Mechanistic Understanding Of Murine Leukemia Virus Glycosylated Gag And Equine Infectious Anemia Virus S2 Mediated Antagonism Of SERINC5 Antiretroviral Activity

SERINC is a family of 5 genes encoding multi-pass transmembrane proteins with unidentified cellular function.Among these SERINC5(Ser5)acts as an efficient retroviral inhibitor.It incorporates into virions and inhibits its penetration to the target cell by an unknown mechanism.Recently,it was reported that retroviral accessory protein of human immunodeficiency virus type 1(HIV-1)Nef excludes Ser5 from virion by downregulating Ser5 via internalization to cytoplasm followed by targeting to lysosomes for degradation.In addition,murine leukemia virus(MLV)glycoGag and equine infectious anemia virus(EIAV)S2 were also discovered as Ser5 antagonizing factors that rescue HIV-1 infectivity.However,for MLV glycoGag and EIAV S2 the Ser5 antagonizing mechanism is still unclear.In the current study,we explored how glycoGag and S2 counteract murine Ser5 protein.We detected that Ser5 incorporates into MLV particles and restrict its infectivity,but glycoGag counters Ser5 restriction by excluding it from virion.The N terminal 189 amino acids of glycoGag named as glycoMA are the minimal region that acquires full-length glycoGag activity was used to investigate the in-depth molecular mechanism of Ser5 antagonism.GlycoMA internalizes Ser5 from the plasma membrane to cytoplasmic compartments and reduces its expression at steady state level.In this process Y36XXL39 motif and two other residues P31 and R63 of glycoMA play a crucial role.GlycoMA interacts with Ser5 and reduces Ser5 cell surface expression via receptor mediated endocytosis.GlycoMA directs the internalized Ser5 to re-localize with Rab5 early,Rab7 late and Rab11 recycling endosomes and then directs to lysosome for degradation.Ser5 polyubiquitination via the K48-and K63-linkage occurs which enhance glycoMA mediated Ser5 downregulation.Although P31,Y36,L39 and R63 are not important for Ser5-glycoMA interactions,but they are essential for Ser5 re-localization to lysosome for destruction.Additionally,Ser1,Ser2 and Ser3 have little influence on HIV-1 infectivity,but glycoMA also target these proteins to lysosomes for degradation.Furthermore,S2 also exploits similar mechanism to counteract Ser5,but S2-Ser5 interaction depends on S2 myristylation site,demonstrating that the interaction occurs at the cell membrane.Moreover,we found that equine Ser5 expresses poorly as compared to other species Ser5 but exhibits strong antiviral activity,which was effectively countered by S2.S2 and glycoMA strongly interact with Ser5 than HIV-1 Nef.Hence,they downregulate Ser5 more efficiently than Nef.GlycoMA and S2 can also downregulate Xenopus tropicalis Ser5(xSer5),but Nef lacks such ability.We concluded that glycoMA and S2 have a broad ability to downregulate SERINC family proteins.Both share similar mechanism and target Ser5 to cellular endosome/lysosome for degradation and play an effective role to enhance viral replication.These mechanistic understandings might provide scientific basis for designing antiviral strategies.