SIV Vpx Is Essential for Macrophage Infection but Not for Development of AIDS

Analysis of rhesus macaques infected with a vpx deletion mutant virus of simian immunodeficiency virus mac239 (SIVΔvpx) demonstrates that Vpx is essential for efficient monocyte/macrophage infection in vivo but is not necessary for development of AIDS. To compare myeloid-lineage cell infection in monkeys infected with SIVΔvpx compared to SIVmac239, we analyzed lymphoid and gastrointestinal tissues from SIVΔvpx-infected rhesus (n = 5), SIVmac239-infected rhesus with SIV encephalitis (7 SIV239E), those without encephalitis (4 SIV239noE), and other SIV mutant viruses with low viral loads (4 SIVΔnef, 2 SIVΔ3). SIV+ macrophages and the percentage of total SIV+ cells that were macrophages in spleen and lymph nodes were significantly lower in rhesus infected with SIVΔvpx (2.2%) compared to those infected with SIV239E (22.7%), SIV239noE (8.2%), and SIV mutant viruses (10.1%). In colon, SIVΔvpx monkeys had fewer SIV+ cells, no SIV+ macrophages, and lower percentage of SIV+ cells that were macrophages than the other 3 groups. Only 2 SIVΔvpx monkeys exhibited detectable virus in the colon. We demonstrate that Vpx is essential for efficient macrophage infection in vivo and that simian AIDS and death can occur in the absence of detectable macrophage infection

Nef Proteins from Diverse Groups of Primate Lentiviruses Downmodulate CXCR4 To Inhibit Migration to the Chemokine Stromal Derived Factor 1

Nef proteins of primate lentiviruses promote viral replication, virion infectivity, and evasion of antiviral immune responses by modulating signal transduction pathways and downregulating expression of receptors at the cell surface that are important for efficient antigen-specific responses, such as CD4, CD28, T-cell antigen receptor, and class I and class II major histocompatibility complex. Here we show that Nef proteins from diverse groups of primate lentiviruses which do not require the chemokine receptor CXCR4 for entry into target cells strongly downmodulate the cell surface expression of CXCR4. In contrast, all human immunodeficiency virus type 1 (HIV-1) and the majority of HIV-2 Nef proteins tested did not have such strong effects. SIVmac239 Nef strongly inhibited lymphocyte migration to CXCR4 ligand, the chemokine stromal derived factor 1 (SDF-1). SIVmac239 Nef downregulated CXCR4 by accelerating the rate of its endocytosis. Downmodulation of CXCR4 was abolished by mutations that disrupt the constitutively strong AP-2 clathrin adaptor binding element located in the N-terminal region of the Nef molecule, suggesting that Nef accelerates CXCR4 endocytosis via an AP-2-dependent pathway. Together, these results point to CXCR4 as playing an important role in simian immunodeficiency virus and possibly also HIV-2 persistence in vivo that is unrelated to viral entry into target cells. We speculate that Nef targets CXCR4 to disrupt ordered trafficking of infected leukocytes between local microenvironments in order to facilitate their dissemination and/or impair the antiviral immune response

HIV-1 Vpr Reprograms CLR4DCAF1 E3 Ubiquitin Ligase to Antagonize Exonuclease 1-Mediated Restriction of HIV-1 Infection

HIV-1 polymerase reverse transcribes the viral RNA genome into imperfectly double-stranded proviral DNA, containing gaps and flaps, for integration into the host cell chromosome. HIV-1 reverse transcripts share characteristics with cellular DNA replication intermediates and are thought to be converted into fully double-stranded DNA by cellular postreplication DNA repair enzymes. Therefore, the finding that the HIV-1 accessory protein Vpr antagonizes select postreplication DNA repair enzymes that can process HIV-1 reverse transcripts has been surprising. Here, we show that one such Vpr-antagonized enzyme, exonuclease 1, inhibits HIV-1 replication in T cells. We identify exonuclease 1 as a member of a new class of HIV-1 restriction factors in T cells and propose that certain modes of DNA “repair” inhibit HIV-1 infection.Viral accessory proteins hijack host cell E3 ubiquitin ligases to antagonize innate/intrinsic defenses and thereby provide a more permissive environment for virus replication. Human immunodeficiency virus type 1 (HIV-1) accessory protein Vpr reprograms CRL4DCAF1 E3 to antagonize select postreplication DNA repair enzymes, but the significance and role of these Vpr interactions are poorly understood. To gain additional insights, we performed a focused screen for substrates of CRL4DCAF1 E3 reprogrammed by HIV-1 Vpr among known postreplication DNA repair proteins and identified exonuclease 1 (Exo1) as a novel direct HIV-1 Vpr target. We show that HIV-1 Vpr recruits Exo1 to the CRL4DCAF1 E3 complex for ubiquitination and subsequent proteasome-dependent degradation and that Exo1 levels are depleted in HIV-1-infected cells in a Vpr-dependent manner. We also show that Exo1 inhibits HIV-1 replication in T cells. Notably, the antagonism of Exo1 is a conserved function of main group HIV-1 and its ancestor Vpr proteins in the simian immunodeficiency virus from chimpanzee (SIVcpz) lineage, further underscoring the relevance of our findings. Overall, our studies (i) reveal that HIV-1 Vpr extensively remodels the cellular postreplication DNA repair machinery by impinging on multiple repair pathways, (ii) support a model in which Vpr promotes HIV-1 replication by antagonizing select DNA repair enzymes, and (iii) highlight the importance of a new class of restrictions placed on HIV-1 replication in T cells by the cellular DNA repair machinery

Double-label immunohistochemistry and <i>in situ</i> hybridization of lymphoid tissues.

<p><b>(A)</b> Representative images of SIV <i>in situ</i> hybridization (ISH, blue) with double-label immunohistochemistry for monocyte/macrophage lineage cell marker Ham56 (DAB, brown) in rhesus macaques in groups SIV239E (left), SIV239noE (middle), and SIVΔ<i>vpx</i> (right). The top images of lymphoid follicles depict Ham-56+ cells morphologically consistent with follicular DCs. Images in the second row depict HAM56+ cells in the red pulp of the spleen (left and center) or paracortex in lymph node (right). Frequent SIV+ HAM56+ macrophages/DCs (Mφ) were observed in tissues from the SIV239E and SIV239noE groups, while only rare SIV+ Ham56+ macrophages/DCs were observed in lymph node from a SIVΔ<i>vpx</i>-infected rhesus (arrow). <b>(B).</b> The overall numbers of infected cells in the spleen and lymph node from the 4 groups of animals were not significantly different. <b>(C)</b> However, there were significantly fewer SIV+ Mφ in SIVΔ<i>vpx</i> monkeys (mean 0.5 SIV+ Mφ) compared to the SIV239E (mean 13.64 SIV+ Mφ), SIV239noE (mean 4.5 SIV+ Mφ) and SIVΔ<i>nef</i>/SIVΔ3 monkeys (mean 6.25 SIV+ Mφ). <b>(D)</b> SIV infected macrophages made up a much lower percentage of all SIV+ cells in SPL and LN of SIVΔ<i>vpx</i>-infected rhesus (mean 2.2%) compared to SIV239E (mean 22.7%), SIV239noE (mean 8.3%), and SIVΔ<i>nef</i>/SIVΔ3 monkeys (10.1%) (p<0.05).</p

Phenotype of SIV+ cells in spleen, peripheral lymph node, and colon of SIVΔ<i>vpx</i>-infected monkeys by double-label <i>in situ</i> hybridization and immunohistochemistry.

<p>Phenotype of SIV+ cells in spleen, peripheral lymph node, and colon of SIVΔ<i>vpx</i>-infected monkeys by double-label <i>in situ</i> hybridization and immunohistochemistry.</p

Double-label immunohistochemistry and <i>in situ</i> hybridization of colon.

<p><b>(A).</b> Representative images of SIV ISH (blue) with double-label immunohistochemistry for macrophage marker Ham56 (DAB, brown) in rhesus macaques in groups SIV239E (left), SIV239noE (middle), and SIVΔ<i>vpx</i> (right). SIV+ Ham56+ macrophages were frequent in SIV239E monkeys (left), but absent in the SIVΔ<i>vpx</i> group (right). <b>(B).</b> SIVΔ<i>vpx</i>-infected monkeys had significantly less virus in the colon (mean 10.5 SIV+ cells) compared to monkeys infected with SIV239 (SIV239E mean 105.3 SIV+ cells, SIV239noE mean 33.5 SIV+ cells), but no difference with animals in the SIVΔ<i>nef</i>/SIVΔ3 group. <b>(C).</b> SIVΔ<i>vpx</i> monkeys (mean 0 cells) had significantly fewer SIV+ Ham56+ macrophages compared to SIV239E (mean 66.7 cells), SIV239noE (mean 9.8 cells), and SIVΔ<i>nef</i>/SIVΔ3 groups (mean 3.3 cells) groups. <b>(D).</b> There was a significantly lower percentage of SIV+ cells that were macrophages in SIVΔ<i>vpx</i>-infected rhesus (mean 0%) compared to both the SIV239E (mean 63.3%) and SIV239noE (mean 22.4%) groups and a trend in the SIVΔ<i>nef</i>/SIVΔ3 group (mean 25.6%).</p

Survival and viral load data.

<p>(<b>A)</b> Survival days post-inoculation (dpi) for rhesus macaques inoculated with SIVΔ<i>vpx</i> compared to animals inoculated with SIVmac239 (SIV239E with encephalitis or SIV239noE without encephalitis) or other mutant viruses (SIVΔ<i>nef</i> or SIVΔ3. SIVΔ<i>vpx</i>-infected macaques survived significantly longer with a slower disease progression compared to SIV239E or SIV239noE animals, but did not differ in survival length compared to SIVΔ<i>nef</i> or SIVΔ3. <b>(B)</b> Plasma viral RNA from SIVΔ<i>vpx</i>-infected rhesus macaques expressed as RNA copy equivalents per ml plasma from chronic disease or near-terminal collections (range 329–1140 dpi, median 730 dpi).</p

Summaryoflentiviral infection of macrophages in spleen, lymph node, anin macaques inoculated intravenously with SIVΔ<i>vpx</i> compared to SIVmac239 (with or without SIV encephalitis) and SIVΔ<i>nef</i> or Δ3 assessed in spleen, lymph node, and colon.

<p>*p<0.05 difference between SIVΔ<i>vpx</i> and both SIVmac239 and SIVΔ<i>nef</i>/Δ3cases.</p