A heterocyclic compound inhibits viral release by inducing cell surface BST2/Tetherin/CD317/HM1.24.

The introduction of combined antiretroviral therapy (cART) has greatly improved the quality of life of human immunodeficiency virus type 1 (HIV-1)-infected individuals. Nonetheless, the ever-present desire to seek out a full remedy for HIV-1 infections makes the discovery of novel antiviral medication compelling. Owing to this, a new late-stage inhibitor, Lenacapavir/Sunlenca, an HIV multi-phase suppressor, was clinically authorized in 2022. Besides unveiling cutting-edge antivirals inhibiting late-stage proteins or processes, newer therapeutics targeting host restriction factors hold promise for the curative care of HIV-1 infections. Notwithstanding, bone marrow stromal antigen 2 (BST2)/Tetherin/CD317/HM1.24, which entraps progeny virions is an appealing HIV-1 therapeutic candidate. In this study, a novel drug screening system was established, using the Jurkat/Vpr-HiBiT T cells, to identify drugs that could obstruct HIV-1 release; the candidate compounds were selected from the Ono Pharmaceutical compound library. Jurkat T cells expressing Vpr-HiBiT were infected with NL4-3, and the amount of virus release was quantified indirectly by the amount of Vpr-HiBiT incorporated into the progeny virions. Subsequently, the candidate compounds that suppressed viral release were used to synthesize the heterocyclic compound, HT-7, which reduces HIV-1 release with less cellular toxicity. Notably, HT-7 increased cell surface BST2 coupled with HIV-1 release reduction in Jurkat cells but not Jurkat/KO-BST2 cells. Seemingly, HT-7 impeded simian immunodeficiency virus (SIV) and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) release. Concisely, these results suggest that the reduction in viral release, following HT-7 treatment, resulted from the modulation of cell surface expression of BST2 by HT-7.

Polypeptides inhibit HIV-1 replication by interfering viral Vpu-mediated tetherin degradation.

Background: HIV-1 Vpu acts by counteracting the tethering function of tetherin and resulting in the release of HIV-1 virion. Disrupting Vpu-tetherin interactions may provide a promising new target for antiretroviral therapy. Methods: Polypeptides that covered the amino acid sequence on the interface of Vpu-tetherin complex were designed. Phenotypic susceptibilities and cellular toxicities to the polypeptides were measured. The mechanisms of the anti-HIV-1 polypeptides were determined by the Western blot analysis and laser confocal scanning. Seven 20-mer polypeptides from wild-type Vpu amino acid sequence were designed. Results: We report the design and identification of 3 novel anti-HIV-1 polypeptides that derived from Vpu sequence which can efficiently inhibit HIV-1 infection. A pilot mechanism study showed that the active polypeptide could counteract Vpu-mediated tetherin downregulation. Laser confocal image scanning study showed that the polypeptides bound on the cell surface with a receptor specific binding manner, which may target tetherin that expressed on cell surface. Conclusion: Our work provided first evidence that counteracting Vpu-mediated tetherin downregulation could be a target for novel anti-HIV-1 drug design. Future works to provide direct evidence of inhibitors interact with tetherin at atomic resolution and the development of small molecules inhibitors targeting Vpu-tetherin interactions may open a new avenue for novel antiretroviral therapy.

Tetherin Restricts SARS-CoV-2 despite the Presence of Multiple Viral Antagonists.

Coronavirus infection induces interferon-stimulated genes, one of which encodes Tetherin, a transmembrane protein inhibiting the release of various enveloped viruses from infected cells. Previous studies revealed that SARS-CoV encodes two Tetherin antagonists: the Spike protein (S), inducing lysosomal degradation of Tetherin, and ORF7a, altering its glycosylation. Similarly, SARS-CoV-2 has also been shown to use ORF7a and Spike to enhance virion release in the presence of Tetherin. Here, we directly compare the abilities and mechanisms of these two viral proteins to counteract Tetherin. Therefore, cell surface and total Tetherin levels upon ORF7a or S expression were investigated using flow cytometry and Western blot analysis. SARS-CoV and SARS-CoV-2 S only marginally reduced Tetherin cell surface levels in a cell type-dependent manner. In HEK293T cells, under conditions of high exogenous Tetherin expression, SARS-CoV-2 S and ORF7a reduced total cellular Tetherin levels much more efficiently than the respective counterparts derived from SARS-CoV. Nevertheless, ORF7a from both species was able to alter Tetherin glycosylation. The ability to decrease total protein levels of Tetherin was conserved among S proteins from different SARS-CoV-2 variants (α, γ, δ, ο). While SARS-CoV-2 S and ORF7a both colocalized with Tetherin, only ORF7a directly interacted with the restriction factor in a two-hybrid assay. Despite the presence of multiple Tetherin antagonists, SARS-CoV-2 replication in Caco-2 cells was further enhanced upon Tetherin knockout. Altogether, our data show that endogenous Tetherin restricts SARS-CoV-2 replication and that the antiviral activity of Tetherin is only partially counteracted by viral antagonists with differential and complementary modes of action.

Blocking of ebolavirus spread through intercellular connections by an MPER-specific antibody depends on BST2/tetherin.

Ebola virus (EBOV) and Bundibugyo virus (BDBV) belong to the family Filoviridae and cause a severe disease in humans. We previously isolated a large panel of monoclonal antibodies from B cells of human survivors from the 2007 Uganda BDBV outbreak, 16 survivors from the 2014 EBOV outbreak in the Democratic Republic of the Congo, and one survivor from the West African 2013-2016 EBOV epidemic. Here, we demonstrate that EBOV and BDBV are capable of spreading to neighboring cells through intercellular connections in a process that depends upon actin and T cell immunoglobulin and mucin 1 protein. We quantify spread through intercellular connections by immunofluorescence microscopy and flow cytometry. One of the antibodies, BDBV223, specific to the membrane-proximal external region, induces virus accumulation at the plasma membrane. The inhibiting activity of BDBV223 depends on BST2/tetherin.

Tetherin antagonism by SARS-CoV-2 ORF3a and spike protein enhances virus release.

The antiviral restriction factor, tetherin, blocks the release of several different families of enveloped viruses, including the Coronaviridae. Tetherin is an interferon-induced protein that forms parallel homodimers between the host cell and viral particles, linking viruses to the surface of infected cells and inhibiting their release. We demonstrate that SARS-CoV-2 infection causes tetherin downregulation and that tetherin depletion from cells enhances SARS-CoV-2 viral titres. We investigate the potential viral proteins involved in abrogating tetherin function and find that SARS-CoV-2 ORF3a reduces tetherin localisation within biosynthetic organelles where Coronaviruses bud, and increases tetherin localisation to late endocytic organelles via reduced retrograde recycling. We also find that expression of Spike protein causes a reduction in cellular tetherin levels. Our results confirm that tetherin acts as a host restriction factor for SARS-CoV-2 and highlight the multiple distinct mechanisms by which SARS-CoV-2 subverts tetherin function.

Independent loss events of a functional tetherin gene in galliform birds.

IMPORTANCE: Birds represent important hosts for numerous viruses, including zoonotic viruses and pathogens with the potential to cause major economic losses to the poultry industry. Viral replication and transmission can be inhibited or blocked by the action of antiviral restriction factors (RFs) encoded by the host. One well-characterized RF is tetherin, a protein that directly blocks the release of newly formed viral particles from infected cells. Here, we describe the evolutionary loss of a functional tetherin gene in two galliform birds, turkey (Meleagris gallopavo) and Mikado pheasant (Syrmaticus mikado). Moreover, we demonstrate that the structurally related protein TMCC(aT) exerts antiviral activity in several birds, albeit by a mechanism different from that of tetherin. The evolutionary scenario described here represents the first documented loss-of-tetherin cases in vertebrates.

Small but Highly Versatile: The Viral Accessory Protein Vpu.

Human and simian immunodeficiency viruses (HIVs and SIVs, respectively) encode several small proteins (Vif, Vpr, Nef, Vpu, and Vpx) that are called accessory because they are not generally required for viral replication in cell culture. However, they play complex and important roles for viral immune evasion and spread in vivo. Here, we discuss the diverse functions and the relevance of the viral protein U (Vpu) that is expressed from a bicistronic RNA during the late stage of the viral replication cycle and found only in HIV-1 and closely related SIVs. It is well established that Vpu counteracts the restriction factor tetherin, mediates degradation of the primary viral CD4 receptors, and inhibits activation of the transcription factor nuclear factor kappa B. Recent studies identified additional activities and provided new insights into the sophisticated mechanisms by which Vpu enhances and prolongs the release of fully infectious viral particles. In addition, it has been shown that Vpu prevents superinfection not only by degrading CD4 but also by modulating DNA repair mechanisms to promote degradation of nuclear viral complementary DNA in cells that are already productively infected.

ATG5 selectively engages virus-tethered BST2/tetherin in an LC3C-associated pathway.

Bone marrow stromal antigen 2 (BST2)/tetherin is a restriction factor that reduces HIV-1 dissemination by tethering virus at the cell surface. BST2 also acts as a sensor of HIV-1 budding, establishing a cellular antiviral state. The HIV-1 Vpu protein antagonizes BST2 antiviral functions via multiple mechanisms, including the subversion of an LC3C-associated pathway, a key cell intrinsic antimicrobial mechanism. Here, we describe the first step of this viral-induced LC3C-associated process. This process is initiated at the plasma membrane through the recognition and internalization of virus-tethered BST2 by ATG5, an autophagy protein. ATG5 and BST2 assemble as a complex, independently of the viral protein Vpu and ahead of the recruitment of the ATG protein LC3C. The conjugation of ATG5 with ATG12 is dispensable for this interaction. ATG5 recognizes cysteine-linked homodimerized BST2 and specifically engages phosphorylated BST2 tethering viruses at the plasma membrane, in an LC3C-associated pathway. We also found that this LC3C-associated pathway is used by Vpu to attenuate the inflammatory responses mediated by virion retention. Overall, we highlight that by targeting BST2 tethering viruses, ATG5 acts as a signaling scaffold to trigger an LC3C-associated pathway induced by HIV-1 infection.

The Feline Immunodeficiency Virus Envelope Signal Peptide Is a Tetherin Antagonizing Protein.

Signal peptides are N-terminal peptides, generally less than 30 amino acids in length, that direct translocation of proteins into the endoplasmic reticulum and secretory pathway. The envelope glycoprotein (Env) of the nonprimate lentivirus feline immunodeficiency virus (FIV) contains the longest signal peptide of all eukaryotic, prokaryotic, and viral proteins (175 amino acids), yet the reason is unknown. Tetherin is a dual membrane-anchored host protein that inhibits the release of enveloped viruses from cells. Primate lentiviruses have evolved three antagonists: the small accessory proteins Vpu and Nef, and in the case of HIV-2, Env. Here, we identify the FIV Env signal peptide (Fsp) as the FIV tetherin antagonist. A short deletion in the central portion of Fsp had no effect on viral replication in the absence of tetherin, but severely impaired virion budding in its presence. Fsp is necessary and sufficient, acting as an autonomous accessory protein with the rest of Env dispensable. In contrast to primate lentivirus tetherin antagonists, its mechanism is to stringently block the incorporation of this restriction factor into viral particles rather than by degrading it or downregulating it from the plasma membrane. IMPORTANCE The study of species- and virus-specific differences in restriction factors and their antagonists has been central to deciphering the nature of these key host defenses. FIV is an AIDS-causing lentivirus that has achieved pandemic spread in the domestic cat. We now identify its tetherin antagonist as the signal sequence of the Envelope glycoprotein, thus identifying the fourth lentiviral anti-tetherin protein and the first new lentiviral accessory protein in decades. Fsp is necessary and sufficient and functions by stringently blocking particle incorporation of tetherin, which differs from the degradation or surface downregulation mechanisms used by primate lentiviruses. Fsp also is a novel example of signal peptide dual function, being both a restriction factor antagonist and a mediator of protein translocation into the endoplasmic reticulum.

A Novel Vpu Adaptive Mutation of HIV-1 Degrades Tetherin in Northern Pig-Tailed Macaques (Macaca leonina) Mainly via the Ubiquitin-Proteasome Pathway and Increases Viral Release.

Tetherin prevents viral cross-species transmission by inhibiting the release of multiple enveloped viruses from infected cells. With the evolution of simian immunodeficiency virus of chimpanzees (SIVcpz), a pandemic human immunodeficiency virus type 1 (HIV-1) precursor, its Vpu protein can antagonize human tetherin (hTetherin). Macaca leonina (northern pig-tailed macaque [NPM]) is susceptible to HIV-1, but host-specific restriction factors limit virus replication in vivo. In this study, we isolated the virus from NPMs infected with strain stHIV-1sv (with a macaque-adapted HIV-1 env gene from simian-human immunodeficiency virus SHIV-KB9, a vif gene replaced by SIVmac239, and other genes originating from HIV-1NL4.3) and found that a single acidic amino acid substitution (G53D) in Vpu could increase its ability to degrade the tetherin of macaques (mTetherin) mainly through the proteasome pathway, resulting in an enhanced release and resistance to interferon inhibition of the mutant stHIV-1sv strain, with no influence on the other functions of Vpu. IMPORTANCE HIV-1 has obvious host specificity, which has greatly hindered the construction of animal models and severely restricted the development of HIV-1 vaccines and drugs. To overcome this barrier, we attempted to isolate the virus from NPMs infected with stHIV-1sv, search for a strain with an adaptive mutation in NPMs, and develop a more appropriate nonhuman primate model of HIV-1. This is the first report identifying HIV-1 adaptations in NPMs. It suggests that while tetherin may limit HIV-1 cross-species transmission, the Vpu protein in HIV-1 can overcome this species barrier through adaptive mutation, increasing viral replication in the new host. This finding will be beneficial to building an appropriate animal model for HIV-1 infection and promoting the development of HIV-1 vaccines and drugs.

Identification of Two Isoforms of Canine Tetherin in Domestic Dogs and Characterization of Their Antiviral Activity against Canine Influenza Virus.

Canine influenza virus (CIV) significantly threatens the canine population and public health. Tetherin, an innate immune factor, plays an important role in the defense against pathogen invasion and has been discovered to restrict the release of various enveloped viruses. Two isoforms of canine tetherin (tetherin-X1 and tetherin-X2) were identified in peripheral blood leukocytes of mixed-breed dogs using reverse transcription polymerase chain reaction (RT-PCR). Amino acid alignment revealed that relative to full-length tetherin (tetherin-X1) and truncated canine tetherin (tetherin-X2) exhibited deletion of 34 amino acids. The deletion occurred at the C-terminus of the coiled-coiled ectodomain and the N-terminus of the glycosylphosphatidylinositol (GPI)-anchor domain. Tetherin-X2 was localized subcellularly at the cell membrane, which was consistent with the localization of tetherin-X1. In addition, canine tetherin-X1 and tetherin-X2 restricted the release of H3N2 CIV. However, canine tetherin-X1 had higher antiviral activity than canine tetherin-X2, indicating that the C-terminus of the coiled-coiled ectodomain and the N-terminus of the GPI-anchor domain of canine tetherin (containing the amino acids deleted in tetherin-X2) are critical for its ability to restrict H3N2 CIV release. This study provides insights for understanding the key functional domains of tetherin that restrict CIV release.

Interleukin-27 Promotes Divergent Effects on HIV-1 Infection in Peripheral Blood Mononuclear Cells through BST-2/Tetherin.

Interleukin-27 (IL-27) is able to inhibit HIV-1 replication in peripheral blood mononuclear cells (PBMCs), macrophages, and dendritic cells. Here, we identify that IL-27 can produce opposing effects on HIV-1 replication in PBMCs and that the HIV-1 restriction factor BST-2/Tetherin is involved in both inhibitory and enhancing effects on HIV-1 infection induced by IL-27. IL-27 inhibited HIV-1 replication when added to cells 2 h after infection, promoting the prototypical BST-2/Tetherin-induced virion accumulation at the cell membrane of HIV-1-infected PBMCs. BST-2/Tetherin gene expression was significantly upregulated in the IL-27-treated PBMCs, with a simultaneous increase in the number of BST-2/Tetherin+ cells. The silencing of BST-2/Tetherin diminished the anti-HIV-1 effect of IL-27. In contrast, IL-27 increased HIV-1 production when added to infected cells 4 days after infection. This enhancing effect was prevented by BST-2/Tetherin gene knockdown, which also permitted IL-27 to function again as an HIV-1 inhibitory factor. These contrasting roles of IL-27 were associated with the dynamic of viral production, since the IL-27-mediated enhancement of virus replication was prevented by antiretroviral treatment of infected cells, as well as by keeping cells under agitation to avoid cell-to-cell contact. Likewise, inhibition of CD11a, an integrin associated with HIV-1 cell-to-cell transmission, abrogated the IL-27 enhancement of HIV-1 production. Our findings illustrate the complexity of the HIV-1-host interactions and may impact the potential therapeutic use of IL-27 and other soluble mediators that induce BST-2/Tetherin expression for HIV-1 infection. IMPORTANCE Here, we describe new findings related to the ability of the cytokine IL-27 to regulate the growth of HIV-1 in CD4+ T lymphocytes. IL-27 has long been considered a potent inhibitor of HIV-1 replication, a notion based on several reports showing that this cytokine controls HIV-1 infection in peripheral blood mononuclear cells (PBMCs), monocyte-derived macrophages, and dendritic cells. However, our present results are contrary to the current knowledge that IL-27 acts only as a powerful downregulator of HIV-1 replication. We observed that IL-27 can either prevent or enhance viral growth in PBMCs, an outcome dependent on when this cytokine is added to the infected cells. We detected that the increase of HIV-1 dissemination is due to enhanced cell-to-cell transmission with the involvement of the interferon-induced HIV-1 restriction factor BST-2/Tetherin and CD11a (LFA-1), an integrin that participates in formation of virological synapse.

Corrigendum: CD317-Positive Immune Stromal Cells in Human "Mesenchymal Stem Cell" Populations.

[This corrects the article DOI: 10.3389/fimmu.2022.903796.].

Unique Evolution of Antiviral Tetherin in Bats.

Bats are recognized as important reservoirs of viruses deadly to other mammals, including humans. These infections are typically nonpathogenic in bats, raising questions about host response differences that might exist between bats and other mammals. Tetherin is a restriction factor which inhibits the release of a diverse range of viruses from host cells, including retroviruses, coronaviruses, filoviruses, and paramyxoviruses, some of which are deadly to humans and transmitted by bats. Here, we characterize the tetherin genes from 27 bat species, revealing that they have evolved under strong selective pressure, and that fruit bats and vesper bats express unique structural variants of the tetherin protein. Tetherin was widely and variably expressed across fruit bat tissue types and upregulated in spleen tissue when stimulated with Toll-like receptor agonists. The expression of two computationally predicted splice isoforms of fruit bat tetherin was verified. We identified an additional third unique splice isoform which includes a C-terminal region that is not homologous to known mammalian tetherin variants but was functionally capable of restricting the release of filoviral virus-like particles. We also report that vesper bats possess and express at least five tetherin genes, including structural variants, more than any other mammal reported to date. These findings support the hypothesis of differential antiviral gene evolution in bats relative to other mammals. IMPORTANCE Bats are an important host of various viruses which are deadly to humans and other mammals but do not cause outward signs of illness in bats. Furthering our understanding of the unique features of the immune system of bats will shed light on how they tolerate viral infections, potentially informing novel antiviral strategies in humans and other animals. This study examines the antiviral protein tetherin, which prevents viral particles from escaping their host cell. Analysis of tetherin from 27 bat species reveals that it is under strong evolutionary pressure, and we show that multiple bat species have evolved to possess more tetherin genes than other mammals, some of which encode structurally unique tetherins capable of activity against different viral particles. These data suggest that bat tetherin plays a potentially broad and important role in the management of viral infections in bats.

Multi-functional BST2/tetherin against HIV-1, other viruses and LINE-1.

Bone marrow stromal cell antigen 2 (BST2), also known as CD317, HM1.24, or tetherin, is a type II transmembrane glycoprotein. Its expression is induced by IFN-I, and it initiates host immune responses by directly trapping enveloped HIV-1 particles onto the cell surface. This antagonistic mechanism toward the virus is attributable to the unique structure of BST2. In addition to its antiviral activity, BST2 restricts retrotransposon LINE-1 through a distinct mechanism. As counteractive measures, different viruses use a variety of proteins to neutralize the function or even stability of BST2. Interestingly, BST2 seems to have both a positive and a negative influence on immunomodulation and virus propagation. Here, we review the relationship between the structural and functional bases of BST2 in anti-HIV-1 and suppressing retrotransposon LINE-1 activation and focus on its dual features in immunomodulation and regulating virus propagation.

Attenuated HIV-1 Nef But Not Vpu Function in a Cohort of Rwandan Long-Term Survivors.

HIV-1 accessory proteins Nef and Vpu enhance viral pathogenesis through partially overlapping immune evasion activities. Attenuated Nef or Vpu functions have been reported in individuals who display slower disease progression, but few studies have assessed the relative impact of these proteins in non-B HIV-1 subtypes or examined paired proteins from the same individuals. Here, we examined the sequence and function of matched Nef and Vpu clones isolated from 29 long-term survivors (LTS) from Rwanda living with HIV-1 subtype A and compared our results to those of 104 Nef and 62 Vpu clones isolated from individuals living with chronic untreated HIV-1 subtype A from the same geographic area. Nef and vpu coding regions were amplified from plasma HIV RNA and cloned. The function of one intact, phylogenetically-validated Nef and Vpu clone per individual was then quantified by flow cytometry following transient expression in an immortalized CD4+ T-cell line. We measured the ability of each Nef clone to downregulate CD4 and HLA class I, and of each Vpu clone to downregulate CD4 and Tetherin, from the cell surface. Results were normalized to reference clones (Nef-SF2 and Vpu-NL4.3). We observed that Nef-mediated CD4 and HLA downregulation functions were lower in LTS compared to the control cohort (Mann-Whitney p=0.03 and p<0.0001, respectively). Moreover, we found a positive correlation between Nef-mediated CD4 downregulation function and plasma viral load in LTS and controls (Spearman ρ= 0.59, p=0.03 and ρ=0.30, p=0.005, respectively). In contrast, Vpu-mediated functions were similar between groups and did not correlate with clinical markers. Further analyses identified polymorphisms at Nef codon 184 and Vpu codons 60-62 that were associated with function, which were confirmed through mutagenesis. Overall, our results support attenuated function of Nef, but not Vpu, as a contributor to slower disease progression in this cohort of long-term survivors with HIV-1 subtype A.

CD317-Positive Immune Stromal Cells in Human "Mesenchymal Stem Cell" Populations.

Heterogeneity of bone marrow mesenchymal stromal cells (MSCs, frequently referred to as "mesenchymal stem cells") clouds biological understanding and hampers their clinical development. In MSC cultures most commonly used in research and therapy, we have identified an MSC subtype characterized by CD317 expression (CD317pos (29.77 ± 3.00% of the total MSC population), comprising CD317dim (28.10 ± 4.60%) and CD317bright (1.67 ± 0.58%) MSCs) and a constitutive interferon signature linked to human disease. We demonstrate that CD317pos MSCs induced cutaneous tissue damage when applied a skin explant model of inflammation, whereas CD317neg MSCs had no effect. Only CD317neg MSCs were able to suppress proliferative cycles of activated human T cells in vitro, whilst CD317pos MSCs increased polarization towards pro-inflammatory Th1 cells and CD317neg cell lines did not. Using an in vivo peritonitis model, we found that CD317neg and CD317pos MSCs suppressed leukocyte recruitment but only CD317neg MSCs suppressed macrophage numbers. Using MSC-loaded scaffolds implanted subcutaneously in immunocompromised mice we were able to observe tissue generation and blood vessel formation with CD317neg MSC lines, but not CD317pos MSC lines. Our evidence is consistent with the identification of an immune stromal cell, which is likely to contribute to specific physiological and pathological functions and influence clinical outcome of therapeutic MSCs.

Substitutions in Nef That Uncouple Tetherin and SERINC5 Antagonism Impair Simian Immunodeficiency Virus Replication in Primary Rhesus Macaque Lymphocytes.

Most simian immunodeficiency viruses (SIVs) use Nef to counteract restriction by the tetherin proteins of their nonhuman primate hosts. In addition to counteracting tetherin, SIV Nef has a number of other functions, including the downmodulation of CD3, CD4, and major histocompatibility complex class I (MHC I) molecules from the surface of SIV-infected cells and the enhancement of viral infectivity by preventing the incorporation of SERINC5 into virions. Although these activities require different surfaces of Nef, they can be difficult to separate because of their dependence on similar interactions with AP-1 or AP-2 for clathrin-mediated endocytosis. We previously observed extensive overlap of the SIV Nef residues required for counteracting tetherin and SERINC5. Here, we define substitutions in Nef that separate anti-tetherin activity from SERINC5 antagonism and other activities of Nef. This information was used to engineer an infectious molecular clone of SIV (SIVmac239nefSA) that is sensitive to tetherin but retains CD3, CD4, MHC I, and SERINC5 downmodulation. In primary rhesus macaque CD4+ T cells, SIVmac239nefSA exhibits impaired replication compared to wild-type SIVmac239 under conditions of interferon-induced upregulation of tetherin. These results demonstrate that tetherin antagonism can be separated from other Nef functions and that resistance to tetherin is essential for optimal replication in primary CD4+ T cells. IMPORTANCE Tetherin is an interferon-inducible transmembrane protein that prevents the detachment of enveloped viruses from infected cells by physically tethering nascent virions to cellular membranes. SIV Nef downmodulates simian tetherin to overcome this restriction in nonhuman primate hosts. Nef also enhances virus infectivity by preventing the incorporation of SERINC5 into virions and contributes to immune evasion by downmodulating other proteins from the cell surface. To assess the contribution of tetherin antagonism to virus replication, we engineered an infectious molecular clone of SIV with substitutions in Nef that uncouple tetherin antagonism from other Nef functions. These substitutions impaired virus replication in interferon-treated macaque CD4+ T cells, revealing the impact of tetherin on SIV replication under physiological conditions in primary CD4+ lymphocytes.

Dual Role of HIV-1 Envelope Signal Peptide in Immune Evasion.

HIV-1 Env signal peptide (SP) is an important contributor to Env functions. Env is generated from Vpu/Env encoded bicistronic mRNA such that the 5' end of Env-N-terminus, that encodes for Env-SP overlaps with 3' end of Vpu. Env SP displays high sequence diversity, which translates into high variability in Vpu sequence. This study aimed to understand the effect of sequence polymorphism in the Vpu-Env overlapping region (VEOR) on the functions of two vital viral proteins: Vpu and Env. We used infectious molecular clone pNL4.3-CMU06 and swapped its SP (or VEOR) with that from other HIV-1 isolates. Swapping VEOR did not affect virus production in the absence of tetherin however, presence of tetherin significantly altered the release of virus progeny. VEOR also altered Vpu's ability to downregulate CD4 and tetherin. We next tested the effect of these swaps on Env functions. Analyzing the binding of monoclonal antibodies to membrane embedded Env revealed changes in the antigenic landscape of swapped Envs. These swaps affected the oligosaccharide composition of Env-N-glycans as shown by changes in DC-SIGN-mediated virus transmission. Our study suggests that genetic diversity in VEOR plays an important role in the differential pathogenesis and also assist in immune evasion by altering Env epitope exposure.

Catch Me if You Can: the Crosstalk of Zika Virus and the Restriction Factor Tetherin.

Zika virus (ZIKV) is a flavivirus that is mainly transmitted by Aedes mosquitos and normally causes mild symptoms. During the outbreak in the Americas in 2015, it was associated with more severe implications, like microcephaly in newborns and the Guillain-Barré syndrome. The lack of specific vaccines and cures strengthens the need for a deeper understanding of the virus life cycle and virus-host interactions. The restriction factor tetherin (THN) is an interferon-inducible cellular protein with broad antiviral properties. It is known to inhibit the release of various enveloped viruses by tethering them to each other and the cell membrane, thereby preventing their further spread. On the other hand, different viruses have developed various escape strategies against THN. Analysis of the cross-talk between ZIKV and THN revealed that, despite a strong induction of THN mRNA expression in ZIKV-infected cells, this is not reflected by an elevated protein level of THN. Contrariwise, the THN protein level is decreased due to a reduced half-life. The increased degradation of THN in ZIKV infected cells involves the endo-lysosomal system but does not depend on the early steps of autophagy. Enrichment of THN by depletion of the ESCRT-0 protein HRS diminishes ZIKV release and spread, which points out the capacity of THN to restrict ZIKV and explains the enhanced THN degradation in infected cells as an effective viral escape strategy. IMPORTANCE Although tetherin expression is strongly induced by ZIKV infection there is a reduction in the amount of tetherin protein. This is due to enhanced lysosomal degradation. However, if the tetherin level is rescued then the release of ZIKV is impaired. This shows that tetherin is a restriction factor for ZIKV, and the induction of an efficient degradation represents a viral escape strategy. To our knowledge, this is the first study that describes and characterizes tetherin as a restriction factor for the ZIKV life cycle.