Synthesis of the Gag-Pol protein of the human immunodeficiency virus type 1 (HIV-1) requires a programmed -1 ribosomal frameshifting when ribosomes translate the unspliced viral messenger RNA. This frameshift occurs at a slippery sequence followed by an RNA structure motif that stimulates frameshifting. This motif is commonly assumed to be a simple stem-loop for HIV-1. In this study, we show that the frameshift stimulatory signal is more complex than believed and consists of a two-stem helix. The upper stem-loop corresponds to the classic stem-loop, and the lower stem is formed by pairing the spacer region following the slippery sequence and preceding this classic stem-loop with a segment downstream of this stem-loop. A three-purine bulge interrupts the two stems. This structure was suggested by enzymatic probing with nuclease V1 of an RNA fragment corresponding to the gag/pol frameshift region of HIV-1. The involvement of the novel lower stem in frameshifting was supported by site-directed mutagenesis. A fragment encompassing the gag/pol frameshift region of HIV-1 was inserted in the beginning of the coding sequence of a reporter gene coding for the firefly luciferase, such that expression of luciferase requires a -1 frameshift. When the reporter was expressed in COS cells, mutations that disrupt the capacity to form the lower stem reduced frameshifting, whereas compensatory changes that allow re-formation of this stem restored the frameshift efficiency near wild-type level. The two-stem structure that we propose for the frameshift stimulatory signal of HIV-1 differs from the RNA triple helix structure recently proposed.

IL-10 is a potent anti-inflammatory cytokine interfering with antigen presentation by inducing the intracellular sequestration of MHC class II (MHC-II) molecules. Here we studied the contribution of membrane-associated RING-CH (MARCH) ubiquitin ligase family members to the IL-10-induced down-regulation of MHC-II molecules. We found that MARCH1 and MARCH8 proteins are the most potent family members for the downregulation of MHC-II surface expression in transfected cells, but only MARCH1 mRNA expression is strongly induced by IL-10 in human primary monocytes. We detected monoand poly-ubiquitinated forms of MHC-II molecules both in IL-10-treated monocytes and in cells transfected with MARCH1. We also show direct interaction between MHC-II and MARCH1 molecules in co-immunoprecipitation assays. Finally, we found that siRNAmediated knockdown of MARCH1 reverses IL-10-induced MHC-II down-regulation in primary monocytes. Thus, the immunosuppressive effect of IL-10 on antigen presentation is mediated through induced expression of MARCH1.

The mitochondrial antiviral signaling (MAVS) protein plays a central role in innate antiviral immunity. Upon recognition of a virus, intracellular receptors of the RIG-I-like helicase family interact with MAVS to trigger a signaling cascade. In this study, we investigate the requirement of the MAVS structure for enabling its signaling by structure-function analyses and resonance energy transfer approaches in live cells. We now report the essential role of the MAVS oligomer in signal transduction and map the transmembrane domain as the main determinant of dimerization. A combination of mutagenesis and computational methods identified a cluster of residues making favorable van der Waals interactions at the MAVS dimer interface. We also correlated the activation of IRF3 and NF-B with MAVS oligomerization rather than its mitochondrial localization. Finally, we demonstrated that MAVS oligomerization is disrupted upon expression of HCV NS3/4A protease, suggesting a mechanism for the loss of antiviral signaling. Altogether, our data suggest that the MAVS oligomer is essential in the formation of a multiprotein membrane-associated signaling complex and enables downstream activation of IRF3 and NF-B in antiviral innate immunity.Viral nucleic acids, potent inducers of the antiviral innate immune response, are recognized at the extracellular level by a subset of endosomal Toll-like receptors and, upon permissive virus infection, at the cytoplasmic level by a family of DexD/H box RNA helicases including RIG-I (retinoic acid inducible gene I) and MDA5 (melanoma differentiation-associated gene 5) (26).The sensor protein RIG-I is believed to be maintained in an auto-inhibited state in resting cells and to undergo a conformational change upon viral RNA binding. This conformational change exposes two N-terminal caspase activation and recruitment domains (CARDs) (36), induces cytoplasmic oligomerization (31), and promotes interaction with mitochondrial antiviral signaling (MAVS) protein (also known as IPS-1, Cardif, and VISA). The interaction between RIG-I and MAVS occurs through the protein-interacting CARDs of both proteins (17,25,32,34) and initiates formation of an as-yet-undefined macromolecular signaling complex to the mitochondrial membrane. Formation of this complex involves the recruitment of multiple signaling components to activate interferon (IFN) regulatory factor 3 (IRF3) and nuclear factor-B (NF-B) transcription factors that are required for production of type-I IFNs (reviewed in reference 26). The RIG-I/MAVS pathway plays an important role in the antiviral host response to hepatitis C virus (HCV) infection, where the uncapped viral RNA of HCV triggers RIG-I signaling (33). However, HCV can counteract the antiviral response; its NS3/4A serine protease cleaves MAVS at cysteine 508, resulting in the loss of mitochondrial localization and the abrogation of signaling function (9,19,20,25). This mechanism has been confirmed to occur in infected cells (22).Here, we applied fluorescence and bioluminescence resonance energ...

The hepatitis C virus (HCV) genome contains an internal ribosome entry site (IRES) followed by a large open reading frame coding for a polyprotein that is cleaved into 10 proteins. An additional HCV protein, the F protein, was recently suggested to result from a +1 frameshift by a minority of ribosomes that initiated translation at the HCV AUG initiator codon of the polyprotein. In the present study, we reassessed the mechanism accounting for the synthesis of the F protein by measuring the expression in cultured cells of a luciferase reporter gene with an insertion encompassing the IRES plus the beginning of the HCV-coding region preceding the luciferase-coding sequence. The insertion was such that luciferase expression was either in the +1 reading frame relative to the HCV AUG initiator codon, mimicking the expression of the F protein, or in-frame with this AUG, mimicking the expression of the polyprotein. Introduction of a stop codon at various positions in-frame with the AUG initiator codon and substitution of this AUG with UAC inhibited luciferase expression in the 0 reading frame but not in the +1 reading frame, ruling out that the synthesis of the F protein results from a +1 frameshift. Introduction of a stop codon at various positions in the +1 reading frame identified the codon overlapping codon 26 of the polyprotein in the +1 reading frame as the translation start site for the F protein. This codon 26(+1) is either GUG or GCG in the viral variants. Expression of the F protein strongly increased when codon 26(+1) was replaced with AUG, or when its context was mutated into an optimal Kozak context, but was severely decreased in the presence of low concentrations of edeine. These observations are consistent with a Met-tRNAi-dependent initiation of translation at a non-AUG codon for the synthesis of the F protein.