The Nef Protein of Human Immunodeficiency Virus Establishes Superinfection Immunity by a Dual Strategy to Downregulate Cell-Surface CCR5 and CD4

SummaryBackground: Viruses frequently render cells refractory to subsequent infection with the same virus. This state of superinfection immunity counteracts potentially detrimental consequences for the infected cell and facilitates high-level replication and viral spread in the host.Results: Here, we show that human immunodeficiency virus (HIV) employs its early gene product Nef to efficiently interfere with superinfection at the viral-entry step. In this context, we identify the downregulation of cell-surface CCR5, the major HIV coreceptor, as a novel and highly conserved activity of Nef. Nef targets the CCR5 coreceptor and the HIV binding receptor CD4 via distinct cellular machineries to enhance the endocytosis rate of both HIV receptor components and to accelerate their degradation. Functionally, these genetically separable actions by Nef synergized to efficiently protect cells from HIV superinfection at the level of fusion of the viral envelope with the plasma membrane.Conclusions: HIV has evolved two independent activities for Nef to downregulate the receptor complex and to facilitate its efficient replication and spread. This evasion strategy likely represents a mechanism by which the pathogenicity factor Nef elevates viral replication in vivo and thus promotes AIDS pathogenesis

Mouse T-cells restrict replication of human immunodeficiency virus at the level of integration

<p>Abstract</p> <p>Background</p> <p>The development of an immunocompetent, genetically modified mouse model to study HIV-1 pathogenesis and to test antiviral strategies has been hampered by the fact that cells from native mice do not or only inefficiently support several steps of the HIV-1 replication cycle. Upon HIV-1 infection, mouse T-cell lines fail to express viral proteins, but the underlying replication barrier has thus far not been unambiguously identified. Here, we performed a kinetic and quantitative assessment of consecutive steps in the early phase of the HIV-1 replication cycle in T-cells from mice and humans.</p> <p>Results</p> <p>Both T-cell lines and primary T-cells from mice harbor a severe post-entry defect that is independent of potential species-specTR transactivation. Reverse transcription occurred efficiently following VSV-G-mediated entry of virions into mouse T-cells, and abundant levels of 2-LTR circles indicated successful nuclear import of the pre-integration complex. To probe the next step in the retroviral replication cycle, i.e. the integration of HIV-1 into the host cell genome, we established and validated a nested real-time PCR to specifically quantify HIV-1 integrants exploiting highly repetitive mouse <it>B1 </it>elements. Importantly, we demonstrate that the frequency of integrant formation is diminished 18- to > 305-fold in mouse T-cell lines compared to a human counterpart, resulting in a largely abortive infection. Moreover, differences in transgene expression from residual vector integrants, the transcription off which is cyclin T1-independent, provided evidence for an additional, peri-integrational deficit in certain mouse T-cell lines.</p> <p>Conclusion</p> <p>In contrast to earlier reports, we find that mouse T-cells efficiently support early replication steps up to and including nuclear import, but restrict HIV-1 at the level of chromosomal integration.</p

β-TrCP is dispensable for Vpu's ability to overcome the CD317/Tetherin-imposed restriction to HIV-1 release

<p>Abstract</p> <p>Background</p> <p>The cellular transmembrane protein CD317/BST-2/HM1.24/Tetherin restricts HIV-1 infection by physically tethering mature virions to the surface of infected cells. HIV-1 counteracts this restriction by expressing the accessory protein Vpu, yet the mechanism of this antagonism is incompletely understood. β-TrCP is the substrate recognition domain of an E3 ubiquitin ligase complex that interacts with the di-serine motif S52/S56 in the cytoplasmic tail of Vpu to target the CD4 receptor for proteasomal degradation. Recently, it has been suggested that β-TrCP is also critically involved in Vpu's ability to overcome the CD317-mediated virion release block.</p> <p>Results</p> <p>To test this model, we analyzed the consequences of several experimental strategies to interfere with the Vpu-β-TrCP protein-protein interaction. Under these conditions, we studied effects of Vpu on expression and localization of CD317 and CD4, as well as on its ability to promote HIV-1 release. Our results demonstrate a strict requirement for Vpu's di-serine motif for degradation of CD4 and also CD317, reduction of cell surface exposure of CD317, and HIV-1 release enhancement. We further show a critical role of β-TrCP2, but not of the structurally related β-TrCP1 isoform, for Vpu-mediated degradation of both receptors. Most importantly, Vpu remained active in downregulating CD317 from the cell surface and in overcoming the HIV-1 release restriction in β-TrCP-depleted cells.</p> <p>Conclusions</p> <p>These results demonstrate that β-TrCP is not strictly required for Vpu's ability to counteract the CD317-imposed virion release block and support the relevance of cell surface down-modulation of the restriction factor as a central mechanism of Vpu antagonism. Moreover, we propose the existence of a critical, yet to be identified cellular factor that interacts with Vpu via its di-serine motif to alter the trafficking of the restriction factor.</p

Post-translational Modification-Based Regulation of HIV Replication

Human immunodeficiency virus (HIV) relies heavily on the host cellular machinery for production of viral progeny. To exploit cellular proteins for replication and to overcome host factors with antiviral activity, HIV has evolved a set of regulatory and accessory proteins to shape an optimized environment for its replication and to facilitate evasion from the immune system. Several cellular pathways are hijacked by the virus to modulate critical steps during the viral life cycle. Thereby, post-translational modifications (PTMs) of viral and cellular proteins gain increasingly attention as modifying enzymes regulate virtually every step of the viral replication cycle. This review summarizes the current knowledge of HIV-host interactions influenced by PTMs with a special focus on acetylation, ubiquitination, and phosphorylation of proteins linked to cellular signaling and viral replication. Insights into these interactions are surmised to aid development of new intervention strategies

HIV-1 Buds Predominantly at the Plasma Membrane of Primary Human Macrophages

HIV-1 assembly and release are believed to occur at the plasma membrane in most host cells with the exception of primary macrophages, for which exclusive budding at late endosomes has been reported. Here, we applied a novel ultrastructural approach to assess HIV-1 budding in primary macrophages in an immunomarker-independent manner. Infected macrophages were fed with BSA-gold and stained with the membrane-impermeant dye ruthenium red to identify endosomes and the plasma membrane, respectively. Virus-filled vacuolar structures with a seemingly intracellular localization displayed intense staining with ruthenium red, but lacked endocytosed BSA-gold, defining them as plasma membrane. Moreover, HIV budding profiles were virtually excluded from gold-filled endosomes while frequently being detected on ruthenium red–positive membranes. The composition of cellular marker proteins incorporated into HIV-1 supported a plasma membrane–derived origin of the viral envelope. Thus, contrary to current opinion, the plasma membrane is the primary site of HIV-1 budding also in infected macrophages

Primary T-cells from human CD4/CCR5-transgenic rats support all early steps of HIV-1 replication including integration, but display impaired viral gene expression

<p>Abstract</p> <p>Background</p> <p><it>In vivo </it>studies on HIV-1 pathogenesis and testing of antiviral strategies have been hampered by the lack of an immunocompetent small animal model that is highly susceptible to HIV-1 infection. Since native rodents are non-permissive, we developed transgenic rats that selectively express the HIV-1 receptor complex, hCD4 and hCCR5, on relevant target cells. These animals display a transient low-level plasma viremia after HIV-1_YU-2infection, demonstrating HIV-1 susceptibility <it>in vivo</it>. However, unlike macrophages, primary CD4 T-cells from double-transgenic animals fail to support viral spread <it>ex vivo</it>. To identify quantitative limitations or absolute blocks in this rodent species, we quantitatively assessed the efficiency of key steps in the early phase of the viral replication cycle in a side-by-side comparison in infected cell lines and primary T-cells from hCD4/hCCR5-transgenic rats and human donors.</p> <p>Results</p> <p>Levels of virus entry, HIV-1 cDNA synthesis, nuclear import, and integration into the host genome were shown to be remarkably similar in cell lines and, where technically accessible, in primary T-cells from both species. In contrast, a profound impairment at the level of early HIV gene expression was disclosed at the single-cell level in primary rat T-cells and most other rat-derived cells. Macrophages were a notable exception, possibly reflecting the unique transcriptional milieu in this evolutionarily conserved target cell of all lentiviruses. Importantly, transient trans-complementation by <it>ex vivo </it>nucleofection with the Tat-interacting protein Cyclin T1 of human origin markedly elevated HIV gene expression in primary rat T-cells.</p> <p>Conclusion</p> <p>This is the first study that has quantitatively determined the efficiency of consecutive steps in the HIV-1 replication cycle in infected primary HIV target cells from a candidate transgenic small animal and compared it to human cells. Unlike cells derived from mice or rabbits, rat cells complete all of the early steps in the HIV-1 replication cycle, including provirus integration <it>in vivo</it>, with high efficiency. A deficiency in gene expression was disclosed at the single cell level and could be counteracted by the human pTEFb transcription complex factor Cyclin T1. Collectively, these results provide the basis for the advancement of this transgenic rat model through strategies aimed at boosting HIV-1 gene expression in primary rat CD4 T-cells, including human Cyclin T1 transgenesis.</p

Secondary zoonotic dog-to-human transmission of SARS-CoV-2 suggested by timeline but refuted by viral genome sequencing

Purpose: The risk of secondary zoonotic transmission of SARS-CoV-2 from pet animals remains unclear. Here, we report on a 44 year old Caucasian male presenting to our clinic with COVID-19 pneumonia, who reported that his dog displayed respiratory signs shortly prior to his infection. The dog tested real-time-PCR (RT-PCR) positive for SARS-CoV-2 RNA and the timeline of events suggested a transmission from the dog to the patient. Methods: RT-PCR and serological assays were used to confirm SARS-CoV-2 infection in the nasopharyngeal tract in the dog and the patient. We performed SARS-CoV-2-targeted amplicon-based next generation sequencing of respiratory samples from the dog and patient for sequence comparisons. Results: SARS-CoV-2 infection of the dog was confirmed by three independent PCR-positive pharyngeal swabs and subsequent seroconversion. Sequence analysis identified two separate SARS-CoV-2 lineages in the canine and the patient’s respiratory samples. The timeline strongly suggested dog-to-human transmission, yet due to the genetic distance of the canine and the patient’s samples paired-transmission was highly unlikely. Conclusion: The results of this case support current knowledge about the low risk of secondary zoonotic dog-to-human transmissions of SARS-CoV-2 and emphasizes the strength of genomic sequencing in deciphering viral transmission chains

Human cyclin T1 expression ameliorates a T-cell-specific transcriptional limitation for HIV in transgenic rats, but is not sufficient for a spreading infection of prototypic R5 HIV-1 strains ex vivo

<p>Abstract</p> <p>Background</p> <p>Cells derived from native rodents have limits at distinct steps of HIV replication. Rat primary CD4 T-cells, but not macrophages, display a profound transcriptional deficit that is ameliorated by transient trans-complementation with the human Tat-interacting protein Cyclin T1 (hCycT1).</p> <p>Results</p> <p>Here, we generated transgenic rats that selectively express hCycT1 in CD4 T-cells and macrophages. hCycT1 expression in rat T-cells boosted early HIV gene expression to levels approaching those in infected primary human T-cells. hCycT1 expression was necessary, but not sufficient, to enhance HIV transcription in T-cells from individual transgenic animals, indicating that endogenous cellular factors are critical co-regulators of HIV gene expression in rats. T-cells from hCD4/hCCR5/hCycT1-transgenic rats did not support productive infection of prototypic wild-type R5 HIV-1 strains <it>ex vivo</it>, suggesting one or more significant limitation in the late phase of the replication cycle in this primary rodent cell type. Remarkably, we identify a replication-competent HIV-1 GFP reporter strain (R7/3 YU-2 Env) that displays characteristics of a spreading, primarily cell-to-cell-mediated infection in primary T-cells from hCD4/hCCR5-transgenic rats. Moreover, the replication of this recombinant HIV-1 strain was significantly enhanced by hCycT1 transgenesis. The viral determinants of this so far unique replicative ability are currently unknown.</p> <p>Conclusion</p> <p>Thus, hCycT1 expression is beneficial to <it>de novo </it>HIV infection in a transgenic rat model, but additional genetic manipulations of the host or virus are required to achieve full permissivity.</p