Toll-like receptor 3 signaling inhibits simian immunodeficiency virus replication in macrophages from rhesus macaques.
ABSTRACT: Toll-like receptor 3 (TLR3) recognizes double-stranded RNA and induces multiple intracellular events responsible for innate antiviral immunity against viral infections. Here we demonstrate that TLR3 signaling of monocyte-derived macrophages (MDM) from rhesus monkeys by poly I:C inhibited simian immunodeficiency virus (SIV) infection and replication. Investigation of the mechanisms showed that TLR3 activation resulted in the induction of type I and type III interferons (IFNs) and IFN-inducible antiviral factors, including APOBEC3G (A3G), tetherin and SAMHD1. In addition, poly I:C-treated macaque macrophages expressed increased levels of CC chemokines including CCL3, CCL4 and CCL5, the ligands for HIV or SIV coreceptor CCR5. Furthermore, TLR3 signaling of macaque macrophages induced the expression of cellular microRNAs (miR-29a, -29b, -146a and -9), the newly identified intracellular SIV restriction factors. TLR3 activation-mediated anti-SIV effect could be compromised by the knockdown of IRF3 and IRF7. These findings indicate that TLR3-mediated induction of multiple viral restriction factors contribute to the inhibition of SIV infection in macaque macrophages, which support future preclinical studies using rhesus macaques to determine whether in vivo TLR3 activation is safe and beneficial for treating people infected with HIV.
Project description:BACKGROUND: The cytidine deaminases APOBEC3G (A3G) and APOBEC3F (A3F) are innate cellular factors that inhibit replication of a number of viruses, including HIV-1. Since antiviral activity of APOBEC3 has been mainly confirmed by in vitro data, we examined their role for disease progression in the SIV/macaque model for AIDS. RESULTS: We quantified A3G and A3F mRNA in PBMC and leukocyte subsets of uninfected and SIVmac-infected rhesus macaques. Compared with uninfected animals, we found increased A3G and A3F mRNA levels in PBMC, purified CD4+ T-cells and CD14+ monocytes as well as lymph node cells from asymptomatic SIV-infected macaques. APOBEC3 mRNA levels correlated negatively with plasma viral load, and highest amounts of APOBEC3 mRNA were detected in long term non-progressors (LTNPs). During acute viremia, A3G mRNA increased in parallel with MxA, a prototype interferon-stimulated gene indicating a common regulation by the initial interferon response. This association disappeared during the asymptomatic stage. CONCLUSION: Our findings suggest a protective effect of APOBEC3 for HIV and SIV in vivo and indicate regulation of APOBEC3 by interferon during early infection and by contribution of other, hitherto undefined factors at later disease stages. Elucidating the regulatory mechanisms leading to increased APOBEC3 mRNA levels in LTNPs could help to develop new therapies against HIV.
Project description:Human immunodeficiency virus (HIV) infections are rarely acquired via an oral route in adults. Previous studies have shown that human whole saliva inhibits HIV infection in vitro, and multiple factors present in human saliva have been shown to contribute to this antiviral activity. Despite the widespread use of simian immunodeficiency virus (SIV)-infected rhesus macaques as models for HIV pathogenesis and transmission, few studies have monitored SIV in the oral cavity of infected rhesus macaques and evaluated the viral inhibitory capacity of macaque saliva. Utilizing a cohort of rhesus macaques infected with SIV(Mac251), we monitored virus levels and genotypic diversity in the saliva throughout the course of the disease; findings were similar to previous observations in HIV-infected humans. An in vitro infectivity assay was utilized to measure inhibition of HIV/SIV infection by normal human and rhesus macaque whole saliva. Both human and macaque saliva were capable of inhibiting HIV and SIV infection. The inhibitory capacity of saliva samples collected from a cohort of animals postinfection with SIV increased over the course of disease, coincident with the development of SIV-specific antibodies in the saliva. These findings suggest that both innate and adaptive factors contribute to inhibition of SIV by whole macaque saliva. This work also demonstrates that SIV-infected rhesus macaques provide a relevant model to examine the innate and adaptive immune responses that inhibit HIV/SIV in the oral cavity.
Project description:APOBEC3G, a member of the double-domain cytidine deaminase (CD) APOBEC, binds RNA to package into virions and restrict HIV-1 through deamination-dependent or deamination-independent inhibition. Mainly due to lack of a full-length double-domain APOBEC structure, it is unknown how CD1/CD2 domains connect and how dimerization/multimerization is linked to RNA binding and virion packaging for HIV-1 restriction. We report rhesus macaque A3G structures that show different inter-domain packing through a short linker and refolding of CD2. The A3G dimer structure has a hydrophobic dimer-interface matching with that of the previously reported CD1 structure. A3G dimerization generates a surface with intensified positive electrostatic potentials (PEP) for RNA binding and dimer stabilization. Unexpectedly, mutating the PEP surface and the hydrophobic interface of A3G does not abolish virion packaging and HIV-1 restriction. The data support a model in which only one RNA-binding mode is critical for virion packaging and restriction of HIV-1 by A3G.
Project description:Molecular adjuvants are a promising method to enhance virus-specific immune responses and protect against HIV-1 infection. Immune activation by ligands for receptors such as CD40 can induce dendritic cell activation and maturation. Here we explore the incorporation of two CD40 mimics, Epstein Barr Virus gene LMP1 or an LMP1-CD40 chimera, into a strain of SIV that was engineered to be limited to a single cycle of infection.Full length LMP1 or the chimeric protein LMP1-CD40 was cloned into the nef-locus of single-cycle SIV. Human and Macaque monocyte derived macrophages and DC were infected with these viruses. Infected cells were analyzed for activation surface markers by flow cytometry. Cells were also analyzed for secretion of pro-inflammatory cytokines IL-1?, IL-6, IL-8, IL-12p70 and TNF by cytometric bead array.Overall, single-cycle SIV expressing LMP1 and LMP1-CD40 produced a broad and potent T(H)1-biased immune response in human as well as rhesus macaque macrophages and DC when compared with control virus. Single-cycle SIV-LMP1 also enhanced antigen presentation by lentiviral vector vaccines, suggesting that LMP1-mediated immune activation may enhance lentiviral vector vaccines against HIV-1.
Project description:Tripartite motif-containing protein 5α (TRIM5α) is considered to be a potential target for cell-based gene modification therapy against human immunodeficiency virus type 1 (HIV-1) infection. In the present study, we used a relevant rhesus macaque model of infection with simian immunodeficiency virus from sooty mangabey (SIVsm) to evaluate the effect of TRIM5α restriction on clinical outcome. For macaques expressing a restrictive TRIM5 genotype, the disease outcomes of those infected with the wild-type TRIM-sensitive SIVsm strain and those infected with a virus with escape mutations in the capsid were compared. We found that TRIM5α restriction significantly delayed disease progression and improved the survival rate of SIV-infected macaques, supporting the feasibility of exploiting TRIM5α as a target for gene therapy against HIV-1. Furthermore, we also found that preservation of memory CD4 T cells was associated with protection by TRIM5α restriction, suggesting memory CD4 T cells or their progenitor cells as an ideal target for gene modification. Despite the significant effect of TRIM5α restriction on survival, SIV escape from TRIM5α restriction was also observed; therefore, this may not be an effective stand-alone strategy and may require combination with other targets.Recent studies suggest that it may be feasible not only to suppress viral replication with antiviral drugs but also potentially to eliminate or "cure" human immunodeficiency virus (HIV) infection. One approach being explored is the use of gene therapy to introduce genes that can restrict HIV replication, including a restrictive version of the host factor TRIM5α. TRIM5 was identified as a factor that restricts HIV replication in macaque cells. The rhesus gene is polymorphic, and some alleles are restrictive for primary SIVsm isolates, although escape mutations arise late in infection. Introduction of these escape mutations into the parental virus conferred resistance to TRIM5 on macaques. The present study evaluated these animals for long-term outcomes and found that TRIM5α restriction significantly delayed disease progression and improved the survival rate of SIV-infected macaques, suggesting that this could be a valid gene therapy approach that could be adapted for HIV.
Project description:Tetherin, also known as BST2, CD317 or HM1.24, was recently identified as an interferon-inducible host-cell factor that interferes with the detachment of virus particles from infected cells. HIV-1 overcomes this restriction by expressing an accessory protein, Vpu, which counteracts tetherin. Since lentiviruses of the SIV(smm/mac)/HIV-2 lineage do not have a vpu gene, this activity has likely been assumed by other viral gene products. We found that deletion of the SIV(mac)239 nef gene significantly impaired virus release in cells expressing rhesus macaque tetherin. Virus release could be restored by expressing Nef in trans. However, Nef was unable to facilitate virus release in the presence of human tetherin. Conversely, Vpu enhanced virus release in the presence of human tetherin, but not in the presence of rhesus tetherin. In accordance with the species-specificity of Nef in mediating virus release, SIV Nef downregulated cell-surface expression of rhesus tetherin, but did not downregulate human tetherin. The specificity of SIV Nef for rhesus tetherin mapped to four amino acids in the cytoplasmic domain of the molecule that are missing from human tetherin, whereas the specificity of Vpu for human tetherin mapped to amino acid differences in the transmembrane domain. Nef alleles of SIV(smm), HIV-2 and HIV-1 were also able to rescue virus release in the presence of both rhesus macaque and sooty mangabey tetherin, but were generally ineffective against human tetherin. Thus, the ability of Nef to antagonize tetherin from these Old World primates appears to be conserved among the primate lentiviruses. These results identify Nef as the viral gene product of SIV that opposes restriction by tetherin in rhesus macaques and sooty mangabeys, and reveal species-specificity in the activities of both Nef and Vpu in overcoming tetherin in their respective hosts.
Project description:A majority of individuals infected with human immunodeficiency virus (HIV) have inadequate access to antiretroviral therapy and ultimately develop debilitating oral infections that often correlate with disease progression. Due to the impracticalities of conducting host-microbe systems-based studies in HIV infected patients, we have evaluated the potential of simian immunodeficiency virus (SIV) infected rhesus macaques to serve as a non-human primate model for oral manifestations of HIV disease. We present the first description of the rhesus macaque oral microbiota and show that a mixture of human commensal bacteria and "macaque versions" of human commensals colonize the tongue dorsum and dental plaque. Our findings indicate that SIV infection results in chronic activation of antiviral and inflammatory responses in the tongue mucosa that may collectively lead to repression of epithelial development and impact the microbiome. In addition, we show that dysbiosis of the lingual microbiome in SIV infection is characterized by outgrowth of Gemella morbillorum that may result from impaired macrophage function. Finally, we provide evidence that the increased capacity of opportunistic pathogens (e.g. E. coli) to colonize the microbiome is associated with reduced production of antimicrobial peptides.
Project description:Tetherin (BST-2 or CD317) is an interferon-inducible transmembrane protein that inhibits virus release from infected cells. To determine the extent of sequence variation and the impact of polymorphisms in rhesus macaque tetherin on simian immunodeficiency virus (SIV) infection, tetherin alleles were sequenced from 146 rhesus macaques, including 68 animals infected with wild-type SIVmac239 and 47 animals infected with SIVmac239?nef Since Nef is the viral gene product of SIV that counteracts restriction by tetherin, these groups afford a comparison of the effects of tetherin polymorphisms on SIV strains that are, and are not, resistant to tetherin. We identified 15 alleles of rhesus macaque tetherin with dimorphic residues at 9 positions. The relationship between these alleles and plasma viral loads was compared during acute infection, prior to the onset of adaptive immunity. Acute viremia did not differ significantly among the wild-type SIV-infected animals; however, differences in acute viral loads were associated with polymorphisms in tetherin among the animals infected with SIV?nef In particular, polymorphisms at positions 43 and 111 (P43 and H111) were associated with lower acute-phase viral loads for SIV?nef infection. These observations reveal extensive polymorphism in rhesus macaque tetherin, maintained perhaps as a consequence of variability in the selective pressure of diverse viral pathogens, and identify tetherin alleles that may have an inherently greater capacity to restrict SIV replication in the absence of Nef.IMPORTANCE As a consequence of ongoing evolutionary conflict with viral pathogens, tetherin has accumulated numerous species-specific differences that represent important barriers to the transmission of viruses between species. This study reveals extensive polymorphism in rhesus macaque tetherin and identifies specific alleles that are associated with lower viral loads during the first few weeks after infection with nef-deleted SIV. These observations suggest that the variable selective pressure of viral pathogens, in addition to driving the diversification of tetherin among species, also operates within certain species to maintain sequence variation in tetherin.
Project description:Prime-boost immunization with heterologous vaccines elicits potent cellular immunity. In this study, we assessed the influence of various TLR ligands on SIV Gag-specific T cell immunity and protection following prime-boost immunization. Rhesus macaques (RMs) were primed with SIV Gag protein emulsified in Montanide ISA51 with or without TLR3 (polyinosinic-polycytidylic acid [poly-IC]), TLR4 (monophosphoryl lipid A), TLR7/8 (3M-012), TLR9 (CpG), or TLR3 (poly-IC) combined with TLR7/8 ligands, then boosted with replication defective adenovirus 5 expressing SIV Gag (rAd5-Gag). After priming, RMs that received SIV Gag protein plus poly-IC developed significantly higher frequencies of SIV Gag-specific CD4(+) Th1 responses in blood and bronchoalveolar lavage (BAL) fluid lymphocytes compared with all other adjuvants, and low-level SIV Gag-specific CD8(+) T cell responses. After the rAd5-Gag boost, the magnitude and breadth of SIV Gag-specific CD8(+) T cell responses were significantly increased in RM primed with SIV Gag protein plus poly-IC, with or without the TLR7/8 ligand, or CpG. However, the anamnestic, SIV Gag-specific CD8(+) T cell response to SIVmac251 challenge was not significantly enhanced by SIV Gag protein priming with any of the adjuvants. In contrast, the anamnestic SIV Gag-specific CD4(+) T cell response in BAL was enhanced by SIV Gag protein priming with poly-IC or CpG, which correlated with partial control of early viral replication after SIVmac251 challenge. These results demonstrate that prime-boost vaccination with SIV Gag protein/poly-IC improves magnitude, breadth, and durability of CD4(+) T cell immune responses, which could have a role in the control of SIV viral replication.
Project description:Simian immunodeficiency virus (SIV) infection in rhesus macaques is often characterized by high viremia and CD4 T cell depletion. By contrast, SIV infection in African nonhuman primate natural hosts is typically nonpathogenic despite active viral replication. Baboons are abundant in Africa and have a geographical distribution that overlaps with natural hosts, but they do not harbor SIVs. Previous work has demonstrated baboons are resistant to chronic SIV infection and/or disease in vivo but the underlying mechanisms remain unknown. Using in vitro SIVmac infections, we sought to identify SIV restriction factors in baboons by comparing observations to the pathogenic rhesus macaque model. SIVmac replicated in baboon PBMC but had delayed kinetics compared to rhesus PBMC. However, SIVmac replication in baboon and rhesus isolated CD4 cells were similar to the kinetics seen for rhesus PBMC, demonstrating intracellular restriction factors do not play a strong role in baboon inhibition of SIVmac replication. Here, we show CD8 T cells contribute to the innate SIV-suppressive activity seen in naïve baboon PBMC. As one mechanism of restriction, we identified higher production of MIP-1?, MIP-1?, and RANTES by baboon PBMC. Contact between CD4 and CD8 T cells resulted in maximum production of these chemokines and suppression of viral replication, whereas neutralization of CCR5-binding chemokines in baboon PBMC increased viral loads. Our studies indicate baboon natural restriction of SIVmac replication is largely dependent on CD4-extrinsinc mechanisms mediated, in part, by CD8 T cells.