Vpx is Critical for SIVmne infection of pigtail macaques

<p>Abstract</p> <p>Background</p> <p>Viral protein X (Vpx) of SIV has been reported to be important for establishing infection <it>in vivo</it>. Vpx has several different activities <it>in vitro</it>, promoting preintegration complex import into the nucleus in quiescent lymphocytes and overcoming a block in reverse transcription in macrophages. Vpx interacts with the DDB1-CUL4-DCAF1 E3 ligase complex, which may or may not be required for the ascribed functions. The goal of the current study was to determine whether these activities of Vpx are important <it>in vivo</it>.</p> <p>Results</p> <p>An infectious, pathogenic clone of SIVmne was used to examine correlations between Vpx functions <it>in vitro </it>and <it>in vivo</it>. Three previously described HIV-2 Vpx mutants that were shown to be important for nuclear import of the preintegration complex in quiescent lymphocytes were constructed in SIVmne: A <it>vpx</it>-deleted virus, a truncation of Vpx at amino acid 102 that deletes the C-terminal proline-rich domain (X(102)), and a mutant with tyrosines 66, 69, and 71 changed to alanine (X(y-a)). All mutant viruses replicated similarly to wild type SIVmne027 in primary pigtail macaque PBMCs, and were only slightly retarded in CEMx174 cells. However, all the <it>vpx </it>mutant viruses were defective for replication in both human and pigtail monocyte-derived macrophages. PCR assays demonstrated that the efficiency of reverse transcription and the levels of viral integration in macrophages were substantially reduced for the <it>vpx </it>mutant viruses. <it>In vitro</it>, the X(y-a) mutant, but not the X(102) mutant lost interaction with DCAF1. The wild type SIVmne027 and the three <it>vpx </it>mutant SIVs were inoculated by the intra-rectal route into pigtail macaques. Peak levels of plasma viremia of the <it>vpx </it>mutant SIVs were variable, but consistently lower than that observed in macaques infected with wild type SIVmne. <it>In situ </it>hybridization for SIV demonstrated that compared to wild type SIVmne infected macaques five of the six animals inoculated with the <it>vpx </it>mutant SIVs had only low levels of SIV-expressing cells in the rectum, most intestinal epithelial tissues, spleen, and mesenteric and peripheral nodes.</p> <p>Conclusions</p> <p>This work demonstrates that the activities of Vpx to overcome restrictions in culture <it>in vitro </it>are also likely to be important for establishment of infection <it>in vivo </it>and suggest that both the nuclear localization and DCAF1-interaction functions of Vpx are critical <it>in vivo</it>.</p

Mucosal Antibodies: Defending Epithelial Barriers against HIV-1 Invasion

The power of mucosal anti-HIV-1 envelope immunoglobulins (Igs) to block virus transmission is underappreciated. We used passive immunization, a classical tool to unequivocally prove whether antibodies are protective. We mucosally instilled recombinant neutralizing monoclonal antibodies (nmAbs) of different Ig classes in rhesus macaques (RMs) followed by mucosal simian&ndash;human immunodeficiency virus (SHIV) challenge. We gave anti-HIV-1 IgM, IgG, and dimeric IgA (dIgA) versions of the same human nmAb, HGN194 that targets the conserved V3 loop crown. Surprisingly, dIgA1 with its wide-open, flat hinge protected 83% of the RMs against intrarectal R5-tropic SHIV-1157ipEL-p challenge, whereas dIgA2, with its narrow hinge, only protected 17% of the animals&mdash;despite identical epitope specificities and in vitro neutralization curves of the two dIgA isotypes (Watkins et al., AIDS 2013 27(9):F13-20). These data imply that factors in addition to neutralization determine in vivo protection. We propose that this underlying protective mechanism is immune exclusion, which involves large nmAb/virion aggregates that prevent virus penetration of mucosal barriers. Future studies need to find biomarkers that predict effective immune exclusion in vivo. Vaccine development strategies against HIV-1 and/or other mucosally transmissible pathogens should include induction of strong mucosal Abs of different Ig classes to defend epithelial barriers against pathogen invasion

Macaques as model hosts for studies of HIV-1 infection

Increasing evidence indicates that the host range of primate lentiviruses is in part determined by their ability to counteract innate restriction factors that are effectors of the type 1 interferon (IFN-1) response. For HIV-1, in vitro experiments have shown that its tropism may be narrow and limited to humans and chimpanzees because its replication in other nonhuman primate species is hindered by factors such as TRIM5α, APOBEC3G, and Tetherin. Based on these data, it has been hypothesized that primate lentiviruses will infect and replicate in a new species if they are able to counteract and evade suppression by the IFN-1 response. Several studies have tested whether engineering HIV-1 recombinants with minimal amounts of SIV sequences would enable replication in CD4+ T-cells of non-natural hosts such as Asian macaques and proposed that infection of these macaque species could be used to study transmission and pathogenesis. Indeed, infection of macaques with these viruses revealed that Vif-mediated counteraction of APOBEC3G function is central to cross-species tropism but that other IFN-induced factors may also play important roles in controlling replication. Further studies of these macaque models of infection with HIV-1 derivatives could provide valuable insights into the interaction of lentiviruses and the innate immune response and how lentiviruses adapt and cause disease

In vivo serial passaging of human–simian immunodeficiency virus clones identifies characteristics for persistent viral replication

We previously reported that a human immunodeficiency virus type 1 with a simian immunodeficiency virus vif substitution (HSIV-vifNL4-3) could replicate in pigtailed macaques (PTMs), demonstrating that Vif is a species-specific tropism factor of primate lentiviruses. However, infections did not result in high-peak viremia or setpoint plasma viral loads, as observed during simian immunodeficiency virus (SIV) infection of PTMs. Here, we characterized variants isolated from one of the original infected animals with CD4 depletion after nearly 4years of infection to identify determinants of increased replication fitness. In our studies, we found that the HSIV-vif clones did not express the HIV-1 Vpr protein due to interference from the vpx open reading frame (ORF) in singly spliced vpr mRNA. To examine whether these viral genes contribute to persistent viral replication, we generated infectious HSIV-vif clones expressing either the HIV-1 Vpr or SIV Vpx protein. And then to determine viral fitness determinants of HSIV-vif, we conducted three rounds of serial in vivo passaging in PTMs, starting with an initial inoculum containing a mixture of CXCR4-tropic [Vpr-HSIV-vifNL4-3 isolated at 196 (C/196) and 200 (C/200) weeks post-infection from a PTM with depressed CD4 counts] and CCR5-tropic HSIV (Vpr+ HSIV-vif derivatives based NL-AD8 and Bru-Yu2 and a Vpx expressing HSIV-vifYu2). Interestingly, all infected PTMs showed peak plasma viremia close to or above 105 copies/ml and persistent viral replication for more than 20weeks. Infectious molecular clones (IMCs) recovered from the passage 3 PTM (HSIV-P3 IMCs) included mutations required for HIV-1 Vpr expression and those mutations encoded by the CXCR4-tropic HSIV-vifNL4-3 isolate C/196. The data indicate that the viruses selected during long-term infection acquired HIV-1 Vpr expression, suggesting the importance of Vpr for in vivo pathogenesis. Further passaging of HSIV-P3 IMCs in vivo may generate pathogenic variants with higher replication capacity, which will be a valuable resource as challenge virus in vaccine and cure studies.Fil: Thippeshappa, Rajesh. Texas Biomedical Research Institute; Estados UnidosFil: Polacino, Patricia. University of Washington; Estados UnidosFil: Chandrasekar, Shaswath S.. Baylor College of Medicine; Estados UnidosFil: Truong, Khanghy. Baylor College of Medicine; Estados UnidosFil: Misra, Anisha. Baylor College of Medicine; Estados UnidosFil: Aulicino, Paula. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Gobierno de la Ciudad de Buenos Aires. Hospital de Pediatría "Juan P. Garrahan". Laboratorio de Biología Celular y Retrovirus; ArgentinaFil: Hu, Shiu-Lok. University of Washington; Estados UnidosFil: Kaushal, Deepak. Texas Biomedical Research Institute; Estados UnidosFil: Kimata, Jason T.. Baylor College of Medicine; Estados Unido