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Human tetherin is a host restriction factor that inhibits replication of enveloped viruses by blocking viral release. Tetherin has an unusual topology that includes an N-terminal cytoplasmic tail, a single transmembrane domain, an extracellular domain, and a C-terminal glycosylphosphatidylinositol anchor. Tetherin is not well conserved across species, so it inhibits viral replication in a species-specific manner. Thus, studies of tetherin activities from different species provide an important tool for understanding its antiviral mechanism. Here, we report cloning of equine tetherin and characterization of its antiviral activity. Equine tetherin shares 53%, 40%, 36%, and 34% amino acid sequence identity with feline, human, simian, and murine tetherins, respectively. Like the feline tetherin, equine tetherin has a shorter N-terminal domain than human tetherin. Equine tetherin is localized on the cell surface and strongly blocks human immunodeficiency virus type 1 (HIV-1), simian immunodeficiency virus (SIV), and equine infectious anemia virus (EIAV) release from virus-producing cells. The antiviral activity of equine tetherin is neutralized by EIAV envelope protein, but not by the HIV-1 accessory protein Vpu, which is a human tetherin antagonist, and EIAV envelope protein does not counteract human tetherin. These results shed new light on our understanding of the species-specific tetherin antiviral mechanism.

Human SERINC5 (SER5) protein is a recently described restriction factor against human immunodeficiency virus-1 (HIV-1), which is antagonized by HIV-1 Nef protein. Other retroviral accessory proteins such as the glycosylated Gag (glycoGag) from the murine leukemia virus (MLV) can also antagonize SER5. In addition, some viruses escape SER5 restriction by expressing a SER5-insensitive envelope (Env) glycoprotein. Here, we studied the activity of human and feline SER5 on HIV-1 and on the two pathogenic retroviruses in cats, feline immunodeficiency virus (FIV) and feline leukemia virus (FeLV). HIV-1 in absence of Nef is restricted by SER5 from domestic cats and protected by its Nef protein. The sensitivity of feline retroviruses FIV and FeLV to human and feline SER5 is considerably different: FIV is sensitive to feline and human SER5 and lacks an obvious mechanism to counteract SER5 activity, while FeLV is relatively resistant to SER5 inhibition. We speculated that similar to MLV, FeLV-A or FeLV-B express glycoGag proteins and investigated their function against human and feline SER5 in wild type and envelope deficient virus variants. We found that the endogenous FeLV recombinant virus, FeLV-B but not wild type exogenous FeLV-A envelope mediates a strong resistance against human and feline SER5. GlycoGag has an additional but moderate role to enhance viral infectivity in the presence of SER5 that seems to be dependent on the FeLV envelope. These findings may explain, why in vivo FeLV-B has a selective advantage and causes higher FeLV levels in infected cats compared to infections of FeLV-A only.

The family of human APOBEC3 (A3) restriction factors is formed by seven different proteins, A3A-D and A3F-H. Among these A3s, A3B harbors strong restriction activity against several retroviruses, such as SIV, and MLV. How lentiviruses and other retroviruses, prevalent in many primate species, counteract A3B is poorly understood. In this study, we found that A3B strongly inhibited SIVmac and HIV-2 infectivity, which was antagonized by their Vif proteins. Both SIVmac and HIV-2 Vifs diminished the protein level of A3B in viral producer cells, and hindered A3B incorporation into viral particles. We observed that HIV-2 Vif binds A3B and induces its degradation by assembly of an A3-Vif-CUL5-ElonginB/C E3-ligase complex. A3B and HIV-2 Vif localize and interact in the nucleus. In addition, we also found that the accessory protein Bet of prototype foamy virus (PFV) significantly antagonized the anti-SIVmac activity of A3B. Like Vif, Bet prevented the incorporation of A3B into viral particles. However, in contrast to Vif Bet did not induce the degradation of A3B. Rather, Bet binds A3B to block formation of high molecular weight A3B complexes and induces A3B cytoplasmic trapping. In summary, these findings indicate that A3B is recognized by diverse retroviruses and counteracted by virus-specific pathways that could be targeted to inhibit A3B mutating activity in cancers.

Feline immunodeficiency virus (FIV) Vif protein counteracts feline APOBEC3s (FcaA3s) restriction factors by inducing their proteasomal degradation. The functional domains in FIV Vif for interaction with FcaA3s are poorly understood. Here, we have identified several motifs in FIV Vif that are important for selective degradation of different FcaA3s. Cats ( Felis catus ) express three types of A3s: single-domain A3Z2, single-domain A3Z3, and double-domain A3Z2Z3. We proposed that FIV Vif would selectively interact with the Z2 and the Z3 A3s. Indeed, we identified two N-terminal Vif motifs (12LF13 and 18GG19) that specifically interacted with the FcaA3Z2 protein but not with A3Z3. In contrast, the exclusive degradation of FcaA3Z3 was regulated by a region of three residues (M24, L25, and I27). Only a FIV Vif carrying a combination of mutations from both interaction sites lost the capacity to degrade and counteract FcaA3Z2Z3. However, alterations in the specific A3s interaction sites did not affect the cellular localization of the FIV Vif protein and binding to feline A3s. Pulldown experiments demonstrated that the A3 binding region localized to FIV Vif residues 50 to 80, outside the specific A3 interaction domain. Finally, we found that the Vif sites specific to individual A3s are conserved in several FIV lineages of domestic cat and nondomestic cats, while being absent in the FIV Vif of pumas. Our data support a complex model of multiple Vif-A3 interactions in which the specific region for selective A3 counteraction is discrete from a general A3 binding domain. IMPORTANCE Both human immunodeficiency virus (HIV) and feline immunodeficiency virus (FIV) Vif proteins counteract their host's APOBEC3 restriction factors. However, these two Vif proteins have limited sequence homology. The molecular interaction between FIV Vif and feline APOBEC3s are not well understood. Here, we identified N-terminal FIV Vif sites that regulate the selective interaction of Vif with either feline APOBEC3Z2 or APOBEC3Z3. These specific Vif sites are conserved in several FIV lineages of domestic cat and nondomestic cats, while being absent in FIV Vif from puma. Our findings provide important insights for future experiments describing the FIV Vif interaction with feline APOBEC3s and also indicate that the conserved feline APOBEC3s interaction sites of FIV Vif allow FIV transmissions in Felidae .

Lentiviruses have evolved the Vif protein to counteract APOBEC3 (A3) restriction factors by targeting them for proteasomal degradation. Previous studies have identified important residues in the interface of human immunodeficiency virus type 1 (HIV-1) Vif and human APOBEC3C (hA3C) or human APOBEC3F (hA3F). However, the interaction between primate A3C proteins and HIV-1 Vif or natural HIV-1 Vif variants is still poorly understood. Here, we report that HIV-1 Vif is inactive against A3Cs of rhesus macaques (rhA3C), sooty mangabey monkeys (smmA3C), and African green monkeys (agmA3C), while HIV-2, African green monkey simian immunodeficiency virus (SIVagm), and SIVmac Vif proteins efficiently mediate the depletion of all tested A3Cs. We identified that residues N/H130 and Q133 in rhA3C and smmA3C are determinants for this HIV-1 Vif-triggered counteraction. We also found that the HIV-1 Vif interaction sites in helix 4 of hA3C and hA3F differ. Vif alleles from diverse HIV-1 subtypes were tested for degradation activities related to hA3C. The subtype F-1 Vif was identified to be inactive for degradation of hA3C and hA3F. The residues that determined F-1 Vif inactivity in the degradation of A3C/A3F were located in the C-terminal region (K167 and D182). Structural analysis of F-1 Vif revealed that impairing the internal salt bridge of E171-K167 restored reduction capacities to A3C/A3F. Furthermore, we found that D101 could also form an internal interaction with K167. Replacing D101 with glycine and R167 with lysine in NL4-3 Vif impaired its counteractivity to A3F and A3C. This finding indicates that internal interactions outside the A3 binding region in HIV-1 Vif influence the capacity to induce degradation of A3C/A3F. IMPORTANCE The APOBEC3 restriction factors can serve as potential barriers to lentiviral cross-species transmissions. Vif proteins from lentiviruses counteract APOBEC3 by proteasomal degradation. In this study, we found that monkey-derived A3C, rhA3C and smmA3C, were resistant to HIV-1 Vif. This was determined by A3C residues N/H130 and Q133. However, HIV-2, SIVagm, and SIVmac Vif proteins were found to be able to mediate the depletion of all tested primate A3C proteins. In addition, we identified a natural HIV-1 Vif (F-1 Vif) that was inactive in the degradation of hA3C/hA3F. Here, we provide for the first time a model that explains how an internal salt bridge of E171-K167-D101 influences Vif-mediated degradation of hA3C/hA3F. This finding provides a novel way to develop HIV-1 inhibitors by targeting the internal interactions of the Vif protein.

Additional file 1: Figure S1 . Cellular localization of feline A3s and FIV Vif. HOS cells were transfected with FcaA3Z2b, FcaA3Z3, or FcaA3Z2Z3 (all with HA-tag), together with FIV Vif-TLQAAA. To detect A3 (green) immunofluorescence, staining was performed with an anti-HA antibody. To detect FIV Vif (red) immunofluorescence, staining was performed with an anti-V5 antibody. Nuclei (blue) were visualized by DAPI staining. Figure S2. Comparison of protein sequences of A3s and Vif. (A, B) The sequence alignment of (A) FcaA3Z2 (FcaA3Z2b), (B) FcaA3Z3 and big cat A3 proteins. The D165-H166 and L40-I41 + A65 domains that are essential for FIV Vif induced degradation are marked by red boxes. (C) Sequence alignment of domestic cat FIV Vif (FIVfca subtype 34TF10) and lion FIV (FIVple subtype E) Vif. The C187 and C190 that are essential for induced FcaA3s degradation and marked the presumed BC box (TLQ/SLQ) marked by red boxes. (D) Sequence alignment of HIV-1 (strain NL4-3) and HIV-2 (strain RodA) Vif. The CUL5 box (HCCH) and BC box (SLQ) were marked by red boxes. Pti, Ple, Lly and Pco represent Panthera tigris corbetti; Panthera leo bleyenberghi; Lynx lynx; Puma concolor. Figure S3. Evolutionary supernetwork of A3 sequences retrieved from carnivores. The network was constructed with SplitsTree_v4 using 1,000 maximum likelihood bootstrapped trees created with RAxML_v8.2. Scale bar is given in substitutions per site. The approximate position of the root obtained using maximum likelihood inference with all A3Z1, A3Z2 and A3Z3 sequences from carnivores is indicated in grey. (A) The evolutionary distances among A3Z3 sequences within the two in-paralogs within Caniformia (upper branches and left branch) and within Feliformia (right branches) are indicated as overall average pairwise nucleotide distance ± bootstrap standard error estimate. For each tip, the actual sequence orthologous to positions 38-44 in the F. catus A3Z3 gene are given in parentheses. The inset displays the evolutionary relationships among the Carnivora species for which we have identified A3Z3 paralogs. (B) The evolutionary distances among A3Z2 sequences within Caniformia (upper branches) and within Feliformia (lower branches) are indicated as overall average pairwise nucleotide distance ± bootstrap standard error estimate. For each tip, the actual sequence orthologous to positions 165-170 in the F. catus A3Z2 genes are given in parentheses. Figure S4. The mutations in FcaA3 that cause resistance to FIV Vif do not alter the cellular distribution of FcaA3s. HOS cells were transfected with FcaA3Z2 (FcaA3Z2b), FcaA3Z2.DH-YN, FcaA3Z3, FcaA3Z3.A65I + LI-AA, FcaA3Z2Z3 or FcaA3Z2Z3-M (all with HA-tag). To detect A3 (green) immunofluorescence, staining was performed with an anti-HA antibody. Nuclei (blue) were visualized by DAPI staining. Figure S5. Sequence alignment of feline APOBEC3. Sequence alignment of feline A3Z2Z3 and human A3G as generated by the TopModel approach. The Z2 and Z3 domains are underlined in yellow and blue, respectively. The sequence of the linker domain is underlined in magenta. Helical regions and β-strands are depicted as red helices and green arrows, respectively. In addition, the alignments of three template structures used to model the structure of the linker domain are given (PDB IDs 2YS9, 2DA4, 2MMB). Figure S6. Expression of CD4 and CCR5 receptors on the surface of HOS (red) and HOS.CD4.CCR5 cells (blue) expressing feline A3Z2Z3 or A3Z2Z3-M. CD4 and CCR5 were detected by flow cytometry and anti-CD4 and anti-CCR5 antibodies. Numbers indicate the percentage of positive cells. HOS cells served as background control. Figure S7. The mutated FcaA3s are encapsidated and inhibit FIV by cytidine deamination. (A) The mutated FcaA3s can inhibit the infectivity of FIVΔvif reporter viruses. 293T cells were co-transfected with plasmids for FIVΔvif luciferase together with FcaA3s. 48 h later, supernatant normalized for reverse transcriptase activity was used to transduce 293T cells. Luciferase activity was determined two days post transduction. Asterisks represent statistically significant differences: ***, p 0.05 [Dunnett t test]. (B) Immunoblot of FIV producer cells and VLPs used for (A). Encapsidation of wild-type and mutated feline A3s into FIVΔvif virus like particles (VLPs), A3 proteins were detected by anti-HA antibody. Tubulin detection for equal loading of cell lysate was done using anti-tubulin, for demonstration of equal loading of FIV VLPs VSV-G and FIV p24 proteins were detected by anti-VSV-G and anti-FIV p24 antibodies separately. (C) Encapsidated wild-type and mutated FcaA3s deaminate cytidines FIV genomes. FIVΔvif was produced in the absence and presence of wild-type and mutant FcaA3s (FcaA3Z3, FcaA3Z3.A65I + LI-AA, FcaA3Z2Z3, FcaA3Z2Z3-M) or HsaA3G. The vector particles were used to infect 293T cells. 12 h later, the total cellular DNA was extracted and differential DNA denaturation PCR (3D-PCR) was performed. Td: denaturing temperature. Figure S8. Vif titration on FcaA3 linker mutants. Co-transfection of increasing amounts of expression plasmids for (A) HIV-2 Vif and (B) SIVmac Vif with constant amounts of the indicated A3 expression plasmids. The expression of FcaA3s and Vifs were analyzed by anti-HA and anti-V5 antibodies, respectively. Cell lysates were also analyzed for equal amounts of total proteins using anti-tubulin antibody. Figure S9. Expression and encapsidation of feline A3 linker mutants using (A) FIVΔvif and (B) SIVmacΔvif. Immunoblots of corresponding experiments shown in Fig. 10C and 10D. Immunoblots of lysates of virus producer cells (cell) and virus particles (VLP). A3s were detected by anti-HA antibodies, cell lysates were also analyzed for equal amounts of total proteins using anti-tubulin antibody and VLP lysates using anti-VSV-G antibody. Figure S10. HIV-1 Vif cannot target the “YYFWDPN/DY” domain in FcaA3. (A) CO-IP of feline A3Z2Z3 (HA tag) with either HIV-1 Vif (V5 tag) or FIV Vif (TLQAAA mutant, V5 tag). A3Z2Z3 immune precipitated and detected by anti-HA antibody, co-precipitated Vif was detected by anti-V5 antibody. (B) Comparison of the “YYFWDPN/DY” domain in HsaA3G and FcaA3Z2Z3 and derived mutations generated in FcaA3Z2Z3, the mutated residues shown in bold. (C) FcaA3Z2Z3 mutants were investigated for being sensitive for degradation by HIV-1 Vif. Expression plasmids of FcaA3Z2Z3 mutants of HsaA3G were co-transfected together with HIV-1 Vif into 293T cells. 48 h later, Cell lysates were used to detect the expression of FcaA3Z2Z3 and HIV-1 Vif by anti-HA and anti-V5 antibodies, respectively. Cell lysates were also analyzed for equal amounts of total proteins using anti-tubulin antibody.

Background Tetherin is an interferon-inducible host cell factor that blocks the viral particle release of the enveloped viruses. Most knowledge regarding the interaction between tetherin and viruses has been obtained using the primate lentiviral system. However, much less is known about the functional roles of tetherin on other lentiviruses. Equine infectious anemia virus (EIAV) is an important macrophage-tropic lentivirus that has been widely used as a practical model for investigating the evolution of the host-virus relationship. The host range of EIAV is reported to include all members of the Equidae family. However, EIAV has different clinical responses in horse and donkey. It’s intriguing to investigate the similarities and differences between the tetherin orthologues encoded by horse and donkey. Results We report here that there are two equine tetherin orthologues. Compared to horse tetherin, there are three valine amino acid deletions within the transmembrane domain and three distinct mutations within the ectodomain of donkey tetherin. However, the antiviral activity of donkey tetherin was not affected by amino acid deletion or substitution. In addition, both tetherin orthologues encoded by horse and donkey are similarly sensitive to EIAV Env protein, and equally activate NF-κB signaling. Conclusion Our data suggest that both tetherin orthologues encoded by horse and donkey showed similar antiviral activities and abilities to induce NF-κB signaling. In addition, the phenomenon about the differential responses of horses and donkeys to infection with EIAV was not related with the differences in the structure of the corresponding tetherin orthologues.

Human tetherin is a host restriction factor that inhibits replication of enveloped viruses by blocking viral release. Tetherin has an unusual topology that includes an N-terminal cytoplasmic tail, a single transmembrane domain, an extracellular domain, and a C-terminal glycosylphosphatidylinositol anchor. Tetherin is not well conserved across species, so it inhibits viral replication in a species-specific manner. Thus, studies of tetherin activities from different species provide an important tool for understanding its antiviral mechanism. Here, we report cloning of equine tetherin and characterization of its antiviral activity. Equine tetherin shares 53%, 40%, 36%, and 34% amino acid sequence identity with feline, human, simian, and murine tetherins, respectively. Like the feline tetherin, equine tetherin has a shorter N-terminal domain than human tetherin. Equine tetherin is localized on the cell surface and strongly blocks human immunodeficiency virus type 1 (HIV-1), simian immunodeficiency virus (SIV), and equine infectious anemia virus (EIAV) release from virus-producing cells. The antiviral activity of equine tetherin is neutralized by EIAV envelope protein, but not by the HIV-1 accessory protein Vpu, which is a human tetherin antagonist, and EIAV envelope protein does not counteract human tetherin. These results shed new light on our understanding of the species-specific tetherin antiviral mechanism.

APOBEC3s (A3s) are potent restriction factors of human immunodeficiency virus type 1/simian immunodeficiency viruses (HIV-1/SIV), and can repress cross-species transmissions of lentiviruses. HIV-1 originated from a zoonotic infection of SIV of chimpanzee (SIVcpz) to humans. However, the impact of human A3s on the replication of SIVcpz remains unclear. By using novel SIVcpz reporter viruses, we identified that human APOBEC3B (A3B) and APOBEC3H (A3H) haplotype II strongly reduced the infectivity of SIVcpz, because both of them are resistant to SIVcpz Vifs. We further demonstrated that human A3H inhibited SIVcpz by deaminase dependent as well independent mechanisms. In addition, other stably expressed human A3H haplotypes and splice variants showed strong antiviral activity against SIVcpz. Moreover, most SIV and HIV lineage Vif proteins could degrade chimpanzee A3H, but no Vifs from SIVcpz and SIV of gorilla (SIVgor) lineages antagonized human A3H haplotype II. Expression of human A3H hapII in human T cells efficiently blocked the spreading replication of SIVcpz. The spreading replication of SIVcpz was also restricted by stable A3H in human PBMCs. Thus, we speculate that stably expressed human A3H protects humans against the cross-species transmission of SIVcpz and that SIVcpz spillover to humans may have started in individuals that harbor haplotypes of unstable A3H proteins., Author summary Cellular cytidine deaminases of the APOBEC3 (A3) family are potent restriction factors that are able to inhibit retroviruses, this A3 restriction is counteracted by lentivirus Vif proteins. Human APOBEC3H (A3H) represents the most evolutionarily divergent A3 gene; it includes seven haplotypes and several splice variants. The polymorphism of human A3H has relevance for HIV-1 infection and AIDS progression. HIV-1 originated from cross-transmission of SIVcpz to humans. However, little is known about how human A3s affect the replication or transmission of SIVcpz. In this study, we comprehensively analyzed the anti-SIVcpz activity of chimpanzee and human A3s. Human A3H haplotype II was identified as strong inhibitor against SIVcpz regardless of Vif. In addition, other stably expressed human A3H haplotypes and splice variants showed strong antiviral activity against SIVcpz. Moreover, based on the recent Great Ape Genome Project, we found that the polymorphism of chimpanzee A3H is lower compared with the diversity of human A3H. And chimpanzee A3H haplotypes identified in this study showed similar anti-SIVcpz activity and Vif sensitivity. Our results provide a model that stably expressed human A3H protects humans against the cross-species transmission of SIVcpz and that SIVcpz spillover to humans may have started in individuals that harbor haplotypes of unstable A3H proteins.