Dual role of the chromatin-binding factor PHF13 in the pre- and post-integration phases of HIV-1 replication.

Viruses interact with multiple host cell factors. Some of these are required to promote viral propagation, others have roles in inhibiting infection. Here, we delineate the function of the cellular factor PHF13 (or SPOC1), a putative HIV-1 restriction factor. Early in the HIV-1 replication cycle PHF13 increased the number of integrated proviral copies and the number of infected cells. However, after HIV-1 integration, high levels of PHF13 suppressed viral gene expression. The antiviral activity of PHF13 is counteracted by the viral accessory protein Vpr, which mediates PHF13 degradation. Altogether, the transcriptional master regulator and chromatin binding protein PHF13 does not have purely repressive effects on HIV-1 replication, but also promotes viral integration. By the functional characterization of the dual role of PHF13 during the HIV-1 replication cycle, we reveal a surprising and intricate mechanism through which HIV-1 might regulate the switch from integration to viral gene expression. Furthermore, we identify PHF13 as a cellular target specifically degraded by HIV-1 Vpr.

T cells with low CD2 levels express reduced restriction factors and are preferentially infected in therapy naïve chronic HIV-1 patients.

INTRODUCTION: Restriction factors (RFs) suppress HIV-1 in cell lines and primary cell models. Hence, RFs might be attractive targets for novel antiviral strategies, but their importance for virus control in vivo is controversial. METHODS: We profiled the expression of RFs in primary blood-derived mononuclear cells (PBMC) from therapy-naïve HIV-1 patients and quantified infection. RESULTS: Overall, there was no correlation between individual RF expression and HIV-1 status in total PBMC. However, we identified a T cell population with low levels of intracellular CD2 and reduced expression of SAMHD1, p21 and SerinC5. CD2low T cells with reduced RF expression were markedly positive for HIV-1 p24. In contrast, CD2+ T cells were less infected and expressed higher levels of RFs. CD2low T cell infection correlated with viral loads and was associated with HIV-1 disease progression. CONCLUSIONS: In untreated therapy naïve chronic HIV-1 patients, RF expression in T cells is associated with CD2 expression and seems to influence viral loads. Our study suggests that RFs help to control HIV-1 infection in certain T cells in vivo and supports the potential for RFs as promising targets for therapeutic intervention.

Ion Channel Activity of Vpu Proteins Is Conserved throughout Evolution of HIV-1 and SIV.

The human immunodeficiency virus type 1 (HIV-1) protein Vpu is encoded exclusively by HIV-1 and related simian immunodeficiency viruses (SIVs). The transmembrane domain of the protein has dual functions: it counteracts the human restriction factor tetherin and forms a cation channel. Since these two functions are causally unrelated it remains unclear whether the channel activity has any relevance for viral release and replication. Here we examine structure and function correlates of different Vpu homologs from HIV-1 and SIV to understand if ion channel activity is an evolutionary conserved property of Vpu proteins. An electrophysiological testing of Vpus from different HIV-1 groups (N and P) and SIVs from chimpanzees (SIVcpz), and greater spot-nosed monkeys (SIVgsn) showed that they all generate channel activity in HEK293T cells. This implies a robust and evolutionary conserved channel activity and suggests that cation conductance may also have a conserved functional significance.

Virion encapsidated HIV-1 Vpr induces NFAT to prime non-activated T cells for productive infection.

The majority of T cells encountered by HIV-1 are non-activated and do not readily allow productive infection. HIV-1 Vpr is highly abundant in progeny virions, and induces signalling and HIV-1 LTR transcription. We hence hypothesized that Vpr might be a determinant of non-activated T-cell infection. Virion-delivered Vpr activated nuclear factor of activated T cells (NFAT) through Ca(2+) influx and interference with the NFAT export kinase GSK3ß. This leads to NFAT translocation and accumulation within the nucleus and was required for productive infection of unstimulated primary CD4(+) T cells. A mutagenesis approach revealed correlation of Vpr-mediated NFAT activation with its ability to enhance LTR transcription and mediate cell cycle arrest. Upon NFAT inhibition, Vpr did not augment resting T-cell infection, and showed reduced G2/M arrest and LTR transactivation. Altogether, Vpr renders unstimulated T cells more permissive for productive HIV-1 infection and stimulates activation of productively infected as well as virus-exposed T cells. Therefore, it could be involved in the establishment and reactivation of HIV-1 from viral reservoirs and might have an impact on the levels of immune activation, which are determinants of HIV-1 pathogenesis.

HIV-1 Nef and Vpu Interfere with L-Selectin (CD62L) Cell Surface Expression To Inhibit Adhesion and Signaling in Infected CD4+ T Lymphocytes.

UNLABELLED: Leukocyte recirculation between blood and lymphoid tissues is required for the generation and maintenance of immune responses against pathogens and is crucially controlled by the L-selectin (CD62L) leukocyte homing receptor. CD62L has adhesion and signaling functions and initiates the capture and rolling on the vascular endothelium of cells entering peripheral lymph nodes. This study reveals that CD62L is strongly downregulated on primary CD4(+) T lymphocytes upon infection with human immunodeficiency virus type 1 (HIV-1). Reduced cell surface CD62L expression was attributable to the Nef and Vpu viral proteins and not due to increased shedding via matrix metalloproteases. Both Nef and Vpu associated with and sequestered CD62L in perinuclear compartments, thereby impeding CD62L transport to the plasma membrane. In addition, Nef decreased total CD62L protein levels. Importantly, infection with wild-type, but not Nef- and Vpu-deficient, HIV-1 inhibited the capacity of primary CD4(+) T lymphocytes to adhere to immobilized fibronectin in response to CD62L ligation. Moreover, HIV-1 infection impaired the signaling pathways and costimulatory signals triggered in primary CD4(+) T cells by CD62L ligation. We propose that HIV-1 dysregulates CD62L expression to interfere with the trafficking and activation of infected T cells. Altogether, this novel HIV-1 function could contribute to virus dissemination and evasion of host immune responses. IMPORTANCE: L-selectin (CD62L) is an adhesion molecule that mediates the first steps of leukocyte homing to peripheral lymph nodes, thus crucially controlling the initiation and maintenance of immune responses to pathogens. Here, we report that CD62L is downmodulated on the surfaces of HIV-1-infected T cells through the activities of two viral proteins, Nef and Vpu, that prevent newly synthesized CD62L molecules from reaching the plasma membrane. We provide evidence that CD62L downregulation on HIV-1-infected primary T cells results in impaired adhesion and signaling functions upon CD62L triggering. Removal of cell surface CD62L may predictably keep HIV-1-infected cells away from lymph nodes, the privileged sites of both viral replication and immune response activation, with important consequences, such as systemic viral spread and evasion of host immune surveillance. Altogether, we propose that Nef- and Vpu-mediated subversion of CD62L function could represent a novel determinant of HIV-1 pathogenesis.

HIV-1 Vpu mediated downregulation of CD155 requires alanine residues 10, 14 and 18 of the transmembrane domain.

HIV-1 NL4-3 Vpu induces downregulation of cell surface CD155, a ligand for the DNAM-1 activating receptor of NK and CD8(+) T cells, to evade NK cell mediated immune response. Here we show that the conserved alanine residues at positions 10, 14 and 18 in the TM domain of Vpu are required for the efficient downregulation of cell surface CD155. In contrast, the CK-2 phosphorylation sites and the second α-helix in the cytoplasmic Vpu domain have no influence on the surface expression of CD155. Thus, compared to Vpu׳s effect on CD4, NTB-A and tetherin, the Vpu mediated downregulation of CD155 is an independent Vpu function. We finally show that in contrast to other lentiviral strains, only Vpu and Nef from HIV-1 M NL4-3 potently interfere with CD155 surface expression. Thus, Vpu seems to subvert NK cell responses against HIV-1 infected T cells by modulation of receptors necessary for NK cell activation.

Analysis of determinants in filovirus glycoproteins required for tetherin antagonism.

The host cell protein tetherin can restrict the release of enveloped viruses from infected cells. The HIV-1 protein Vpu counteracts tetherin by removing it from the site of viral budding, the plasma membrane, and this process depends on specific interactions between the transmembrane domains of Vpu and tetherin. In contrast, the glycoproteins (GPs) of two filoviruses, Ebola and Marburg virus, antagonize tetherin without reducing surface expression, and the domains in GP required for tetherin counteraction are unknown. Here, we show that filovirus GPs depend on the presence of their authentic transmembrane domains for virus-cell fusion and tetherin antagonism. However, conserved residues within the transmembrane domain were dispensable for membrane fusion and tetherin counteraction. Moreover, the insertion of the transmembrane domain into a heterologous viral GP, Lassa virus GPC, was not sufficient to confer tetherin antagonism to the recipient. Finally, mutation of conserved residues within the fusion peptide of Ebola virus GP inhibited virus-cell fusion but did not ablate tetherin counteraction, indicating that the fusion peptide and the ability of GP to drive host cell entry are not required for tetherin counteraction. These results suggest that the transmembrane domains of filoviral GPs contribute to tetherin antagonism but are not the sole determinants.

HIV-1 Vpu affects the anterograde transport and the glycosylation pattern of NTB-A.

HIV-1 Vpu induces downregulation of cell surface NTB-A to evade lysis of HIV-1 infected cells by NK cells. Here we show that Vpu affects the anterograde transport and the glycosylation pattern of NTB-A by a mechanism that is distinct from the Vpu induced downregulation of CD4 and tetherin. In the presence of Vpu, only the high mannose form of NTB-A was detectable, suggesting that Vpu prevented the formation of the mature form of NTB-A. This phenomenon is associated with the ability of Vpu to downregulate cell surface NTB-A by retention of NTB-A within the Golgi-compartment. Furthermore, the Vpu-mediated effect on NTB-A glycosylation is highly conserved among Vpu proteins derived from HIV-1 and SIV and corresponds to the level of downregulation of NTB-A. Together, these results suggest that the reduction of NTB-A from the cell surface is associated with the Vpu-mediated effect on the glycosylation pattern of newly synthesized NTB-A molecules.

Ion channel activity of HIV-1 Vpu is dispensable for counteraction of CD317.

While the C-terminal domain of HIV-1 Vpu is critical for CD4 degradation, the transmembrane domain (TM) mediates ion channel activity, enhances virus release and is essential for counteracting CD317/Bst-2/Tetherin. Here we analyzed whether the ion channel activity of Vpu is required to antagonize CD317-mediated restriction of virion release. We examined TM-mutants of three conserved residues: the S23A mutation, which was previously shown to abrogate ion channel function, did not affect Vpu mediated augmentation of virus release. In contrast, the A14N and A18N mutation did not affect ion channel activity of Vpu, but substantially reduced its ability to support virus release and to down-regulate CD317 from the cell surface. Altogether, our data suggest that not the ion channel activity of Vpu, but its ability to remove CD317 from the cell surface is required to augment HIV-1 release.