IFN-α is constitutively expressed in the human thymus, but not in peripheral lymphoid organs

Type I interferons have been typically studied for their effects in the context of bacterial or viral infections. However in this report, we provide evidence that Interferon-alpha (IFN-α) expressing cells are present in the thymus in the absence of infection. We show that pDC express the highest level of IFN-α and that MxA, which is exclusively expressed after engagement of the type I IFN receptor by IFN-α/β, is expressed in normal fetal and post-natal thymus, but not in the periphery. The highest level of MxA is expressed in mature thymocytes and pDC located in the medulla and at the cortico-medullary junction. The anti-microbial peptide LL-37, which is expressed in the thymus, when complexed with eukaryotic nucleic acids, induces the secretion of IFN-α by thymic pDC. This results in the upregulation of MxA expression in responsive thymocytes. We propose that the secretion of IFN-α in the thymus may function to regulate the rate of T cell development and modulate the requirements for the selection of developing T cells.     

Homology-based identification of capsid determinants that protect HIV1 from human TRIM5α restriction

The tropism of retroviruses relies on their ability to exploit cellular factors for their replication as well as to avoid host-encoded inhibitory activities such as TRIM5α. N-tropic murine leukemia virus is sensitive to human TRIM5α (huTRIM5α) restriction, whereas human immunodeficiency virus type 1 (HIV1) escapes this antiviral factor. We previously revealed that mutation of four critical amino acid residues within the capsid can render murine leukemia virus resistant to huTRIM5α. Here, we exploit the high degree of conservation in the tertiary structure of retroviral capsids to map the corresponding positions on the HIV1 capsid. We then demonstrated that, when changes were introduced at some of these positions, HIV1 becomes sensitive to huTRIM5α restriction, a phenomenon reinforced by additionally mutating the nearby cyclophilin A binding loop of the viral protein. These results indicate that retroviruses have evolved similar mechanisms to escape TRIM5α restriction via the interference of structurally homologous determinants in the viral capsid.     

Molecular characterization of the type I IFN receptor in two woodchuck species and detection of its expression in liver samples from woodchucks infected with woodchuck hepatitis virus (WHV)

Type I interferons (IFN-α/β) serve as the first line of defense against viral infection and share the same type I IFN receptor (IFNAR) complex, which is composed of IFNAR1 and -2. The Eastern woodchuck (Marmota monax) and Chinese woodchuck (Marmota himalayana) are suitable for studying hepatitis B virus (HBV) infection. Here, the complete or partial sequences of the IFNARs of both species were obtained and analyzed. Small interference RNAs targeting wIFNAR1 and -2 specifically down-regulated the expression of wIFNAR1 and -2 and the IFN-stimulated gene MxA in a woodchuck cell line, respectively. IFNAR2 was significantly up-regulated in primary woodchuck hepatocytes stimulated with IFN-α or -γ. The expression of woodchuck IFNAR1 and -2 was decreased in woodchucks chronically infected with woodchuck hepatitis virus (WHV). These results are essential for studying type I IFN-related innate immunity and therapy in hepadnaviral infection in the woodchuck model.     

Human tripartite motif 5alpha domains responsible for retrovirus restriction activity and specificity

The tripartite motif 5alpha protein (TRIM5alpha) is one of several factors expressed by mammalian cells that inhibit retrovirus replication. Human TRIM5alpha (huTRIM5alpha) inhibits infection by N-tropic murine leukemia virus (N-MLV) but is inactive against human immunodeficiency virus type 1 (HIV-1). However, we show that replacement of a small segment in the carboxy-terminal B30.2/SPRY domain of huTRIM5alpha with its rhesus macaque counterpart (rhTRIM5alpha) endows it with the ability to potently inhibit HIV-1 infection. The B30.2/SPRY domain and an additional domain in huTRIM5alpha, comprising the amino-terminal RING and B-box components of the TRIM motif, are required for N-MLV restriction activity, while the intervening coiled-coil domain is necessary and sufficient for huTRIM5alpha multimerization. Truncated huTRIM5alpha proteins that lack either or both the N-terminal RING/B-Box or the C-terminal B30.2/SPRY domain form heteromultimers with full-length huTRIM5alpha and are dominant inhibitors of its N-MLV restricting activity, suggesting that homomultimerization of intact huTRIM5alpha monomers is necessary for N-MLV restriction. However, localization in large cytoplasmic bodies is not required for inhibition of N-MLV by huTRIM5alpha or for inhibition of HIV-1 by chimeric or rhTRIM5alpha.     

Inhibitory effect of human TRIM5α on HIV-1 production

Tripartite motif-containing 5 isoform-α (TRIM5α), a host restriction factor, blocks infection of some retroviruses at a post-entry, pre-integration stage in a species-specific manner. A recent report by Sakuma et al. describes a second antiretroviral activity of rhesus macaque TRIM5α, which blocks HIV-1 production through rapid degradation of HIV-1 Gag polyproteins. Here, we find that human TRIM5α limits HIV-1 production. Transient expression of TRIM5α decreased HIV-1 production, whereas knockdown of TRIM5α in human cells increased virion release. A single amino acid substitution (R437C) in the SPRY domain diminished the restriction effect. Moderate levels of human wild-type TRIM5α and a little amount of R437C mutant were incorporated into HIV-1 virions. The R437C mutant also lost restriction activity against N-tropic murine leukemia virus infection. However, the corresponding R to C mutation in rhesus macaque TRIM5α had no effect on the restriction ability. Our findings suggest human TRIM5α is an intrinsic immunity factor against HIV-1 infection. The importance of arginine at 437 aa in SPRY domain for the late restriction is species-specific.     

HIV-1 but not SIVmac239 induces higher interferon-α antiviral state in chronic infected northern pig-tailed macaques (Macaca leonina)

Studies have shown that interferon (IFN)-α has an inhibitory effect on human immunodeficiency virus type 1 (HIV-1) replication in the acute infection stage, but its role in chronic infection is still unclear. We previously established a nonpathogenic HIV-1 and pathogenic simian immunodeficiency virus (SIV) model in northern pig-tailed macaques (NPMs, Macaca leonina). In the current study, we detected viral RNA and DNA in various tissues (axillary lymph nodes (LNs), inguinal LNs, and spleen) in HIV-1_NL4-3- and SIV_mac239-infected NPM during the chronic stage of infection. Results indicated that the levels of viral DNA and RNA were higher in the tested tissues (LNs and spleen) of the SIV_mac239-infected NPMs than in the HIV-1_NL4-3 infected NPMs. Furthermore, IFN-α expression was higher in the HIV-infected tissues than in the SIV-infected controls. The HIV restriction factors induced by IFN-α (i.e., tetherin and MX2), as well as inflammatory factors IFN-γ, tumor necrosis factor-α (TNF-α), and interleukin 6 (IL-6), were analyzed using real-time polymerase chain reaction (PCR) and immunofluorescence staining assays. Results showed that their expression levels were much higher in the HIV-infected tissues than in the SIV-infected controls. These findings were confirmed by in vitro experiments on healthy NPM peripheral blood mononuclear cells infected with HIV-1_NL4-3, which showed lower viral replication, higher IFN-α expression, and an antiviral status. This study demonstrated that HIV-1 infection, but not SIV_mac239 infection, in NPMs caused higher expression of IFN-α and induced a higher antiviral status. This may be one of the reasons why HIV-1 cannot replicate at a high level or develop into AIDS in NPMs.     

Gag-CA Q110D mutation elicits TRIM5-independent enhancement of HIV-1mt replication in macaque cells

HIV-1 is strictly adapted to humans, and cause disease-inducing persistent infection only in humans. We have generated a series of macaque-tropic HIV-1 (HIV-1mt) to establish non-human primate models for basic and clinical studies. HIV-1mt clones available to date grow poorly in macaque cells relative to SIVmac239. In this study, viral adaptive mutation in macaque cells, G114E in capsid (CA) helix 6 of HIV-1mt, that enhances viral replication was identified. Computer-assisted structural analysis predicted that another Q110D mutation in CA helix 6 would also increase viral growth potential. A new proviral construct MN4Rh-3 carrying CA-Q110D exhibited exquisitely enhanced growth property specifically in macaque cells. Susceptibility of MN4Rh-3 to macaque TRIM5α/TRIMCyp proteins was examined by their expression systems. HIV-1mt clones so far constructed already completely evaded TRIMCyp restriction, and further enhancement of TRIMCyp resistance by Q110D was not observed. In addition, Q110D did not contribute to evasion from TRIM5α restriction. However, the single-cycle infectivity of MN4Rh-3 in macaque cells was enhanced relative to the other HIV-1mt clones. Our results here indicate that CA-Q110D accelerates viral growth in macaque cells irrelevant to TRIM5 proteins restriction.     

TRIM79α, an interferon-stimulated gene product, restricts tick-borne encephalitis virus replication by degrading the viral RNA polymerase

In response to virus infection, type I interferons (IFNs) induce several genes, most of whose functions are largely unknown. Here, we show that the tripartite motif (TRIM) protein, TRIM79α, is an IFN-stimulated gene (ISG) product that specifically targets tick-borne encephalitis virus (TBEV), a Flavivirus that causes encephalitides in humans. TRIM79α restricts TBEV replication by mediating lysosome-dependent degradation of the flavivirus NS5 protein, an RNA-dependent RNA polymerase essential for virus replication. NS5 degradation was specific to tick-borne flaviviruses, as TRIM79α did not recognize NS5 from West Nile virus (WNV) or inhibit WNV replication. In the absence of TRIM79α, IFN-β was less effective in inhibiting tick-borne flavivirus infection of mouse macrophages, highlighting the importance of a single virus-specific ISG in establishing an antiviral state. The specificity of TRIM79α for TBEV reveals?a remarkable ability of the innate IFN response to discriminate between closely related flaviviruses.     

Ribavirin enhances IFN-α signalling and MxA expression: a novel immune modulation mechanism during treatment of HCV

The nucleoside analogue Ribavirin significantly increases patient response to IFN-α treatment of HCV, by directly inhibiting viral replication. Recent studies indicate that Ribavirin also regulates immunity and we propose that Ribavirin enhances specific interferon sensitive gene (ISG) expression by amplifying the IFN-α-JAK/STAT pathway. We found that IFN-α-induced STAT1 and STAT3 phosphorylation was increased in hepatocytes co-treated with Ribavirin and IFN-α, compared to IFN-α alone. Ribavirin specifically enhanced IFN-α induced mRNA and protein of the anti-viral mediator MxA, which co-localised with HCV core protein. These novel findings indicate for the first time that Ribavirin, in addition to its viral incorporation, also enhances IFN-α-JAK/STAT signalling, leading to a novel MxA-mediated immuno-modulatory mechanism that may enhance IFN-α anti-viral activity against HCV.     

Genetic association of the antiviral restriction factor TRIM5alpha with human immunodeficiency virus type 1 infection

The innate antiviral factor TRIM5alpha restricts the replication of some retroviruses through its interaction with the viral capsid protein, leading to abortive infection. While overexpression of human TRIM5alpha results in modest restriction of human immunodeficiency virus type 1 (HIV-1), this inhibition is insufficient to block productive infection of human cells. We hypothesized that polymorphisms within TRIM5 may result in increased restriction of HIV-1 infection. We sequenced the TRIM5 gene (excluding exon 5) and the 4.8-kb 5' putative regulatory region in genomic DNA from 110 HIV-1-infected subjects and 96 exposed seronegative persons, along with targeted gene sequencing in a further 30 HIV-1-infected individuals. Forty-eight single nucleotide polymorphisms (SNPs), including 20 with allele frequencies of >1.0%, were identified. Among these were two synonymous and eight nonsynonymous coding polymorphisms. We observed no association between TRIM5 polymorphism in HIV-1-infected subjects and their set-point viral load after acute infection, although one TRIM5 haplotype was weakly associated with more rapid CD4(+) T-cell loss. Importantly, a TRIM5 haplotype containing the nonsynonymous SNP R136Q showed increased frequency among HIV-1-infected subjects relative to exposed seronegative persons, with an odds ratio of 5.49 (95% confidence interval = 1.83 to 16.45; P = 0.002). Nonetheless, we observed no effect of individual TRIM5alpha nonsynonymous mutations on the in vitro HIV-1 susceptibility of CD4(+) T cells. Therefore, any effect of TRIM5alpha polymorphism on HIV-1 infection in primary lymphocytes may depend on combinations of SNPs or on DNA sequences in linkage disequilibrium with the TRIM5alpha coding sequence.     

Interferon-α and -γ Differentially Reduce Rapid Immature T-Cell Death by Contact with HIV-1 Carrier Cell Clones In Vitro

The non-antigen specific rapid cytotoxic (CT) death of immature TdT⁺CD4⁺CD8⁺ T cells due to contact with HIV-1 carrier T-cell clones we have found recently is a novel phenomenon. The effects of interferons (IFN) on this CT reaction were studied in vitro. Treatment of the HIV-1 carrier clones, referred to as “effectors,” with IFN-α but not IFN-γ, or of the susceptible immature TdT⁺CD4⁺CD8⁺ T cells, referred to as “targets,” with IFN-γ but not IFN-α, for 24 hr prior to CT testing was found to reduce the CT reaction. Simultaneously, a down-regulated CD8 expression and an up-regulated antigen expression of both major histocompatibility antigen complex class I (MHC-I) and HIV-1 gp120/gp160 in the IFN-α treated effector (gp120⁺CD8⁺ HPB-ALL/HIV), and/or simultaneously up-regulated antigen expression of both CD8 and MHC-I in the IFN-γ treated target (CD4⁺CD8⁺ HPB-ALL) were found to be associated with reduced CT reaction. However, altered antigen expression in the IFN-γ treated effectors or IFN-α treated targets did not affect the ultimate degree of CT reaction. This study thus suggests a possible therapeutic efficacy of IFN by reducing the direct elimination of the T-cell precursors in HIV-1 infection.