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.

Autophagy-related protein 5 (ATG5) interacts with bone marrow stromal cell antigen 2 (BST2) to stimulate HBV replication through antagonizing the antiviral activity of BST2.

Hepatitis B virus (HBV) infection is a major global health burden with 820 000 deaths per year. In our previous study, we found that the knockdown of autophagy-related protein 5 (ATG5) significantly upregulated the interferon-stimulated genes (ISGs) expression to exert the anti-HCV effect. However, the regulation of ATG5 on HBV replication and its underlying mechanism remains unclear. In this study, we screened the altered expression of type I interferon (IFN-I) pathway genes using RT² Profiler™ PCR array following ATG5 knock-down and we found the bone marrow stromal cell antigen 2 (BST2) expression was significantly increased. We then verified the upregulation of BST2 by ATG5 knockdown using RT-qPCR and found that the knockdown of ATG5 activated the Janus kinase/signal transducer and activator of transcription (JAK-STAT) signaling pathway. ATG5 knockdown or BST2 overexpression decreased Hepatitis B core Antigen (HBcAg) protein, HBV DNA levels in cells and supernatants of HepAD38 and HBV-infected NTCP-HepG2. Knockdown of BST2 abrogated the anti-HBV effect of ATG5 knockdown. Furthermore, we found that ATG5 interacted with BST2, and further formed a ternary complex together with HBV-X (HBx). In conclusion, our finding indicates that ATG5 promotes HBV replication through decreasing BST2 expression and interacting with it directly to antagonize its antiviral function.

IFNα-induced BST2+ tumor-associated macrophages facilitate immunosuppression and tumor growth in pancreatic cancer by ERK-CXCL7 signaling.

Pancreatic ductal adenocarcinoma (PDAC) features an immunosuppressive tumor microenvironment (TME) that resists immunotherapy. Tumor-associated macrophages, abundant in the TME, modulate T cell responses. Bone marrow stromal antigen 2-positive (BST2+) macrophages increase in KrasG12D/+; Trp53R172H/+; Pdx1-Cre mouse models during PDAC progression. However, their role in PDAC remains elusive. Our findings reveal a negative correlation between BST2+ macrophage levels and PDAC patient prognosis. Moreover, an increased ratio of exhausted CD8+ T cells is observed in tumors with up-regulated BST2+ macrophages. Mechanistically, BST2+ macrophages secrete CXCL7 through the ERK pathway and bind with CXCR2 to activate the AKT/mTOR pathway, promoting CD8+ T cell exhaustion. The combined blockade of CXCL7 and programmed death-ligand 1 successfully decelerates tumor growth. Additionally, cGAS-STING pathway activation in macrophages induces interferon (IFN)α synthesis leading to BST2 overexpression in the PDAC TME. This study provides insights into IFNα-induced BST2+ macrophages driving an immune-suppressive TME through ERK-CXCL7 signaling to regulate CD8+ T cell exhaustion in PDAC.

Neuronal BST2: A Pruritic Mediator alongside Protease-Activated Receptor 2 in the IL-27-Driven Itch Pathway.

Chronic itch is a common and complex symptom often associated with skin diseases such as atopic dermatitis (AD). Although IL-27 is linked to AD, its role and clinical significance in itch remain undefined. We sought to investigate IL-27 function in itch using tissue-specific transgenic mice, various itch models, behavior scoring, RNA sequencing, and cytokine/kinase array. Our findings show that IL-27 receptors were overexpressed in human AD skin. Intradermal IL-27 injection failed to directly induce itch in mice but upregulated skin protease-activated receptor 2 (PAR2) transcripts, a key factor in itch and AD. IL-27 activated human keratinocytes, increasing PAR2 transcription and activity. Coinjection of SLIGRL (PAR2 agonist) and IL-27 in mice heightened PAR2-mediated itch. In addition, IL-27 boosted BST2 transcription in sensory neurons and keratinocytes. BST2 was upregulated in AD skin, and its injection in mice induced itch-like response. BST2 colocalized with sensory nerve branches in AD skin from both human and murine models. Sensory neurons released BST2, and mice with sensory neuron-specific BST2 knockout displayed reduced itch responses. Overall, this study provides evidence that skin IL-27/PAR2 and neuronal IL-27/BST2 axes are implicated in cutaneous inflammation and pruritus. The discovery of neuronal BST2 in pruritus shed light on BST2 in the itch cascade.

Antiviral factors and their counteraction by HIV-1: many uncovered and more to be discovered.

Extensive studies on HIV-1 have led to the discovery of a variety of structurally and functionally diverse innate defense factors that target various steps of the retroviral replication cycle. Some of them, such as APOBEC3, tetherin, and SERINC5, are well established. Their importance is evident from the fact that HIV-1 uses its accessory proteins Vif, Vpu, and Nef to counteract them. However, the list of antiviral factors is constantly increasing, and accumulating evidence suggests that innate defense mechanisms, which restrict HIV-1 and/or are counteracted by viral proteins, remain to be discovered. These antiviral factors are relevant to diseases other than HIV/AIDS, since they are commonly active against various viral pathogens. In this review, we provide an overview of recently reported antiretroviral factors and viral countermeasures, present the evidence suggesting that more innate defense mechanisms remain to be discovered, and discuss why this is a challenging but rewarding task.

Bone marrow stromal cell antigen 2: Tumor biology, signaling pathway and therapeutic targeting (Review).

Bone marrow stromal cell antigen 2 (BST2) is a type II transmembrane protein that serves critical roles in antiretroviral defense in the innate immune response. In addition, it has been suggested that BST2 is highly expressed in various types of human cancer and high BST2 expression is related to different clinicopathological parameters in cancer. The molecular mechanism underlying BST2 as a potential tumor biomarker in human solid tumors has been reported on; however, to the best of our knowledge, there has been no review published on the molecular mechanism of BST2 in human solid tumors. The present review focuses on human BST2 expression, structure and functions; the molecular mechanisms of BST2 in breast cancer, hepatocellular carcinoma, gastrointestinal tumor and other solid tumors; the therapeutic potential of BST2; and the possibility of BST2 as a potential marker. BST2 is involved in cell membrane integrity and lipid raft formation, which can activate epidermal growth factor receptor signaling pathways, providing a potential mechanistic link between BST2 and tumorigenesis. Notably, BST2 may be considered a universal tumor biomarker and a potential therapeutical target.

BST2 promotes gastric cancer metastasis under the regulation of HOXD9 and PABPC1.

Gastric cancer (GC) constitutes substantial cancer mortality worldwide. Several cancer types aberrantly express bone marrow stromal cell antigen 2 (BST2), yet its functional and underlying mechanisms in GC progression remain unknown. In our study, RNA sequencing data revealed that BST2 was transcriptionally activated by homeobox D9 (HOXD9). BST2 was significantly upregulated in GC tissues and promoted epithelial-mesenchymal transition and metastasis of GC. BST2 knockdown reversed HOXD9's oncogenic effect on GC metastasis. Moreover, BST2 messenger RNA stability could be enhanced by poly(A) binding protein cytoplasmic 1 (PABPC1) through the interaction between BST2 3'-UTR and PABPC1 in GC cells. PABPC1 promoted GC metastasis, which BST2 silencing attenuated in vitro and in vivo. In addition, positive correlations among HOXD9, BST2, and PABPC1 were established in clinical samples. Taken together, increased expression of BST2 induced by HOXD9 synergizing with PABPC1 promoted GC cell migration and invasion capacity.

Mutations accumulated in the Spike of SARS-CoV-2 Omicron allow for more efficient counteraction of the restriction factor BST2/Tetherin.

BST2/Tetherin is a restriction factor with broad antiviral activity against enveloped viruses, including coronaviruses. Specifically, BST2 traps nascent particles to membrane compartments, preventing their release and spread. In turn, viruses have evolved multiple mechanisms to counteract BST2. Here, we examined the interactions between BST2 and SARS-CoV-2. Our study shows that BST2 reduces SARS-CoV-2 virion release. However, the virus uses the Spike (S) protein to downregulate BST2. This requires a physical interaction between S and BST2, which routes BST2 for lysosomal degradation in a Clathtin- and ubiquitination-dependent manner. By surveying different SARS-CoV-2 variants of concern (Alpha-Omicron), we found that Omicron is more efficient at counteracting BST2, and that mutations in S account for its enhanced anti-BST2 activity. Mapping analyses revealed that several surfaces in the extracellular region of BST2 are required for an interaction with the Spike, and that the Omicron variant has changed its patterns of association with BST2 to improve its counteraction. Therefore, our study suggests that, besides enhancing receptor binding and evasion of neutralizing antibodies, mutations accumulated in the Spike afford more efficient counteraction of BST2, which highlights that BST2 antagonism is important for SARS-CoV-2 infectivity and spread.

Higher BST2 Expression Promotes the Anti-HBV Effect of IFN-α and BST2 Genetic Variant Predicts PegIFNα Treatment Response of HBeAg-Positive Chronic Hepatitis B Patients.

We previously reported that an interferon (IFN)-inducible protein, BST2, was regulated by the JAK-STAT pathway activated by CD40, and subsequently suppressing hepatitis B virus (HBV) repliaction and transcription. The current research attempted to assess the impact of BST2 on the IFN-treated anti-HBV effect, and explore BST2 variants for predicting pegylated IFN alpha (PegIFNα) therapy response of patients with hepatitis B e antigen (HBeAg)-positive chronic hepatitis B (CHB). Using an HBV-transfected cell model, the function of BST2 on HBV DNA replication and transcription driven by IFN was studied. The potentially functional BST2 variants were selected through a strategy of gene-wide screening. The associations of BST2 variants and polygenic score (PGS) model, which was used to quantify the combined influence of several genetic variants, with treatment response were examined in 2 separate PegIFNα-treated cohorts of 238 and 707 patients with CHB, respectively. We found that overexpression of BST2 improved the anti-HBV activity triggered by IFN-α. Among PegIFNα-treated patients with CHB, BST2_rs9576 was screened out to be significantly correlated with combined response (CR; i.e., HBeAg seroconversion along with HBV DNA level <3.3log10 IU/mL, P = 7.12 × 10-5 ). Additionally, there was a strong correlation between the PGS incorporating BST2_rs9576 and other 5 genetic variations (previously described predictors of therapy response to PegIFNα) and CR (P = 1.81 × 10-13 ), hepatitis B surface antigen (HBsAg) level (P = 0.004), as well as HBsAg decline (P = 0.017). In conclusion, higher BST2 expression responded better to IFN-α treatment. BST2_rs9576 is an effective indicator to forecast therapy response of PegIFNα-treated patients with CHB. The PGS possesses the potential to boost the ability of PegIFNα therapy response.

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.

Human BST2 inhibits rabies virus release independently of cysteine-linked dimerization and asparagine-linked glycosylation.

The innate immune response is a first-line defense mechanism triggered by rabies virus (RABV). Interferon (IFN) signaling and ISG products have been shown to confer resistance to RABV at various stages of the virus's life cycle. Human tetherin, also known as bone marrow stromal cell antigen 2 (hBST2), is a multifunctional transmembrane glycoprotein induced by IFN that has been shown to effectively counteract many viruses through diverse mechanisms. Here, we demonstrate that hBST2 inhibits RABV budding by tethering new virions to the cell surface. It was observed that release of virus-like particles (VLPs) formed by RABV G (RABV-G VLPs), but not RABV M (RABV-G VLPs), were suppressed by hBST2, indicating that RABV-G has a specific effect on the hBST2-mediated restriction of RABV. The ability of hBST2 to prevent the release of RABV-G VLPs and impede RABV growth kinetics is retained even when hBST2 has mutations at dimerization and/or glycosylation sites, making hBST2 an antagonist to RABV, with multiple mechanisms possibly contributing to the hBST2-mediated suppression of RABV. Our findings expand the knowledge of host antiviral mechanisms that control RABV infection.

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.

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.

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.

CD317 maintains proteostasis and cell survival in response to proteasome inhibitors by targeting calnexin for RACK1-mediated autophagic degradation.

Unbalanced protein homeostasis (proteostasis) networks are frequently linked to tumorigenesis, making cancer cells more susceptible to treatments that target proteostasis regulators. Proteasome inhibition is the first licensed proteostasis-targeting therapeutic strategy, and has been proven effective in hematological malignancy patients. However, drug resistance almost inevitably develops, pressing for a better understanding of the mechanisms that preserve proteostasis in tumor cells. Here we report that CD317, a tumor-targeting antigen with a unique topology, was upregulated in hematological malignancies and preserved proteostasis and cell viability in response to proteasome inhibitors (PIs). Knocking down CD317 lowered Ca2+ levels in the endoplasmic reticulum (ER), promoting PIs-induced proteostasis failure and cell death. Mechanistically, CD317 interacted with calnexin (CNX), an ER chaperone protein that limits calcium refilling via the Ca2+ pump SERCA, thereby subjecting CNX to RACK1-mediated autophagic degradation. As a result, CD317 decreased the level of CNX protein, coordinating Ca2+ uptake and thus favoring protein folding and quality control in the ER lumen. Our findings reveal a previously unrecognized role of CD317 in proteostasis control and imply that CD317 could be a promising target for resolving PIs resistance in the clinic.

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.

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.

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.

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.