Release Of Human Immunodeficiency Virus Type 1 Particles Research Articles

The role of BST2/tetherin in feline retrovirus infection

Pathogenic retroviral infections of mammals have induced the evolution of cellular anti-viral restriction factors and have shaped their biological activities. This intrinsic immunity plays an important role in controlling viral replication and imposes a barrier to viral cross-species transmission. Well-studied examples of such host restriction factors are TRIM5α, an E3 ubiquitin ligase that binds incoming retroviral capsids in the cytoplasm via its C-terminal PRY/SPRY (B30.2) domain and targets them for proteasomal degradation, and APOBEC3 proteins, cytidine deaminases that induce hypermutation and impair viral reverse transcription. Tetherin (BST-2, CD317) is an interferon-inducible transmembrane protein that potently inhibits the release of nascent retrovirus particles in single-cycle replication assays. However, whether the primary biological activity of tetherin in vivo is that of a restriction factor remains uncertain as recent studies on human tetherin suggest that it is unable to prevent spreading infection of human immunodeficiency virus type 1 (HIV-1). The feline tetherin homologue resembles human tetherin in amino acid sequence, protein topology and anti-viral activity. Transiently expressed feline tetherin displays potent inhibition of feline immunodeficiency virus (FIV) and HIV-1 particle release. However, stable ectopic expression of feline tetherin in a range of feline cell lines has no inhibitory effect on the growth of either primary or cell culture-adapted strains of FIV. By comparing and contrasting the activities of the felid and primate tetherins against their respective immunodeficiency-causing lentiviruses we may gain insight into the contribution of tetherins to the control of lentiviral replication and the evolution of lentiviral virulence.

CD4 downregulation by the human immunodeficiency virus type 1 Nef protein is dispensable for optimal output and functionality of viral particles in primary T cells

Nef is a multifunctional protein of the human immunodeficiency virus type 1 (HIV-1) required for high viral replication and disease progression. Several findings indicate that the capacity of Nef to downregulate surface CD4 is essential for the protein's pathogenic activity, although the mechanisms that link the two functions are yet unclear. It is believed that, by reducing surface CD4 levels, Nef counteracts the receptor's negative effects on virion infectivity and release. Here, we show that, in 293T cells co-expressing CD4 and HIV-1, the capacity of Nef to enhance the virion incorporation of Env products and release of viral particles was mediated by retention-degradation of neo-synthesized CD4 rather than by accelerated receptor endocytosis. Different results were observed in primary CD4(+) T lymphocytes in which Nef-mediated CD4 downregulation occurs primarily by accelerated internalization. In HIV-infected T cell cultures, Nef was crucial for the removal of surface CD4 at the beginning of the infection, while later on maximal CD4 downregulation was achieved in a Nef-independent manner. Moreover, by means of in vivo selected Nef mutants, we observed that CD4 downregulation is not essential for Nef ability to enhance Env incorporation into virions and increase viral infectivity or replication in CD4(+) T lymphocytes. Notably, Nef expression itself was dispensable for efficient release of HIV-1 particles by T cells. In conclusion, we propose that the CD4 downregulation activity of Nef plays a role before the late productive phases of HIV-1 replication.

High Natural Permissivity of Primary Rabbit Cells for HIV-1, with a Virion Infectivity Defect in Macrophages as the Final Replication Barrier

An immunocompetent, permissive, small-animal model would be valuable for the study of human immunodeficiency virus type 1 (HIV-1) pathogenesis and for the testing of drug and vaccine candidates. However, the development of such a model has been hampered by the inability of primary rodent cells to efficiently support several steps of the HIV-1 replication cycle. Although transgenesis of the HIV receptor complex and human cyclin T1 have been beneficial, additional late-phase blocks prevent robust replication of HIV-1 in rodents and limit the range of in vivo applications. In this study, we explored the HIV-1 susceptibility of rabbit primary T cells and macrophages. Envelope-specific and coreceptor-dependent entry of HIV-1 was achieved by expressing human CD4 and CCR5. A block of HIV-1 DNA synthesis, likely mediated by TRIM5, was overcome by limited changes to the HIV-1 gag gene. Unlike with mice and rats, primary cells from rabbits supported the functions of the regulatory viral proteins Tat and Rev, Gag processing, and the release of HIV-1 particles at levels comparable to those in human cells. While HIV-1 produced by rabbit T cells was highly infectious, a macrophage-specific infectivity defect became manifest by a complex pattern of mutations in the viral genome, only part of which were deamination dependent. These results demonstrate a considerable natural HIV-1 permissivity of the rabbit species and suggest that receptor complex transgenesis combined with modifications in gag and possibly vif of HIV-1 to evade species-specific restriction factors might render lagomorphs fully permissive to infection by this pathogenic human lentivirus.

BST-2 Diminishes HIV-1 Infectivity

Bone marrow stromal cell antigen 2 (BST-2, also known as tetherin/CD317/HM1.24) inhibits the release of human immunodeficiency virus type 1 (HIV-1) and other enveloped viruses by tethering virus particles to the cell surface. In this study, we provide evidence not only that the yield of cell-free HIV-1 particles is significantly reduced by BST-2 but also that the infectivity of these progeny virions is severely impaired. The lowered virion infectivity is due to the accumulation of pr55 Gag precursor and the p40Gag intermediates as well as to the loss of a mature core in the majority of HIV-1 particles. These data suggest that, in addition to impeding the release of HIV-1 particles from host cells, BST-2 may also interfere with the activation of viral protease and, as a result, impairs viral Gag processing as well as maturation of HIV-1 particles.

Broad-Spectrum Inhibition of Retroviral and Filoviral Particle Release by Tetherin

The expression of many putative antiviral genes is upregulated when cells encounter type I interferon (IFN), but the actual mechanisms by which many IFN-induced gene products inhibit virus replication are poorly understood. A recently identified IFN-induced antiretroviral protein, termed tetherin (previously known as BST-2 or CD317), blocks the release of nascent human immunodeficiency virus type 1 (HIV-1) particles from infected cells, and an HIV-1 accessory protein, Vpu, acts as a viral antagonist of tetherin. Here, we show that tetherin is capable of blocking not only the release of HIV-1 particles but also the release of particles assembled using the major structural proteins of a variety of prototype retroviruses, including members of the alpharetrovirus, betaretrovirus, deltaretrovirus, lentivirus, and spumaretrovirus families. Moreover, we show that the release of particles assembled using filovirus matrix proteins from Marburg virus and Ebola virus is also sensitive to inhibition by tetherin. These findings indicate that tetherin is a broadly specific inhibitor of enveloped particle release, and therefore, inhibition is unlikely to require specific interactions with viral proteins. Nonetheless, tetherin colocalized with nascent virus-like particles generated by several retroviral and filoviral structural proteins, indicating that it is present at, or recruited to, sites of particle assembly. Overall, tetherin is potentially active against many enveloped viruses and likely to be an important component of the antiviral innate immune defense.

Structural and Functional Characterization of Human SGT and Its Interaction with Vpu of the Human Immunodeficiency Virus Type 1,

The small glutamine-rich tetratricopeptide repeat protein (SGT) belongs to a family of cochaperones that interacts with both Hsp70 and Hsp90 via the so-called TPR domain. Here, we present the crystal structure of the TPR domain of human SGT (SGT-TPR), which shows that it contains typical features found in the structures of other TPR domains. Previous studies show that full-length SGT can bind to both Vpu and Gag of human immunodeficiency virus type 1 (HIV-1) and the overexpression of SGT in cells reduces the efficiency of HIV-1 particle release. We show that SGT-TPR can bind Vpu and reduce the amount of HIV-1 p24, which is the viral capsid, secreted from cells transfected with the HIV-1 proviral construct, albeit at a lower efficiency than full-length SGT. This indicates that the TPR domain of SGT is sufficient for the inhibition of HIV-1 particle release but the N- and/or C-terminus also have some contributions. The SGT binding site in Vpu was also identified by using peptide array and confirmed by GST pull-down assay.

Multiple Restrictions of Human Immunodeficiency Virus Type 1 in Feline Cells

The productive replication of human immunodeficiency virus type 1 (HIV-1) occurs exclusively in defined cells of human or chimpanzee origin, explaining why heterologous animal models for HIV replication, pathogenesis, vaccination, and therapy are not available. This lack of an animal model for HIV-1 studies prompted us to examine the susceptibility of feline cells in order to evaluate the cat (Felis catus) as an animal model for studying HIV-1. Here, we report that feline cell lines harbor multiple restrictions with respect to HIV-1 replication. The feline CD4 receptor does not permit virus infection. Feline T-cell lines MYA-1 and FeT-1C showed postentry restrictions resulting in low HIV-1 luciferase reporter activity and low expression of viral Gag-Pol proteins when pseudotyped vectors were used. Feline fibroblastic CrFK and KE-R cells, expressing human CD4 and CCR5, were very permissive for viral entry and HIV-long terminal repeat-driven expression but failed to support spreading infection. KE-R cells displayed a profound block with respect to release of HIV-1 particles. In contrast, CrFK cells allowed very efficient particle production; however, the CrFK cell-derived HIV-1 particles had low specific infectivity. We subsequently identified feline apolipoprotein B-editing catalytic polypeptide 3 (feAPOBEC3) proteins as active inhibitors of HIV-1 particle infectivity. CrFK cells express at least three different APOBEC3s: APOBEC3C, APOBEC3H, and APOBEC3CH. While the feAPOBEC3C did not significantly inhibit HIV-1, the feAPOBEC3H and feAPOBEC3CH induced G to A hypermutations of the viral cDNA and reduced the infectivity approximately 10- to approximately 40-fold.

The C-Terminal Portion of the Hrs Protein Interacts with Tsg101 and Interferes with Human Immunodeficiency Virus Type 1 Gag Particle Production

The human immunodeficiency virus type 1 (HIV-1) Gag protein recruits Tsg101 to facilitate HIV-1 particle budding and release. In uninfected cells, the Hrs protein recruits the ESCRT-I complex to the endosome, also through an interaction with Tsg101, to promote the sorting of host proteins into endosomal vesicles and multivesicular bodies. Here, we show that the overexpression of the C-terminal fragment of Hrs (residues 391 to 777) or Hrs mutants lacking either the N-terminal FYVE domain (mutant dFYVE) or the PSAP (residues 348 to 351) motif (mutant ASAA) all efficiently inhibit HIV-1 Gag particle production. Expression of the dFYVE or ASAA mutants of Hrs had no effect on the release of Moloney murine leukemia virus. Coimmunoprecipitation analysis showed that the expression of Hrs mutant dFYVE or ASAA significantly reduced or abolished the HIV-1 Gag-Tsg101 interaction. Yeast-two hybrid assays were used to identify two new and independent Tsg101 binding sites, one in the Hrs coiled-coil domain and one in the proline/glutamic acid-rich domain. Scanning electron microscopy of HeLa cells expressing HIV-1 Gag and the Hrs ASAA mutant showed viral particles arrested in "lump-like" structures that remained attached to the cell surface. Together, these data indicate that fragments of Hrs containing the C-terminal portion of the protein can potently inhibit HIV-1 particle release by efficiently sequestering Tsg101 away from the Gag polyprotein.

The Role of LIP5 and CHMP5 in Multivesicular Body Formation and HIV-1 Budding in Mammalian Cells

We examined the function of LIP5 in mammalian cells, because the yeast homologue Vta1p was recently identified as a protein required for multivesicular body (MVB) formation. LIP5 is predominantly a cytosolic protein. Depletion of LIP5 by small inhibitory RNA (siRNA) does not affect the distribution or morphology of early endosomes, lysosomes, or Golgi but does reduce the degradation of internalized epidermal growth factor receptor (EGFR), with EGFR accumulating in intracellular vesicles. Depletion of LIP5 by siRNA also decreases human immunodeficiency virus type 1 (HIV-1) budding by 70%. We identify CHMP5 as a LIP5-binding protein and show that CHMP5 is primarily cytosolic. Depletion of CHMP5 by siRNA does not affect the distribution or morphology of early endosomes, lysosomes, or Golgi but does result in reduced degradation of the EGFR similar to silencing of LIP5. Surprisingly, CHMP5 depletion results in an increase in the release of infectious HIV-1 particles. Overexpression of CHMP5 with a large carboxyl-terminal epitope affects the distribution of both early and late endocytic compartments, whereas overexpression of LIP5 does not alter the endocytic pathway. Comparison of overexpression and siRNA phenotypes suggests that the roles of these proteins in MVB formation may be more specifically addressed using RNA interference and that both LIP5 and CHMP5 function in MVB sorting, whereas only LIP5 is required for HIV release.

Transfer of endoplasmic reticulum and Golgi retention signals to human immunodeficiency virus type 1 gp160 inhibits intracellular transport and proteolytic processing of viral glycoprotein but does not influence the cellular site of virus particle budding.

In this study, specific signals known to mediate endoplasmic reticulum or Golgi localization of transmembrane proteins have been transferred to the human immunodeficiency virus type 1 (HIV-1) env gene product. The intracellularly retained recombinant glycoproteins were not proteolytically processed to gp120 and gp41, which is further evidence that this process occurs at a later stage in the transport pathway, presumably within or near the trans-Golgi network. Since the subcellular localization of the viral glycoproteins of enveloped viruses can be one of the factors determining the cellular site of particle assembly and release, experiments were performed to determine if this property was altered by coexpression of the recombinant HIV-1 glycoproteins. When wild-type virus was compared to mutant virus encoding the intracellularly retained glycoproteins, the extent of HIV-1 particle release into the extracellular medium remained unaffected, and electron-microscopic analysis did not reveal any significant alteration in the cellular sites of particle assembly and budding. Thus, in COS-7 cells, altered subcellular localization of the viral glycoprotein does not exert a dominant influence on the assembly site of the HIV-1 particle.

Obstruction of HIV-1 Particle Release by Interferon-α Occurs Before Viral Protease Processing and Is Independent of Envelope Glycoprotein

The effect of human interferon-alpha (Hu-IFN-alpha) on the maturation process of the human immunodeficiency virus type 1 (HIV-1) has been studied using stable cell lines that produce nonenveloped particles. These cell lines secrete particles devoid of the viral envelope proteins gp120 and gp41. The CH-1 cells produce active viral protease that correctly processes its natural substrates, whereas the CH-1kww cell line expresses an enzymatically inactive viral protease, thus producing immature viral capsids. A block in the secretion of particles was observed in both cell lines when treated with 100-1000 U/ml Hu-IFN-alpha, as judged by measurements of encapsidated gag proteins. Electron microscopy shows that Hu-IFN-alpha-treated CH-1 cells are decorated with assembled immature particles at the cell surface. These results suggest that the observed block in particle release on Hu-IFN-alpha treatment is independent of viral envelope expression and occurs before capsid polyprotein processing. In addition, particles remaining attached to the cell fail to mature into structures with condensed cores. Viral gag proteins from IFN-treated and untreated CH-1 cells were analyzed by 2-D gel electrophoresis. Results suggest a change in posttranslational modifications of gag proteins, as IFN treatment allowed the detection of more basic forms of p55, p39, and p24. Further analysis of cellular or viral protein alterations induced by Hu-IFN-alpha treatment may identify the mechanism of action by which particle maturation is obstructed.

Multiple Effects of Interferon on HIV-1 Replication

Journal of Interferon ResearchVol. 11, No. 6 Multiple Effects of Interferon on HIV-1 ReplicationPAULA M. PITHAPAULA M. PITHASearch for more papers by this authorPublished Online:5 May 2009https://doi.org/10.1089/jir.1991.11.313AboutSectionsPDF/EPUB ToolsPermissionsDownload CitationsTrack CitationsAdd to favorites Back To Publication ShareShare onFacebookTwitterLinked InRedditEmail FiguresReferencesRelatedDetailsCited ByThe Role of Cytokines in Viral Infections15 March 2010Interferon-induced exonuclease ISG20 exhibits an antiviral activity against human immunodeficiency virus type 1Journal of General Virology, Vol. 86, No. 8Current status of gene therapy strategies to treat HIV/AIDSMolecular Therapy, Vol. 11, No. 6Structure of the mitochondrial ATP synthase by electron cryomicroscopyThe EMBO Journal, Vol. 22, No. 23Trans-Activated Interferon-α2 Delivered to T Cells by SV40 Inhibits Early Stages in the HIV-1 Replicative Cycle9 July 2004 | Journal of Hematotherapy & Stem Cell Research, Vol. 11, No. 5Immunologic Profile of Highly Exposed Yet HIV Type 1-Seronegative Men5 July 2004 | AIDS Research and Human Retroviruses, Vol. 18, No. 14Low-Dose IFN-α Monotherapy in Treatment-Naive Individuals with HIV-1 Infection: Evidence of Potent Suppression of Viral Replication7 July 2004 | Journal of Interferon & Cytokine Research, Vol. 21, No. 10AIDS-Associated MalignanciesRegulation of chemokine/cytokine network during in vitro differentiation and HIV‐1 infection of human monocytes: possible importance in the pathogenesis of AIDS1 September 2000 | Journal of Leukocyte Biology, Vol. 68, No. 3A Randomized Trial of Interferon alpha Therapy for HIV Type 1 Infection5 July 2004 | AIDS Research and Human Retroviruses, Vol. 16, No. 3Phase II, Randomized, Open-Label, Community-Based Trial to Compare the Safety and Activity of Combination Therapy With Recombinant Interferon-α2b and Zidovudine Versus Zidovudine Alone in Patients With Asymptomatic to Mildly Symptomatic HIV InfectionJournal of Acquired Immune Deficiency Syndromes and Human Retrovirology, Vol. 20, No. 3Molecular Mechanisms of Interferon Resistance Mediated by Viral-Directed Inhibition of PKR, the Interferon-Induced Protein KinasePharmacology & Therapeutics, Vol. 78, No. 1Regulation of Human Immunodeficiency Virus Replication by 2′,5′-Oligoadenylate-Dependent RNase LJournal of Virology, Vol. 72, No. 2Interferon-α and -γ Differentially Reduce Rapid Immature T-Cell Death by Contact with HIV-1 Carrier Cell Clones In Vitro14 November 2013 | Microbiology and Immunology, Vol. 41, No. 9Obstruction of HIV-1 Particle Release by Interferon-α Occurs Before Viral Protease Processing and Is Independent of Envelope Glycoprotein28 March 2009 | Journal of Interferon & Cytokine Research, Vol. 17, No. 5What Happens Inside Lentivirus or Influenza Virus Infected Cells: Insights into Regulation of Cellular and Viral Protein SynthesisMethods, Vol. 11, No. 4Interferon-betaHIV-permissive cells from solid tissues: Cytokine induction and effectsPerspectives in Drug Discovery and Design, Vol. 5, No. 1Anti-HIV Potential of a New Interferon, Interferon-τ (Trophoblastin)Journal of Acquired Immune Deficiency Syndromes and Human Retrovirology, Vol. 11, No. 3IMMUNE-BASED THERAPY FOR HIVNursing Clinics of North America, Vol. 31, No. 1Regulation of human immunodeficiency virus type 1 and cytokine gene expression in myeloid cells by NF-kappa B/Rel transcription factorsMicrobiological Reviews, Vol. 59, No. 3Selective decrease in human immunodeficiency virus type 1 (HIV-1)-induced alpha interferon production by peripheral blood mononuclear cells during HIV-1 infectionClinical Diagnostic Laboratory Immunology, Vol. 2, No. 2Further Evaluation of Soluble CD4 as an Anti-HIV Type 1 Gene Therapy: Demonstration of Protection of Primary Human Peripheral Blood Lymphocytes from Infection by HIV Type 116 March 2009 | AIDS Research and Human Retroviruses, Vol. 10, No. 11Specific inhibition of viral protein synthesis in HIV-infected cells in response to interferon treatment.Journal of Biological Chemistry, Vol. 269, No. 37Regulation of HIV Replication in Monocytes by InterferonIncreased Efficacy of Human Natural Interferon α (IFN-αn3) Versus Human Recombinant IFN-α2 for Inhibition of HIV-1 Replication in Primary Human Monocytes16 March 2009 | AIDS Research and Human Retroviruses, Vol. 9, No. 11HIV-1 reverse transcriptase: Inhibition by 2',5'-oligoadenylates1 May 2002 | Biochemistry, Vol. 32, No. 45Preclinical evaluation of antiviral activity and toxicity of Abbott A77003, an inhibitor of the human immunodeficiency virus type 1 proteaseAntimicrobial Agents and Chemotherapy, Vol. 37, No. 1The War Against the Interferon-induced dsRNA-Activated Protein Kinase: Can Viruses Win?5 May 2009 | Journal of Interferon Research, Vol. 12, No. 4 Volume 11Issue 6Dec 1991 To cite this article:PAULA M. PITHA.Multiple Effects of Interferon on HIV-1 Replication.Journal of Interferon Research.Dec 1991.313-318.http://doi.org/10.1089/jir.1991.11.313Published in Volume: 11 Issue 6: May 5, 2009PDF download