patient isolate YU2 . As a control, we also included a Vif variant from the clade B transmitted founder virus CH470   . Consist4-3 Vif) . Depleti4-3 Vif) .To further assess the function of these variants against other APOBEC3 and PPP2R5 family members, we again adopted a TMT-based functional proteomic approach . As wellIn this experiment, we identified 8265 proteins , includiAs a functional readout, we tested the ability of the same panel of Vif variants to cause cell cycle arrest. Consistent with previous reports  and corrUnlike individual mutations of residues 31 or 128, combined I31A and RI127/128YR mutations in NL4-3 Vif (Vif AYR) completely abolished the depletion of all PPP2R5 family subunits in our proteomic analysis , withoutWe next sought to confirm these results in the context of viral infection by introducing the same mutations into the NL4-3-based HIV-AFMACS molecular clone . This EnTo assess the function of Vif AYR against APOBEC3 and PPP2R5 family members during viral infection, we first adopted a TMT-based functional proteomic approach to compare mock-infected cells with cells infected with ΔVpr-Vif WT, ΔVpr-ΔVif or ΔVpr-Vif AYR viruses . CEM-T4sIn this experiment, we identified 6297 proteins , includiIf the ability of Vif to antagonise PP2A is maintained by selection pressure in vivo, combinations of unfavourable (less active against PPP2R5A-E) mutations in residues 31 and 128  should be rare amongst naturally occurring HIV-1 Vif variants. Furthermore, if effects on viral fitness are synergistic, such combinations should occur less frequently than predicted by chance. We therefore examined covariance of polymorphisms of residues 31 and 128 across the clade B HIV-1 Vif sequences available from the Los Alamos National Laboratory Web Alignments database .Amongst these sequences, 21.8% encode 31V (less active) and 33.6% encode R128 (less active). By chance, combinations of 31V and 128R would therefore be expected in 7.3% of sequences. Conversely, this combination is observed in only 5.8% of sequences. Whist this difference appears modest, the association between these polymorphisms is highly statistically significant , we constructed phylogenetic trees of all Vif variants based on Vif  or Nef, Taken together, these data therefore provide evidence of a functional interaction between residues 31/128, and suggest significant in vivo selection pressure to maintain the ability of Vif to antagonise PP2A.whether they are important, and why they are important.The study of cellular proteins and processes targeted by HIV has provided critical insights into the host-virus interaction. Typically, these targets have been identified piecemeal, using candidate approaches. In contrast, we have recently adopted unbiased proteomic approaches to identify novel substrates of HIV accessory proteins . A key cIn this study, we sought to address these questions for Vif targets PPP2R5A-E. By demonstrating that depletion of PPP2R5 family subunits by Vif is separable from targeting of APOBEC3 family proteins, we formally prove that PP2A antagonism is neither required for, nor an epiphenomenon of, APOBEC3 family protein depletion. Combined with evidence of conservation across HIV-1 viruses and the broader lentiviral lineage , these oStrikingly, the critical residues for PPP2R5 depletion identified in our screen included several previously determined to be important for Vif-dependent cell cycle arrest in other, independent studies  . As wellWe were initially puzzled because some Vif point mutants were markedly impaired in their ability to cause cell cycle arrest, yet retained the ability to deplete at least some PPP2R5 family subunits. Furthermore, the ability of Vif to cause cell cycle arrest did not appear to correlate with depletion of any one, specific PPP2R5 subunit. In fact, because efficient depletion of all PPP2R5 subunits is required to halt cell cycle progression, these are not paradoxes at all. This same model also suggests explanations for two related phenomena.First, expression of HIV-1 Vif in mouse or COS cells results in depletion of PPP2R5D, but does not cause cell cycle arrest . As withAmongst all HIV-1 Vif sequences analysed here, the commonest single combination of residues at positions 31 and 128 was 31I/128I, accounting for approximately 30% of Vif variants. Most of these sequences also encode 33R or 33K and, like NL4-3 and CH470 Vif, are therefore expected to efficiently degrade PPP2R5A-E, and cause cell cycle arrest. Conversely, only approximately 5% encode 31V/128R, and are therefore expected to be severely impaired for PPP2R5A-E degradation. It is likely that most of the remaining Vif variants, like YU2 and HXB2, are active against at least some PPP2R5 family members, but may be variably attenuated in their ability to cause cell cycle arrest. Interestingly, naturally occurring Vif variants have also been shown to exhibit a spectrum of activity against APOBEC3 family proteins, including variants which fail to neutralise one or more APOBEC3 family proteins .Mechanistically, hyperphosphorylation of aurora kinase substrates is expected to contribute significantly to Vif-dependent cell cycle arrest . NonetheMany RNA and DNA viruses cause and are thought to benefit from cell cycle arrest . In HIV Functional redundancy with Vpr may also help explain why Vif-dependent cell cycle arrest is not more strictly conserved across naturally occurring HIV-1 Vif variants. In addition, key polymorphic residues which regulate PPP2R5 antagonism may be subject to balancing selection pressures. First, the requirement to maintain the Exonic Splicing Silencer of Vpr (ESSV) at the RNA level limits the sequence variability tolerated at position 128 . Second,Vif-dependent cell cycle arrest correlates with viral cytopathicity , and wasAs importantly, the ability to assess APOBEC3-independent effects of Vif on viral fitness in vitro has hitherto been limited to comparisons between WT and Vif-deficient viruses on an APOBEC3-negative background, such as the CEM-SS cell line. In contrast, the point mutants identified in this study maintain the ability to antagonise APOBEC3 family proteins, and will therefore allow the assessment of Vif-dependent PP2A antagonism by the community in a full range of cell types, including primary and myeloid cells, as well as providing a mechanistic framework to interpret the results.5 to 2 × 106 cells/ml in RPMI supplemented with 10% fetal calf serum (FCS), 100 units/ml penicillin and 0.1 mg/ml streptomycin at 37°C in 5% CO2. HeLa cells and HEK 293T cells (authenticated by STR profiling [CEM-T4 T cells  and THP-rofiling ) were obPrimary human CD4+ T cells were isolated from peripheral blood by density gradient centrifugation over Lympholyte-H (Cedarlane Laboratories) and negative selection using the Dynabeads Untouched Human CD4 T Cells kit (Invitrogen) according to the manufacturer’s instructions. Purity was assessed by flow cytometry for CD3 and CD4 and routinely found to be ≥95%. Cells were activated using Dynabeads Human T-Activator CD3/CD28 beads (Invitrogen) according to the manufacturer’s instructions and cultured in RPMI supplemented with 10% FCS, 30 U/ml recombinant human IL-2 (PeproTech), 100 units/ml penicillin and 0.1 mg/ml streptomycin at 37°C in 5% CO2.Ethical permission for this study was granted by the University of Cambridge Human Biology Research Ethics Committee (HBREC.2017.20). Written informed consent was obtained from all volunteers prior to providing blood samples.Sequences for Vif variants from NL4-3 (AF324493.2), HXB2 (K03455.1), YU2 (GenBank: M93258.1) and CH470 (JX972238-JX972249) viruses were obtained from GenBank. The CH470 transmitted founder (TF) Vif sequence was inferred as previously described .E-P2A-Vif-W vectors). In these vectors, Vif variants are expressed from the Friend spleen focus-forming virus (SFFV) promoter as EGFP-P2A-Vif, downstream of EGFP and a ‘self-cleaving’ Porcine teschovirus-1 2A (P2A) peptide.For co-expression of codon optimised Vif variants with EGFP, gBlocks (IDT) encoding NL4-3, HXB2, YU2 or CH470 Vif were incorporated into pHRSIN-SE-P2A-SBP-ΔLNGFR-W  in placeComplete sequences for all gBlocks are included in The parental vector (in which EGFP and the SBP-ΔLNGFR cell surface selection marker are expressed from the SFFV promoter as EGFP-SBP-ΔLNGFR) was used here as a control. Where indicated, pHRSIN-SE-W-pSV40-puro (in which EGFP is expressed from the SFFV promoter as a single transgene) was used as an alternative control .To generate stable 293T cell lines for our flow cytometric screen, N-terminal 4xHA-tagged PPP2R5B and C-terminal 4xHA-tagged APOBEC3G were expressed using pHRSIN-S-W-pGK-puro exactly as previously described . HeLas sTo generate a library of Vif point mutants, a PCR and Gibson assembly-based approach was used to modify codon-optimised NL4-3 Vif directly in pHRSIN-SE-P2A-Vif-W .Briefly, forward and reverse primers encoding each point mutation were designed with ~15 bp fully complementary flanking sequences. These mutation-specific primers were used in pairwise PCR reactions in conjunction with common primers complementary to the vector backbone, which was cut between XhoI/KpnI sites. The two PCR products were then assembled into the vector using the NEBuilder HiFi DNA Assembly Master Mix (NEB).Sequences for all primers used are tabulated in HIV-AFMACS  has been previously described . To intrTo generate ΔVpr-Vif WT virus , a silent mutation was introduced into Vif codon 173 , eliminating the Vpr start codon in the +two reading frame. Additional point mutations were introduced to generate ΔVpr-Vif AYR virus  and ΔVpr-ΔVif virus .Final Vif coding sequences for each virus are included in For the flow cytometric screen, 293T cells stably expressing HA-tagged PPP2R5B or APOBEC3G were transfected with 200 ng/well control or Vif expression vector in 24-well plates using FuGENE 6 (Promega). After 36 hr, cells were harvested with trypsin-EDTA and analysed by flow cytometry.For RNAi-mediated knockdown, HeLa cells were transfected with custom siRNA duplexes (Sigma) using transfected using Lipofectamine RNAiMAX (Invitrogen) according to the manufacturer’s instructions.5 cells/well were seeded in 6-well plates 24 hr prior to transfection with a total of 50 pmol/well siRNA . Knockdown was verified by real-time PCR or immunoblot 24 hr post-transfection, and cells were re-seeded prior to cell cycle analysis 48 hr post-transfection (target 50% confluency).Briefly, 2 × 10All siRNA target sequences used are tabulated in VSVg-pseudotyped lentivector stocks were generated by co-transfection of 293Ts with pHRSIN-based lentivector, p8.91 and pMD.G at a ratio of 2:1:1 (μg) DNA and a DNA:FuGENE 6 ratio of 1 μg:3 μl. Media was changed the next day and viral supernatants harvested and filtered (0.45 μm) at 48 hr prior to concentration with Lenti-X Concentrator (Clontech) and storage at −80°C.VSVg-pseudotyped HIV-AFMACS viral stocks were generated by co-transfection of 293Ts with HIV-AFMACS molecular clones and pMD.G at a ratio of 9:1 (μg) DNA and a DNA:FuGENE 6 ratio of 1 μg:6 μl. Viral supernatants were harvested, filtered, concentrated and stored as per pHRSIN-based lentivector stocks.-MOI). Typically, a dilution series of each stock was tested, and titre determined by linear regression of -ln(1-f) on volume of stock.Lentivector/viral stocks were titrated by transduction/infection of known numbers of relevant target cells with known volumes of stocks under standard experimental conditions, followed by flow cytometry for EGFP (GFP-expressing lentivectors) or SBP-ΔLNGFR and CD4 (HIV-AFMACS viruses) at 48 hr to identify the fraction of transduced/infected cells (f) containing at least one transcriptionally active provirus (EGFP positive or SBP-ΔLNGFR positive/CD4 low). The number of transducing/infectious units present was then calculated by assuming a Poisson distribution , 2 mM L-glutamine, 2 mM EDTA and 10 mM HEPES) at 107 cells/ml and incubated with Dynabeads Biotin Binder (Invitrogen) at a bead-to-total cell ratio of 4:1 for 30 min at 4°C. Bead-bound cells expressing SBP-ΔLNGFR were selected using a DynaMag-2 magnet (Invitrogen), washed to remove uninfected cells, then released from the beads by incubation in complete RPMI with 2 mM biotin for 15 min at room temperature (RT). Enrichment was assessed by flow cytometry pre- and post-selection.AFMACS-based selection of CEM-T4s using the streptavidin-binding SBP-ΔLNGFR affinity tag was carried out essentially as previously described . BrieflyFor TMT-based whole cell proteomic analysis of transduced or infected CEM-T4s, washed cell pellets were lysed in 50 mM HEPES pH 8 with 5% SDS followed by 10 min (30 s on/off) sonication in a Bioruptor Pico sonicator (Diagenode) at 18°C. Lysates were quantified by BCA assay (Thermo Scientific) and 25 μg  or 10 μg  total protein/condition used for further analysis.3PO4 was added and samples mixed by vortexing briefly. 6 volumes of 90% methanol HEPES pH 7.1 (loading buffer) were then added and pipette-mixed before loading onto columns using a vacuum manifold.Sample volumes were equalised with lysis buffer and proteins reduced and alkylated by addition of 10 mM TCEP and 20 mM iodoacetamide followed by incubation at RT for 30 min, protected from light. Samples were then processed using S-Trap micro columns (Protifi). To each sample 10% v/v HSamples were then washed with 4 × 150 μl loading buffer. A 1:25 enzyme:protein ratio of LysC/trypsin mix (Promega) was added to each column in 30 μl of 50 mM HEPES pH 8 with 0.1% sodium deoxycholate (SDC). Columns were placed into microcentrifuge tubes and incubated for 6 hr at 37°C in a Thermomixer S (Eppendorf) without shaking. Open tubes of water were placed in empty positions and the Thermomixer lid used to minimise evaporation.After incubation, peptides were eluted in three stages: 40 μl 10 mM HEPES pH 8; 35 μl 0.2% formic acid (FA); then 35 μl 0.2% FA in 50% Acetonitrile (ACN). Samples were dried for a short period in a vacuum centrifuge to evaporate ACN and then acidified with FA to precipitate SDC. Samples were then made up to ~100 μl with water, then 600 μl ethyl acetate was added and samples vortexed vigorously. After centrifugation at 15000 g for 5 min the lower phase (containing peptides) was retained and the upper phase (containing SDC and ethyl acetate) was discarded.After drying fully in a vacuum centrifuge, samples were resuspended in 21 μl 100 mM HEPES pH 8, to which was added 0.2 mg of TMT label dissolved in 9 μl ACN. After 1 hr incubation at RT samples were analysed by LCMS to ensure complete labelling, then pooled and dried by ~50% in a vacuum centrifuge. The pooled sample was made up to ~1 ml in a final concentration of 0.1% triflouracetic acid (TFA) and the pH was adjusted to <2 with FA. The samples were then subjected to C18 SPE clean-up using 500 mg Sep-Pak tC18 cartridges (Waters). Columns were wetted with 1 ml ACN and equilibrated with 3 ml 0.1% TFA before loading the sample, washing with 2 ml 0.1% TFA and eluting with 250 μl 40% ACN, 250 μl 80% ACN and 250 μl 80% ACN. The eluates were dried in a vacuum centrifuge.HpRP fractionation was conducted on an Ultimate 3000 UHPLC system (Thermo Scientific) equipped with a 2.1 mm ×15 cm, 1.7 µm Kinetex EVO C18 column (Phenomenex). Solvent A was 3% ACN, Solvent B was 100% ACN, and solvent C was 200 mM ammonium formate (pH 10). Throughout the analysis solvent C was kept at a constant 10%. The flow rate was 400 µl/min and UV was monitored at 280 nm. Samples were loaded in 90% A for 10 min before a gradient elution of 0–10% B over 10 min (curve 3), 10–34% B over 21 min (curve 5), 34–50% B over 5 min (curve 5) followed by a 10 min wash with 90% B. 15 s (100 µl) fractions were collected throughout the run. Peptide-containing fractions were orthogonally recombined into 24  or 12  fractions, dried in a vacuum centrifuge and stored at −20°C prior to analysis.5 AGC target, 50 msec maximum injection time, ions injected for all parallelisable time. MS2: quadrupole isolation at an isolation width of m/z 0.7, CID fragmentation (NCE 35) with the ion trap scanning out in rapid mode from m/z 120, 8 × 103 AGC target, 70 msec maximum injection time, ions accumulated for all parallelisable time. In synchronous precursor selection mode the top 10 MS2 ions were selected for HCD fragmentation (65NCE) and scanned out in the orbitrap at 50000 resolution with an AGC target of 2 × 104 and a maximum accumulation time of 120 msec, ions were not accumulated for all parallelisable time. The entire MS/MS/MS cycle had a target time of 3 s. Dynamic exclusion was set to + /− 10 ppm for 90 s, MS2 fragmentation was trigged at 5 × 103 ions.Data were acquired on an Orbitrap Fusion mass spectrometer (Thermo Scientific) coupled to an Ultimate 3000 RSLC nano UHPLC (Thermo Scientific). Solvent A was 0.1% FA and solvent B was ACN/0.1% FA. HpRP fractions were resuspended in 20 µl 5% DMSO 0.5% TFA and 10 µl injected. Fractions were loaded at 10 μl/min for 5 min on to an Acclaim PepMap C18 cartridge trap column  in 0.1% TFA. After loading, a linear gradient of 3–32% B over 3 hr was used for sample separation over a column of the same stationary phase  before washing with 90% B and re-equilibration. An SPS/MS3 acquisition was used for all samples and was run as follows. MS1: quadrupole isolation, 120000 resolution, 5 × 10Spectra were searched using Mascot within Proteome Discoverer 2.2 in two rounds. The first search was against the UniProt human reference proteome, a custom HIV proteome (adjusted to include the exact protein coding sequences used) and a compendium of common contaminants . The second search took all unmatched spectra from the first search and searched against the human trEMBL database. The following search parameters were used. MS1 tol: 10 ppm; MS2 tol: 0.6 Da; fixed mods: carbamidomethyl (C) and TMT ; var mods: oxidation (M); enzyme: trypsin (/P). MS3 spectra were used for reporter ion based quantitation with a most confident centroid tolerance of 20 ppm. PSM FDR was calculated using Mascot percolator and was controlled at 0.01% for ‘high’ confidence PSMs and 0.05% for ‘medium’ confidence PSMs. Normalisation was automated and based on total s/n in each channel.The mass spectrometry proteomic data have been deposited to the ProteomeXchange consortium via the PRIDE , partnerAbundances or proteins/peptides satisfying at least a ‘medium’ FDR confidence were subjected to further analysis in Excel 2016 (Microsoft) and R v.3.6.1 .t-scores) for each condition:  according to the manufacturer’s instructions. Permeabilised cells were stained with AF647-conjugated rabbit anti-HA antibody (Abcam) for 20 min at RT, washed, and analysed with an LSR Fortessa flow cytometer (BD Biosciences). Doublets were excluded by comparing SSC-W with SSC-H. Depletion of HA-tagged PPP2R5B, APOBEC3F, or APOBEC3G was quantified by the ratio of median AF647 fluorescence in GFP+ /GFP-  cells for each condition, after deducting background fluorescence of control 293Ts (no HA-tagged protein expression).5 washed CEM-T4s were stained with fluorochrome-conjugated anti-LNGFR and anti-CD4 for 15 min at 4°C then fixed in PBS/1% paraformaldehyde and analysed as above. For titration of lentivectors, GFP fluorescence was quantified without antibody staining.For titration of HIV-AFMACS viruses, typically 2 × 106 cells/condition (CEM-T4s) or a 50% confluent 6-well/condition (HeLas) were washed with PBS, then fixed for 30 min with ice-cold 90% methanol. Fixed cells were stained with 7-AAD at 25 μg/ml for 30 mins at 37°C, then analysed with an LSR Fortessa flow cytometer (BD Biosciences). Doublets were excluded by comparing SSC-W with SSC-H. The FlowJo cell cycle platform was used to determine the fraction of cells in each phase of cell cycle. G2/M cell cycle arrest was quantified by the ratio of cells in G2/M for each condition, compared with mock-transduced/transfected cells.For cell cycle analysis in transduced CEM-T4s and transduced/transfected HeLas, 1 × 10For cell cycle analysis in CEM-T4s infected with HIV-AFMACS, cells were first stained with FITC-conjugated anti-LNGFR (BioLegend), then washed, fixed and stained with 7-AAD and analysed as above. G2/M cell cycle arrest was quantified by the ratio of cells in G2/M for LNGFR+ /LNGFR- (uninfected) cells for each condition.For cell cycle analysis in primary human CD4+ T cells infected with HIV-AFMACS, CD3/CD28 Dynabeads were first removed using a DynaMag-2 magnet (Invitrogen). Cells were stained with FITC-conjugated anti-LNGFR (BioLegend) and BV421-conjugated anti CD4 (BioLegend), then washed, fixed, stained with 7-AAD and analysed as above. G2/M cell cycle arrest was quantified by the ratio of cells in G2/M for LNGFR+ CD4- /LNGFR- CD4+ (uninfected) cells for each condition.Washed cell pellets were lysed in PBS/2% SDS supplemented with Halt Protease and Phosphatase Inhibitor Cocktail (Thermo Scientific) and benzonase (Sigma) for 10 min at RT. Post-nuclear supernatants were heated in Laemelli Loading Buffer for 5 min at 95 °C, separated by SDS-PAGE and transferred to Immobilon-P membrane (Millipore). Membranes were blocked in PBS/5% non-fat dried milk (Marvel)/0.2% Tween and probed with the indicated primary antibody overnight at 4 °C. Reactive bands were visualised using HRP-conjugated secondary antibodies and SuperSignal West Pico or Dura chemiluminescent substrates (Thermo Scientific). Typically 10–20 μg total protein was loaded per lane.For quantification of PPP2R5 subunit mRNA levels, total RNA was extracted using TRIzol reagent (Invitrogen), followed by DNase I treatment. cDNA was synthesised using the Superscript III First-Strand Synthesis System (Invitrogen) with Oligo(dT) (Invitrogen), and 10 ng/test subjected to real-time PCR using the primers tabulated in To control for PCR efficiency and allow comparison of abundances between different PPP2R5 subunits, standard curves were generated using plasmids encoding PPP2R5A, PPP2R5B, PPP2R5C, PPP2R5D and PPP2R5E , together with housekeeping gene Tata Binding Protein . Abundances of different PPP2R5 subunits for each cell type or condition were derived from the corresponding standard curves, and expressed as mRNA copy numbers relative to TBP as an endogenous reference.The previously determined structure of Vif in complex with CUL5, CBFβ, and ELOB/C (PDB ID: 4N9F) was used to identify solvent-exposed residues to be mutated in this study . Structuhttp://www.hiv.lanl.gov/). The following server options were selected: Alignment type, Web ; Organism, HIV-1/SIVcpz; Subtype, M group without recombinants (A-K); DNA/Protein, protein; Year, 2018; Format, FASTA.Protein sequence Web Alignments for Vif, Env, Gag and Nef were downloaded from the Los Alamos HIV Sequence Alignments Database  using the information in the sequence name for example To examine amino acid polymorphism in naturally occurring Vif variants at positions corresponding to residues 31, 33/34 and 128 of NL4-3 Vif, sequence logos were generated using WebLogo . FurtherTo construct phylogenetic trees, only viruses with protein sequences available for all of Vif, Env, Gag and Nef were included. This enabled direct comparison of trees based on different viral proteins. Multiple sequence alignments and phylogenetic tree data (in Newick format) for each viral protein were generated using the Clustal Omega web server , then vihttps://github.com/annaprotasio/Marelli_et_al_HIV_Vif .All alignment and sequence files, scripts and details of the bioinformatic analyses described here are available at: t-tests , Fisher’s exact tests (two-tailed) and 95% confidence intervals were calculated using Prism 7.0 (GraphPad). General data manipulation was conducted using Excel 2016 (Microsoft).Where indicated, Student’s  In the interests of transparency, eLife publishes the most substantive revision requests and the accompanying author responses.Acceptance summary:In addition to inducing degradation of APOBEC3G, Vif also triggers proteasomal degradation of PPP2R5 family regulatory subunits of the host phosphatase PP2A. In the present study, the authors identified mutations in Vif separating these activities and provide evidence that the degradation of PPP2R5 family members accounts for the cell cycle arrest caused by certain Vif proteins. Thus, the findings provide new insights on how HIV-1 manipulates its host cells.Decision letter after peer review:eLife. Your article has been reviewed by two peer reviewers, and the evaluation has been overseen by Frank Kirchhoff as the Reviewing Editor and Päivi Ojala as the Senior Editor. The following individual involved in review of your submission have agreed to reveal their identity: Linda Chelico (Reviewer #1).Thank you for submitting your article "Antagonism of PP2A is an independent and conserved function of HIV-1 Vif and causes cell cycle arrest" for consideration by The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.Summary:In the present study, Matheson, Lehner and coworkers followed up on their previous work showing that, in addition to inducing degradation of APOBEC3G, Vif also triggers the proteasomal degradation of all PPP2R5 family regulatory subunits of the host phosphatase PP2A. Here, they identified mutations in Vif separating these activities and provide evidence that the degradation of PPP2R5 family members accounts for the cell cycle arrest caused by certain Vif proteins. They also show that efficient depletion of all PPP2R5 subunits seems required for the Vif-dependent cell cycle arrest, providing an explanation for why HIV-1 Vif variants that antagonize some but not all PPP2R5 subunits do not cause cell cycle arrest.The study addresses an important issue and for most part the data are clearly presented and convincing. Limitations are that the significance of this Vif activity for HIV-1 replication in primary cells and in vivo remain elusive although the authors provide some evidence for selective pressure to maintain this Vif function. Altogether, the findings provide relevant insights into the molecular biology of HIV-1 but as outlined below some additional experiments should be performed to further increase its significance.Essential revisions:1) Figure 1 and Vif mutants. An immunoblot of the Vif mutants would assist in determining their stabilities by observing steady state levels. Later in manuscript there appears to be some differences in Vif mutant stabilities that should be addressed directly in Figure 1, rather than citing that they are not predicted to interfere with stability.2) In Figure 1, only APOBEC3G is used as a control, but since Vif binds APOBEC3F and APOBEC3H in different regions, it would be more convincing if all 3 APOBECs were used to determine the extent of overlap. This is partially addressed in later figures and so showing the data in Figure 1 for the 3 main APOBEC3s that have anti-HIV activity would make the later data, which shows some overlap in activity for some APOBEC3s, less contradictory to this figure.3) The rationale for the cutoff of different amino acids in Figure 1C is not clear; why was R15A not chosen? Why is only PPP2R5B examined by flow cytometry in Figure 1C when PPP2R5D is shown in immunoblots in Figure 1A and Figure 2A? Figure needs to be more consistent or have consistent reasoning for data shown.4) Figure 4: Conclusion that all PPP2R5B subunits are required; yet HeLa cells don't express the PPP2R5B subunit . Can the authors transfect PPP2R5B to supplement the HeLa cells to confirm that all subunits need to be degraded to see effect? PPP2R5B is used as the major example in all the mechanistic data; but lacks the key functional evidence. In supplementary figure for Figure 4, there is more PPP2R5B level than the other PPP2R5s, yet authors state that the mRNA is low to not detectable by qRT-PCR. The levels and importance of PPP2R5B needs to be better tested/presented. Especially since a recent article, Salamango et al., Cell Reports, 2019 implicates only PPP2R5A, C, and D in cell cycle arrest. Essential revisions:1) Figure 1 and Vif mutants. An immunoblot of the Vif mutants would assist in determining their stabilities by observing steady state levels. Later in manuscript there appears to be some differences in Vif mutant stabilities that should be addressed directly in Figure 1, rather than citing that they are not predicted to interfere with stability.Thank you for the suggestion, we have included the requested immunoblot as an additional figure supplement , and altered the main text accordingly. In summary, when overexpressed in 293Ts, steady state levels of the Vif mutants we found to be defective for PPP2R5B depletion  were comparable to wildtype.Results:“We discovered several Vif mutants to be defective for PPP2R5B depletion . Vif protein expression levels were similar …”Please also note that, the aim of our flow cytometric screen was to identify Vif variants which lacked the ability to deplete PPP2R5B, but retained the ability to deplete APOBEC3G. A functional test of Vif stability was therefore built into the screen i.e. “stable enough to deplete APOBEC3G”.This is important, because it is a direct test of the biologically relevant endpoint, controls for changes in recruitment of the CUL5 E3 ligase complex as well as steady state Vif levels, and avoids potential confounding by changes to anti-Vif antibody binding affinity caused by the point mutations. We make this point in the text.Results:“As well as indicating preserved APOBEC3 family substrate recruitment, the ability to deplete APOBEC3G served as a control for unanticipated effects on Vif expression or stability, or assembly of the Vif-CUL5 complex.”We would like to take the opportunity to emphasise that, we have not observed any differences in Vif stability for the critical Vif I31 and I128 mutants which form the focus of this manuscript, based on either immunoblot or proteomic measurements of steady state levels, or flow cytometric or proteomic assays of APOBEC3 family depletion.Conversely, as described in the Results and illustrated in Figure 2, mutations in R33/K34 were typically associated with somewhat lower Vif levels in CEM-T4s, accompanied by partial loss of activity against APOBEC3 family members, particularly APOBEC3F. This is why we chose to focus instead on residues 31 and 128.2) In Figure 1, only APOBEC3G is used as a control, but since Vif binds APOBEC3F and APOBEC3H in different regions, it would be more convincing if all 3 APOBECs were used to determine the extent of overlap. This is partially addressed in later figures and so showing the data in Figure 1 for the 3 main APOBEC3s that have anti-HIV activity would make the later data, which shows some overlap in activity for some APOBEC3s, less contradictory to this figure.Thank you for the suggestion, we have included the requested flow cytometric screen against APOBEC3F as an additional figure supplement , and altered the main text accordingly. In summary, APOBEC3F depletion by the Vif mutants we found to be defective for PPP2R5B  was preserved, with the exception of the R15A mutant (as previously reported) and the RK33/34AA mutant.Results:“First, we tested the ability of Vif mutants lacking the ability to deplete PPP2R5B to deplete HA-tagged APOBEC3F in 293Ts, similar to our initial flow cytometry screen . As previously reported , mutation of R15 resulted in loss of activity against APOBEC3F. The RK33/34AA mutant was also partially impaired, but other mutants retained full activity against APOBEC3F.”Please note that, these results concord precisely with our proteomic data . In fact, a major strength of our study is the use of a functional proteomic approach to assess Vif activity against endogenous APOBEC3 and PPP2R5 family members in T cells, avoiding the possibility of over-expression artefacts, or the need for protein tags.Since APOBEC3F was not quantitated in our second proteomic experiment , however, we took the opportunity to use the APOBEC3F flow cytometric screen to formally test the activity of our critical “Vif AYR” double mutant (combined I31/I128 mutations). This data is now included in an expanded figure supplement , and we have altered the main text accordingly. In summary, and as predicted, Vif AYR retained activity against APOBEC3F.Results:“Since PPP2R5B and APOBEC3F were not quantitated in the proteomic experiment, we tested the ability of Vif AYR to deplete these subunits by flow cytometry in 293Ts, including APOBE3G as a control . As expected, Vif AYR was able to deplete APOBEC3F and APOBEC3G but not PPP2R5B, and failed to cause cell cycle arrest .”Please also note that, we did not screen against APOBEC3H haplotype II, because wildtype NL4-3 Vif (on which our mutant library was based) is known to be unable to deplete this APOBEC3 family member. For clarity, we now include a better overview of the AOBEC3 family, and explicitly make this point in the main text.Results:“As well as APOBEC3G, other APOBEC3 family members (such as APOBEC3F and APOBEC3H haplotype II) are also able to restrict HIV replication , and Vif recruits different APOBEC3 family members for degradation using distinct binding surfaces .”Results:“APOBEC3H haplotype II was not examined, because wildtype NL4-3 Vif (on which our Vif mutant library was based) is unable to deplete this APOBEC3 family member .”3) The rationale for the cutoff of different amino acids in Figure 1C is not clear; why was R15A not chosen? Why is only PPP2R5B examined by flow cytometry in Figure 1C when PPP2R5D is shown in immunoblots in Figure 1A and Figure 2A? Figure needs to be more consistent or have consistent reasoning for data shown.Thank you for the comment/questions, we are sorry that these points were not clear to the reviewer. In brief: first, we did not evaluate the R15A mutant further because it was already known to be defective for APOBEC3F depletion , and we were seeking Vif mutants which lacked the ability to deplete PPP2R5 family subunits, but retained the ability to deplete APOBEC3 family members.Second, we chose PPP2R5B as a representative PPP2R5 subunit for our flow cytometric screen because we had previously shown that, amongst the PPP2R5 family subunits, depletion of PPP25B was most conserved across Vif variants from different primate lentiviruses.Third, we chose PPP2R5D as a representative PPP2R5 subunit for our immunoblots because, to our knowledge, no validated PPP2R5B antibody is available, and, amongst the other PPP2R5 family subunits, this is (in our experience) the most reliable antibody reagent.To ensure that our reasoning is clear, we have reviewed and in some cases rewritten the main text to emphasise the overall strategy, and explicitly address these points.Results:“We discovered several Vif mutants to be defective for PPP2R5B depletion . Vif protein expression levels were similar , but some mutations affected residues already known to be required for depletion of APOBEC3G   or APOBEC3C/F (R15)  . Conversely, Vif variants with mutations in residues Y30/I31, R33/K34 and I128 were defective for PPP2R5B depletion, retained the ability to antagonise APOBEC3G, and had not been implicated in APOBEC3C/F depletion. These residues are grouped in three similarly orientated patches on the Vif surface . Aiming to identify Vif variants specifically defective for PPP2R5 subunit depletion, we therefore focused on mutations in residues I128, I31 and R33/K34 for further evaluation, including representatives from each patch.”Results:“Amongst the five PPP2R5 family subunits, we previously showed that depletion of PPP2R5B is most conserved across Vif variants from HIV-1/2 and the non-human primate lentiviruses . We therefore transfected our library into HEK 293T cells (293Ts) stably expressing HA-tagged PPP2R5B or APOBEC3G, and used flow cytometry to quantify PPP2R5B and APOBEC3G depletion by each Vif variant…”Results:“Levels of an indicative PPP2R5 subunit for which a reliable antibody is available  were then measured by immunoblot .”4) Figure 4: Conclusion that all PPP2R5B subunits are required; yet HeLa cells don't express the PPP2R5B subunit . Can the authors transfect PPP2R5B to supplement the HeLa cells to confirm that all subunits need to be degraded to see effect? PPP2R5B is used as the major example in all the mechanistic data; but lacks the key functional evidence. In supplementary figure for Figure 4, there is more PPP2R5B level than the other PPP2R5s, yet authors state that the mRNA is low to not detectable by qRT-PCR. The levels and importance of PPP2R5B needs to be better tested/presented. Especially since a recent article, Salamango et al., Cell Reports, 2019 implicates only PPP2R5A, C, and D in cell cycle arrest.+ T cells , and altered the main text accordingly. These revisions are described in more detail below.Thank you for the suggestions, we have included the requested PPP2R5B over-expression and qRT-PCR experiments as an additional figure supplement , added a further figure supplement examining Vif-dependent cell cycle arrest in primary human CD4First, to control for PCR efficiency and allow formal comparison of abundances between different PPP2R5 subunits, we have repeated all our qRT-PCR data using standard curves generated from plasmids encoding PPP2R5A-E and TBP. Copies of mRNA  for PPP2R5A-E in CEM-T4s and HeLas are now shown in Figure 4—figure supplement 2A. In summary, these data confirm that expression of PPP2R5B is detectable in both cell types, but lower than other PPP2R5 subunits, particularly in HeLas.Results:“First, we confirmed that, as in CEM-T4s, expression of WT NL4-3 Vif in HeLas causes cell cycle arrest . Likewise, mRNA expression levels of individual PPP2R5 subunits were determined by quantitative real-time PCR (qRT-PCR), and found to be similar between cell types, with PPP2R5B much lower than other subunits .”Please note that, we have also repeated our qRT-PCR test of siRNA knockdown efficiency for each subunit, using the same method . In this figure, relative mRNA abundance for each subunit is shown for siRNA-transfected vs control cells. Because expression of PPP2R5B in wildtype cells is lower than other subunits, expression falls below the linear range of the assay in cells transfected with siRNA.Second, we over-expressed PPP2R5B in HeLas, resulting in elevated PPP2R5B mRNA levels comparable to other subunits . As predicted by the reviewer, supplementation with PPP2R5B rescued cell cycle progression in the presence of PPP2R5A, C, D and E depletion by siRNA .Results:“That depletion of PPP2R5B is neither sufficient  nor required  in this setting may reflect low expression of PPP2R5B , consistent with previous protein-level data from HeLas . To test this hypothesis, we generated HeLa cells expressing exogenous PPP2R5B at similar levels to other PPP2R5 subunits . As predicted, transfection of a pool of siRNA targeting PPP2R5A, C D and E (but not B) caused cell cycle arrest in wildtype HeLas, but not HeLas over-expressing PPP2R5B .”Please also note the reasons described above for focussing on PPP2R5B as a representative PPP2R5 subunit for our flow cytometric screen. In fact, our data suggest that efficient depletion of all PPP2R5 subunits is required to cause cell cycle arrest. Critically, this explains why Vif variants with impaired activity against any PPP2R5 subunit (rather than one particular PPP2R5 subunit) are defective for this phenotype . We elaborate on this in our Discussion (paragraph three).+ T cells, we infected cells from 2 donors with WT, ΔVif or Vif AYR viruses (on a ΔVpr background), and confirmed that the combined I31/I128 mutations in Vif AYR did indeed abolish Vif-dependent cell cycle arrest .Finally, to confirm that the effects we describe in CEM-T4s and HeLas are relevant to HIV-1 infection of primary human CD4Results:+ T cells .”“To confirm a functional effect on PP2A, we then used these viruses to infect CEM-T4 T cells, and measured their effect on cell cycle progression. Again, only ΔVpr-Vif WT virus, but not ΔVpr-ΔVif or ΔVpr-Vif AYR viruses, was able to induce G2/M cell cycle arrest . A similar effect was observed in HIV-infected primary human CD4


Animal studies suggest vital roles of sphingolipids, especially ceramides, in the pathogenesis of type 2 diabetes (T2D) via pathways involved in insulin resistance, β-cell dysfunction, and inflammation, but human studies are limited. We aimed to evaluate the associations of circulating sphingolipids with incident T2D and to explore underlying mechanisms.n = 5,731). During the 6-year period, 529 participants developed T2D. Eleven novel and 3 reported sphingolipids, namely ceramides , saturated sphingomyelins , unsaturated sphingomyelins , hydroxyl-sphingomyelins , and a hexosylceramide (d18:1/20:1), were positively associated with incident T2D , after multivariate adjustment including lifestyle characteristics and BMI. Network analysis further identified 5 modules, and 2 modules containing saturated sphingomyelins showed the strongest associations with increased T2D risk . Mediation analysis suggested that the detrimental associations of 13 sphingolipids with T2D were largely mediated through β-cell dysfunction, as indicated by HOMA-B . Moreover, Mendelian randomization evidenced a positive association between a genetically instrumented ceramide (d18:1/20:1) and T2D . Main limitations in the current study included potential undiagnosed cases and lack of an independent population for replication.The current study included 826 men and 1,148 women who were aged 50–70 years, from Beijing and Shanghai, and without T2D in 2005 and who were resurveyed in 2011. Cardiometabolic traits were measured at baseline and follow-up surveys. A total of 76 sphingolipids were quantified using high-coverage targeted lipidomics. Summary data for 2-sample Mendelian randomization were obtained from genome-wide association studies of circulating sphingolipids and the China Health and Nutrition Survey  through inducing insulin resistance, impairing β-cell function, and promoting inflammation.Sphingolipids represent a class of structurally and functionally diverse lipids that may exert different impacts on T2D outcome. However, the majority of available studies covered only 4 to 15 species and may have missed certain T2D-associated sphingolipid species or subclasses.To our knowledge, no epidemiological study has examined the relationships between genetically predicted sphingolipids and T2D risk so far.Most previous studies of sphingolipid–T2D associations were conducted in Western populations. It remains largely unknown whether the associations differ between Western and Asian populations, and to what extent they are mediated by β-cell dysfunction or insulin resistance.By applying high-coverage targeted lipidomics to measure 76 sphingolipids in 1,974 Chinese men and women, 11 novel  and 3 previously reported sphingolipids were identified to be positively associated with incident T2D, independent of conventional risk factors including lifestyle characteristics and BMI.We conducted a network analysis to determine the collective effects of the sphingolipids on the onset of T2D. Of 5 identified groupings (modules) of sphingolipids, 2 modules containing saturated sphingomyelins showed the strongest associations with incident T2D.Mediation analysis showed that the detrimental associations of 13 out of the 14 sphingolipids with risk of T2D were largely mediated through β-cell dysfunction.We performed genome-wide association studies for the sphingolipids and also conducted 2-sample Mendelian randomization analysis. A positive association between a genetically instrumented ceramide (d18:1/20:1) and T2D was evidenced.Our study is the first to our knowledge that simultaneously discovered the largest number of T2D-associated sphingolipid species—which covered 3 subclasses, including ceramides, sphingomyelins, and a hexosylceramide—and also identified predominantly monounsaturated ceramides associated with T2D in the Chinese population, rather than saturated ceramides as reported in Western populations.High-coverage targeted lipidomics in our study revealed comprehensive details about how and to what extent altered absolute concentrations of sphingolipids with specific structures were associated with T2D development, raising attention to structurally diverse sphingolipids and the fact that they might have different associations with T2D.Our results highlight a stronger mediation effect of β-cell dysfunction than of insulin resistance in the sphingolipid–T2D associations in Chinese individuals.We found a positive association between a genetically instrumented ceramide (d18:1/20:1) and T2D. Together, our findings underscore potential applications of sphingolipids as early biomarkers or intervention targets for prevention and control of T2D. The recent epidemic of type 2 diabetes (T2D) and related complications have contributed enormously to global burdens of mortality and disability . Though Sphingolipids represent a class of structurally and functionally diverse lipid molecules , includiWith recently advanced lipidomic technology, over 600 sphingolipids were detected in humans . HoweverBy adopting a high-coverage targeted lipidomic approach in a well-characterized Chinese cohort, we aimed to investigate (1) the associations of different sphingolipids with incident T2D, (2) specific sphingolipid networks linked to incident T2D, and (3) possible mediators and relationships of genetically instrumented sphingolipids with T2D.2).The current study was based on the Nutrition and Health of Aging Population in China (NHAPC) study, a well-designed prospective cohort investigating environmental and genetic factors and their interactions with cardiometabolic diseases. The study design has been described in detail elsewhere ,19. Brien = 274) or without baseline sphingolipid profile (n = 281). The analysis plan was drafted in August 2018  after excluding those with diabetes at baseline , insulin, total cholesterol, low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C), triglycerides (TGs), high-sensitivity C-reactive protein (hsCRP), and adiponectin were measured as previously described . Th. Th38]. In conclusion, we identified a panel of novel sphingolipids with unique structures that were significantly associated with elevated risk of T2D, and these associations appeared to be largely mediated through β-cell dysfunction among Chinese individuals. Our findings suggested that specific sphingolipids could be promising early biomarkers and intervention targets beyond traditional ones in T2D prevention and control in future clinical settings. Certainly, more studies are merited to confirm our findings and further identify their determinants and potential applications in other populations.S1 STROBE Checklist(DOCX)Click here for additional data file.S1 Fign denotes the number of sphingolipids identified in the current study. Sphingoid base denotes sphingosine, generally defined as a carbon chain, di-hydroxylated at positions 1 and 3 and with a double bond at position 4, which varies in chain length (C16–C19), number of double bonds (0–2), and number of hydroxyls (0–3). The fatty acid residue also varies substantially with respect to chain length (C14–C26), number of double bonds (saturated or unsaturated), and number of hydroxyl groups (0–1).For each graph, (TIF)Click here for additional data file.S2 Fig(TIF)Click here for additional data file.S3 Fig(TIF)Click here for additional data file.S4 Fig1c ≥ 6.5% to define T2D. Model was adjusted for age, sex, region (Beijing or Shanghai), residence , educational attainment , current smoking (yes or no), current alcohol drinking (yes or no), physical activity , family history of diabetes (yes or no), and BMI.M1: incident T2D was defined with self-reported doctor-diagnosed diabetes, taking antidiabetic medications, or fasting glucose ≥ 7.0 mmol/l. M2: further adding HbA(TIF)Click here for additional data file.S5 FigR2 ≥ 0.80 was chosen. (C). Spearman correlations were calculated between module eigengenes and metabolic traits.Scale-free topology parameters in WGCNA are shown in (A) and (B). The smallest soft power (7) with (TIF)Click here for additional data file.S6 FigP values calculated using linear regression analysis under an additive genetic model are presented in the figure. (A) Cer(d18:1/20:0); (B) Cer(d18:1/20:1); (C) SM C34:0; (D) SM C36:0; (E) SM C34:1; (F) SM C42:3; (G) HexCer C20:1. The red lines in Manhattan plots represent genome-wide significant level (P < 5 × 10−8).The −log10 (TIF)Click here for additional data file.S7 Fig(TIF)Click here for additional data file.S8 Fig(TIF)Click here for additional data file.S9 FigCeramides can be produced by (a) a de novo pathway initiated from serine and palmitoyl precursors, (b) synthesis via sphingosine-1-phosphate (S-1-P), namely the “salvage pathway,” and (c) degradation of sphingomyelins through sphingomyelinase. As important signal molecules, ceramides can (d) induce pancreatic β-cell apoptosis through increasing endoplasmic reticulum stress, producing reactive oxygen species, (e) promote the development of insulin resistance via activating either protein phosphatase 2 or protein kinase C ζ, leading to attenuated serine/threonine protein kinase, and (f) activate NLR family 3 inflammasome and produce more cytokines. Solid lines represent a 1-step or certain process, whereas dotted lines represent multiple-step or uncertain processes. CDase, ceramidase; CS, ceramide synthase; DES, dihydroceramide synthase; GlcCer, glucosylceramide; GCase, glucosylceramidase; GCS, glucosylceramide synthase; HexCer, hexosylceramide; LacCer, lactosylceramide; S1PP, S-1-P phosphatase; SK, sphingosine kinase; SMase, sphingomyelinase; SMS, sphingomyelin synthase; T2D, type 2 diabetes.(TIF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.S4 Table(DOCX)Click here for additional data file.S5 Table(DOCX)Click here for additional data file.S6 Table(DOCX)Click here for additional data file.S7 Table(DOCX)Click here for additional data file.S8 Table(DOCX)Click here for additional data file.S9 Table(DOCX)Click here for additional data file.S1 Text(DOCX)Click here for additional data file.S2 Text(DOCX)Click here for additional data file.