Multiple Homozygous Variants in the STING-Encoding TMEM173 Gene in HIV Long-Term Nonprogressors.

Among HIV-infected individuals, long-term nonprogressor (LTNP) patients experience slow CD4 T cell decline and almost undetectable viral load for several years after primary acquisition of HIV. Type I IFN has been suggested to play a pathogenic role in HIV pathogenesis, and therefore diminished IFN responses may underlie the LTNP phenotype. In this study, we examined the presence and possible immunological role of multiple homozygous single-nucleotide polymorphisms in the stimulator of IFN genes (STING) encoding gene TMEM173 involved in IFN induction and T cell proliferation in HIV LTNP patients. We identified LTNPs through the Danish HIV Cohort and performed genetic analysis by Sanger sequencing, covering the R71H-G230A-R293Q (HAQ) single-nucleotide polymorphisms in TMEM173 This was followed by investigation of STING mRNA and protein accumulation as well as innate immune responses and proliferation following STING stimulation and infection with replication-competent HIV in human blood-derived cells. We identified G230A-R293Q/G230A-R293Q and HAQ/HAQ homozygous TMEM173 variants in 2 out of 11 LTNP patients. None of the 11 noncontrollers on antiretroviral treatment were homozygous for these variants. We found decreased innate immune responses to DNA and HIV as well as reduced STING-dependent inhibition of CD4 T cell proliferation, particularly in the HAQ/HAQ HIV LTNP patients, compared with the age- and gender-matched noncontrollers on antiretroviral treatment. These findings suggest that homozygous HAQ STING variants contribute to reduced inhibition of CD4 T cell proliferation and a reduced immune response toward DNA and HIV, which might result in reduced levels of constitutive IFN production. Consequently, the HAQ/HAQ TMEM173 genotype may contribute to the slower disease progression characteristic of LTNPs.

Mucosal stromal fibroblasts markedly enhance HIV infection of CD4+ T cells.

Understanding early events of HIV transmission within mucosal tissues is vital for developing effective prevention strategies. Here, we report that primary stromal fibroblasts isolated from endometrium, cervix, foreskin, male urethra, and intestines significantly increase HIV infection of CD4+ T cells-by up to 37-fold for R5-tropic HIV and 100-fold for X4-tropic HIV-without themselves becoming infected. Fibroblasts were more efficient than dendritic cells at trans-infection and mediate this response in the absence of the DC-SIGN and Siglec-1 receptors. In comparison, mucosal epithelial cells secrete antivirals and inhibit HIV infection. These data suggest that breaches in the epithelium allow external or luminal HIV to escape an antiviral environment to access the infection-favorable environment of the stromal fibroblasts, and suggest that resident fibroblasts have a central, but previously unrecognized, role in HIV acquisition at mucosal sites. Inhibiting fibroblast-mediated enhancement of HIV infection should be considered as a novel prevention strategy.

The 2'-5'-oligoadenylate synthetase 3 enzyme potently synthesizes the 2'-5'-oligoadenylates required for RNase L activation.

UNLABELLED: The members of the oligoadenylate synthetase (OAS) family of proteins are antiviral restriction factors that target a wide range of RNA and DNA viruses. They function as intracellular double-stranded RNA (dsRNA) sensors that, upon binding to dsRNA, undergo a conformational change and are activated to synthesize 2'-5'-linked oligoadenylates (2-5As). 2-5As of sufficient length act as second messengers to activate RNase L and thereby restrict viral replication. We expressed human OAS3 using the baculovirus system and purified it to homogeneity. We show that recombinant OAS3 is activated at a substantially lower concentration of dsRNA than OAS1, making it a potent in vivo sensor of dsRNA. Moreover, we find that OAS3 synthesizes considerably longer 2-5As than previously reported, and that OAS3 can activate RNase L intracellularly. The combined high affinity for dsRNA and the capability to produce 2-5As of sufficient length to activate RNase L suggests that OAS3 is a potent activator of RNase L. In addition, we provide experimental evidence to support one active site of OAS3 located in the C-terminal OAS domain and generate a low-resolution structure of OAS3 using SAXS. IMPORTANCE: We are the first to purify the OAS3 enzyme to homogeneity, which allowed us to characterize the mechanism utilized by OAS3 and identify the active site. We provide compelling evidence that OAS3 can produce 2'-5'-oligoadenylates of sufficient length to activate RNase L. This is contrary to what is described in the current literature but agrees with recent in vivo data showing that OAS3 harbors an antiviral activity requiring RNase L. Thus, our work redefines our understanding of the biological role of OAS3. Furthermore, we used a combination of mutagenesis and small-angle X-ray scattering to describe the active site and low-resolution structure of OAS3.

Extracellular 2'-5' oligoadenylate synthetase stimulates RNase L-independent antiviral activity: a novel mechanism of virus-induced innate immunity.

The 2'-5' oligoadenylate synthetase (OAS) proteins are traditionally considered intracellular antiviral proteins. However, several studies demonstrate a correlation between the concentration of freely circulating OAS protein in sera from hepatitis C patients and their clinical prognosis. Here we demonstrate that extracellular OAS1 enters into cells and possesses a strong antiviral activity, both in vitro and in vivo, which is independent of RNase L. The OAS protein directly inhibits viral proliferation and does not require the activation of known antiviral signaling pathways. We propose that OAS produced by cells infected with viruses is released to the extracellular space, where it acts as a paracrine antiviral agent. Thus, the OAS protein represents the first direct antiviral compound released by virus-infected cells.

Cell-Extrinsic Priming Increases Permissiveness of CD4+ T Cells to Human Immunodeficiency Virus Infection by Increasing C-C Chemokine Receptor Type 5 Co-receptor Expression and Cellular Activation Status.

The chemokine receptor CCR5 is expressed on multiple cell types, including macrophages, dendritic cells, and T cells, and is the major co-receptor used during HIV transmission. Using a standard αCD3/CD28 in vitro stimulation protocol to render CD4+ T cells from PBMCs permissive to HIV infection, we discovered that the percentage of CCR5+ T cells was significantly elevated in CD4+ T cells when stimulated in the presence of peripheral blood mononuclear cells (PBMCs) as compared to when stimulated as purified CD4+ T cells. This indicated that environmental factors unique to the T-PBMCs condition affect surface expression of CCR5 on CD4+ T cells. Conditioned media from αCD3/CD28-stimulated PBMCs induced CCR5 expression in cultures of unstimulated cells. Cytokine profile analysis of these media suggests IL-12 as an inducer of CCR5 expression. Mass cytometric analysis showed that stimulated T-PBMCs exhibited a uniquely activated phenotype compared to T-Pure. In line with increased CCR5 expression and activation status in stimulated T-PBMCs, CD4+ T cells from these cultures were more susceptible to infection by CCR5-tropic HIV-1 as compared with T-Pure cells. These results suggest that in order to increase ex vivo infection rates of blood-derived CD4+ T cells, standard stimulation protocols used in HIV infection studies should implement T-PBMCs or purified CD4+ T cells should be supplemented with IL-12.

Rapid Uptake and Inhibition of Viral Propagation by Extracellular OAS1.

The oligoadenylate synthetase (OAS) proteins are traditionally considered intracellular antiviral proteins that mediate antiviral activity through the synthesis of 2'-5'-linked oligoadenylates and subsequent activation of the endoribonuclease RNase L. However, we have recently demonstrated that exogenous recombinant OAS1 is taken up by cells and reduces viral replication both in cell culture and in vivo, independent of RNase L. These results demonstrate a novel paracrine antiviral activity of OAS working in parallel with the classical RNase L pathway. In this study, we investigate the uptake kinetics of recombinant porcine OAS1 and show that it is rapidly and efficiently internalized in a manner that can be blocked by heparin. Heparin, furthermore, abolishes the antiviral activity of OAS1, demonstrating the requirement of the intracellular localization of OAS1 to inhibit the virus. In addition, we demonstrate that exogenous OAS1 affects an early step of the viral replication cycle.