Equine Tetherin Blocks Retrovirus Release and Its Activity Is Antagonized by Equine Infectious Anemia Virus Envelope Protein

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Viral infectivity factor (Vif) is required for lentivirus fitness and pathogenicity, except in equine infectious anemia virus (EIAV).Vif enhances viral infectivity by a Cullin5-Elongin B/C E3 complex to inactivate the host restriction factor APOBEC3. Corebinding factor subunit beta (CBF-␤) is a cell factor that was recently shown to be important for the primate lentiviral Vif function. Non-primate lentiviral Vif also degrades APOBEC3 through the proteasome pathway. However, it is unclear whether CBF-␤ is required for the non-primate lentiviral Vif function. In this study, we demonstrated that the Vifs of non-primate lentiviruses, including feline immunodeficiency virus (FIV), bovine immunodeficiency virus (BIV), caprine arthritis encephalitis virus (CAEV), and maedi-visna virus (MVV), do not interact with CBF-␤. In addition, CBF-␤ did not promote the stability of FIV, BIV, CAEV, and MVV Vifs. Furthermore, CBF-␤ silencing or overexpression did not affect non-primate lentiviral Vif-mediated APOBEC3 degradation. Our results suggest that non-primate lentiviral Vif induces APOBEC3 degradation through a different mechanism than primate lentiviral Vif. IMPORTANCEThe APOBEC3 protein family members are host restriction factors that block retrovirus replication. Vif, an accessory protein of lentivirus, degrades APOBEC3 to rescue viral infectivity by forming Cullin5-Elongin B/C-based E3 complex. CBF-␤ was proved to be a novel regulator of primate lentiviral Vif function. In this study, we found that CBF-␤ knockdown or overexpression did not affect FIV Vif's function, which induced polyubiquitination and degradation of APOBEC3 by recruiting the E3 complex in a manner similar to that of HIV-1 Vif. We also showed that other non-primate lentiviral Vifs did not require CBF-␤ to degrade APOBEC3. CBF-␤ did not interact with non-primate lentiviral Vifs or promote their stability. These results suggest that a different mechanism exists for the Vif-APOBEC interaction and that non-primates are not suitable animal models for exploring pharmacological interventions that disrupt Vif-CBF-␤ interaction.

“…The transfections accomplished in 293T cells used the standard calcium phosphate method as previously described (50,51). The cells were harvested 2 days after transfection.…”

Section: Cbf-␤-kd 293t Cell Generationmentioning

confidence: 99%

Core-Binding Factor Subunit Beta Is Not Required for Non-Primate Lentiviral Vif-Mediated APOBEC3 Degradation

Zhu

Wang

et al. 2014

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“…Polymorphisms in the ϩ1 ATG site have been observed three times so far in different mammalian species. Both the domestic cat (50,51) and horse (52) tetherin genes lack the ϩ1 ATG, and a similar defect exists in at least one inbred mouse strain, NZW (53). In the latter case, this has been shown to lead to higher tetherin expression levels on lymphocytes and correlates with a better control of murine leukemia virus viremia (53).…”

Section: Discussionmentioning

confidence: 99%

Differential Sensitivities of Tetherin Isoforms to Counteraction by Primate Lentiviruses

Weinelt

Neil

2014

The mammalian antiviral membrane protein tetherin (BST2/CD317) can be expressed as two isoforms derived from differential translational initiation. The shorter isoform of the human protein (S-tetherin) lacks the first 12 amino acids of the longer (Ltetherin) cytoplasmic tail, which includes a tyrosine motif that acts as both an endocytic recycling signal and a determinant of virus-induced NF-B activation. S-tetherin is also reported to be less sensitive to the prototypic viral antagonist human immunodeficiency virus type 1 (HIV-1) Vpu. Here we analyzed the relative sensitivities of L-and S-tetherins to primate lentiviral countermeasures. We show that the reduced sensitivity of S-tetherin to HIV-1 Vpu is a feature of all group M proteins, including those of transmitted founder viruses, primarily because it cannot be targeted for endosomal degradation owing to the truncation of its cytoplasmic tail. In contrast, both isoforms of the human and rhesus macaque tetherins display the same sensitivity to nondegradative lentiviral countermeasures of HIV-2 and SIVmac, respectively. Surprisingly, however, the Vpu proteins encoded by simian immunodeficiency viruses (SIVs) of African guenons, as well as that from recently isolated highly pathogenic HIV-1 group N, do not discriminate between tetherin isoforms. Together, these data suggest that the group M HIV-1 Vpu primarily adapted to target L-tetherin upon zoonotic transmission from chimpanzees, and further, we speculate that functions specifically associated with this isoform, such as proinflammatory signaling, play key roles in human tetherin's antiviral function in vivo. IMPORTANCEThe ability of HIV-1 and related viruses to counteract a host antiviral protein, tetherin, is strictly maintained. The adaptation of the HIV-1 Vpu protein to counteract human tetherin is thought to have been one of the key events in the establishment of the HIV/AIDS pandemic. Recent evidence shows that tetherin is expressed as two isoforms and that Vpu preferentially targets the longer form. Here we show that unlike other virus-encoded countermeasures, such as those from primate viruses related to HIV-1, the enhanced ability to counteract the long tetherin isoform is conserved among HIV-1 strains that make up the majority of the human pandemic. This correlates with the ability of Vpu to induce long tetherin degradation. We speculate that functions associated with the human version of this isoform, such as an inflammatory signaling capacity, selected for Vpu's enhanced targeting of long tetherin during its adaptation to humans.

“…A luciferase-expressing EIAV reporter virus was constructed by packaging the viral genome with either vesicular stomatitis virus glycoprotein (VSV-G) or EIAV Env. HEK293T cells were cotransfected with 9 g of pONY8.1-LUC, pEIAV-GagPol, and 1 g of pMD2.0G (for VSV-G) or the EIAV envelope (Env) expression plasmid (21). The VSV-G or EIAV Env packaged virus was collected 48 h posttransfection (hpt) and used to infect the HEK293T/ELR1 cells seeded in 48-well plates.…”

Section: Methodsmentioning

confidence: 99%

“…Tat was amplified from EIAV-CMV-3-8 and cloned into the pEGFP-N1 mammalian expression vector (Clontech, USA) with a hemagglutinin (HA) tag. The Rev expression plasmid and the Env expression plasmid (non-codon-optimized for packaging the EIAV reporter) were constructed as previously described (21). The sequences of the PCR primers are summarized in Table S1 in the supplemental material.…”

Section: Methodsmentioning

confidence: 99%

Equine Viperin Restricts Equine Infectious Anemia Virus Replication by Inhibiting the Production and/or Release of Viral Gag, Env, and Receptor via Distortion of the Endoplasmic Reticulum

Tang

Lei

Zhu

et al. 2014

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Viperin is an endoplasmic reticulum (ER)-associated multifunctional protein that regulates virus replication and possesses broad antiviral activity. In many cases, viperin interferes with the trafficking and budding of viral structural proteins by distorting the membrane transportation system. The lentivirus equine infectious anemia virus (EIAV) has been studied extensively. In this study, we examined the restrictive effect of equine viperin (eViperin) on EIAV replication and investigated the possible molecular basis of this restriction to obtain insights into the effect of this cellular factor on retroviruses. We demonstrated that EIAV infection of primary equine monocyte-derived macrophages (eMDMs) upregulated the expression of eViperin. The overexpression of eViperin significantly inhibited the replication of EIAV in eMDMs, and knockdown of eViperin transcription enhanced the replication of EIAV in eMDMs by approximately 45.8%. Further experiments indicated that eViperin restricts EIAV at multiple steps of viral replication. The overexpression of eViperin inhibited EIAV Gag release. Both the ␣-helix domain and radical S-adenosylmethionine (SAM) domain were required for this activity. However, the essential motifs in SAM were different from those reported for the inhibition of HIV-1 Gag by human viperin. Furthermore, eViperin disrupted the synthesis of both EIAV Env and receptor, which consequently inhibited viral production and entry, respectively, and this disruption was dependent on the eViperin ␣-helix domain. Using immunofluorescence assays and electron microscopy, we demonstrated that the ␣-helix domain is responsible for the distortion of the endoplasmic reticulum (ER). Finally, EIAV did not exhibit counteracting eViperin at the protein level. IMPORTANCEIn previous studies, viperin was indicated as restricting virus replications primarily by the inhibition of virus budding. Here, we show that viperin may have multiple antiviral mechanisms, including the reduction of EIAV Gag budding and Env expression, and these activities are dependent on different viperin domains. We especially demonstrate that the overexpression of viperin inhibits EIAV entry by decreasing the level of virus receptor. Therefore, viperin restriction of viruses is determined largely by the dependence of virus on the cellular membrane transportation system.