The interferon (IFN) system is a major effector of the innate immunity that allows time for the subsequent establishment of an adaptive immune response against a wide-range of pathogens. Their diverse biological actions are thought to be mediated by the products of specific but usually overlapping sets of cellular genes induced in the target cells. Ubiquitin ligase members of the tripartite motif (TRIM) protein family have emerged as IFN-induced proteins involved in both innate and adaptive immunity. In this report, we provide evidence that TRIM22 is a functional E3 ubiquitin ligase that is also ubiquitinated itself. We demonstrate that TRIM22 expression leads to a viral protection of HeLa cells against encephalomyocarditis virus infections. This effect is dependent upon its E3 ubiquitinating activity, since no antiviral effect was observed in cells expressing a TRIM22-deletion mutant defective in ubiquitinating activity. Consistent with this, TRIM22 interacts with the viral 3C protease (3C PRO ) and mediates its ubiquitination. Altogether, our findings demonstrate that TRIM22 E3 ubiquitin ligase activity represents a new antiviral pathway induced by IFN against picornaviruses. INTRODUCTIONPicornaviruses include important human pathogens such as rhinovirus, poliovirus and hepatitis A virus, as well as significant insect, plant and agricultural pathogens, such as foot-and-mouth disease virus (Whitton et al., 2005). Encephalomyocarditis virus (EMCV) is the prototype of the cardiovirus subgroup of picornaviruses. Its genome consists of a single-strand of messenger-sense RNA. Viral proteins are expressed from a large open reading frame encoding a giant precursor polyprotein (approx. 255 kDa), which is processed through a series of proteolytic cleavages to produce both capsid and non-structural proteins (Palmenberg, 1990). The majority of the processing reactions are carried out by the 3C protease (3C PRO ), which acts both inter-and intramolecularly to produce polyprotein precursors as well as the mature 3C PRO polypeptide (Palmenberg et al., 1979). Cleavage of the polyprotein normally occurs between glutamine-glycine or glutamine-serine amino acid pairs, which are usually flanked by proline residues (Palmenberg et al., 1984). Besides its commitment to the processing of the viral polyprotein, picornaviral 3C PRO can also facilitate virus replication by altering fundamental cellular processes such as transcription and translation of host cell mRNA (Barral et al., 2007;Ehrenfeld, 1982;Kuyumcu-Martinez et al., 2004; Neznanov et al., 2005;Shen et al., 1996;Yalamanchili et al., 1996Yalamanchili et al., , 1997. Because the regulated proteolysis mediated by 3C PRO is a critical feature in both the processing of picornaviral polyprotein and the inhibition of cellular defences, 3C PRO constitutes an important target for host antiviral innate pathways. Consistent with this, the degradation of EMCV 3C PRO by the ubiquitin/26S proteasome system has been reported (Losick et al., 2003;Schlax et al., 2007). Although the precise ro...
The human immunodeficiency virus type 1 (HIV-1) Vpr protein binds to the cellular uracil–DNA glycosylase UNG2 and induces its degradation through the assembly with the DDB1-CUL4 ubiquitin ligase complex. This interaction counteracts the antiviral activity exerted by UNG2 on HIV-1 gene transcription, as previously reported by us. In this work, we show that Vpr expression in the context of HIV-1 infection markedly decreases UNG2 expression in transformed or primary CD4+ T lymphocytes. We demonstrate for the first time that Vpr-UNG2 interaction significantly impairs the uracil excision activity of infected cells. The loss of uracil excision activity coincides with a significant accumulation of uracilated bases in the genome of infected cells without changes in cell division. Although UNG2 expression and uracil–DNA glycosylase activity are recovered after the peak of retroviral replication, the mutagenic effect of transient DNA uracilation in cycling cells should be taken into account. Therefore, the possible consequences of Vpr-mediated temporary depletion of endogenous nuclear UNG2 and subsequent alteration of the genomic integrity of infected cells need to be evaluated in the physiopathogenesis of HIV infection.
Beyond their role in cellular RNA metabolism, DExD/H-box RNA helicases are hijacked by various RNA viruses in order to assist replication of the viral genome. Here, we identify the DExH-box RNA helicase 9 (DHX9) as a binding partner of chikungunya virus (CHIKV) nsP3 mainly interacting with the C-terminal hypervariable domain. We show that during early CHIKV infection, DHX9 is recruited to the plasma membrane, where it associates with replication complexes. At a later stage of infection, DHX9 is, however, degraded through a proteasome-dependent mechanism. Using silencing experiments, we demonstrate that while DHX9 negatively controls viral RNA synthesis, it is also required for optimal mature nonstructural protein translation. Altogether, this study identifies DHX9 as a novel cofactor for CHIKV replication in human cells that differently regulates the various steps of CHIKV life cycle and may therefore mediate a switch in RNA usage from translation to replication during the earliest steps of CHIKV replication. IMPORTANCE The reemergence of chikungunya virus (CHIKV), an alphavirus that is transmitted to humans by Aedes mosquitoes, is a serious global health threat. In the absence of effective antiviral drugs, CHIKV infection has a significant impact on human health, with chronic arthritis being one of the most serious complications. The molecular understanding of host-virus interactions is a prerequisite to the development of targeted therapeutics capable to interrupt viral replication and transmission. Here, we identify the host cell DHX9 DExH-Box helicase as an essential cofactor for early CHIKV genome translation. We demonstrate that CHIKV nsP3 protein acts as a key factor for DHX9 recruitment to replication complexes. Finally, we establish that DHX9 behaves as a switch that regulates the progression of the viral cycle from translation to genome replication. This study might therefore have a significant impact on the development of antiviral strategies.
SummaryIn search of key enzymes in Plasmodium phospholipid metabolism, we demonstrate the presence of a parasite-encoded phosphatidylserine decarboxylase (PSD) in the membrane fraction of Plasmodium falciparum -infected erythrocytes. PSD cDNA, encoding phosphatidylserine decarboxylase ( PfPSD ), was cloned by screening a directional cDNA library derived from the trophozoite erythrocytic stage. The corresponding PfPSD gene is located on chromosome 9 of P. falciparum , contains one intron of 938 nucleotides and is transcribed into a 3.7 kb mRNA. PfPSD cDNA encodes a putative protein of 362 amino acids, with a predicted molecular mass of 42.6 kDa, which clearly belongs to the type I PSD family. Only a 35 kDa polypeptide was detected in the parasite using a specific rabbit antiserum. PfPSD has a 314VGSS317 sequence near its carboxyl-terminus that is related to the Escherichia coli , yeast and human LGST motif, which is the site of proenzyme processing. PSD enzyme was expressed in E. coli with a K M of 63 ± ± ± ± 19 m m m m M and a V MAX of 680 ± ± ± ± 49 nmol of phosphatidylethanolamine formed h ----1 mg ----1 protein. Sitedirected mutagenesis of the VGSS active site demonstrated that the PfPSD proenzyme was processed into two non-identical subunits ( a a a a and b b b b ) and revealed the crucial role played by each residue in enzyme processing and activity. Using indirect immunofluorescence, PfPSD labelling was co-localized with an endoplasmic reticulum marker, but not with a mitochondrial vital dye. This P. falciparum PSD is the first type I PSD identified in the endoplasmic reticulum compartment.
We have previously shown that ISG20, an interferon (IFN)-induced gene, encodes a 3' to 5' exoribonuclease member of the DEDD superfamily of exonucleases. ISG20 specifically degrades single-stranded RNA. In this report, using immunofluorescence analysis, we demonstrate that in addition to a diffuse cytoplasmic and nucleoplasmic localization, the endogenous ISG20 protein was present in the nucleus both in the nucleolus and in the Cajal bodies (CBs). In addition, we show that the ectopic expression of the CBs signature protein, coilin, fused to the red fluorescent protein (coilin-dsRed) increased the number of nuclear dots containing both ISG20 and coilin-dsRed. Using electron microcopy analysis, ISG20 appeared principally concentrated in the dense fibrillar component of the nucleolus, the major site for rRNA processing. We also present evidences that ISG20 was associated with survival of motor neuron (SMN)-containing macromolecular nuclear complexes required for the biogenesis of various small nuclear ribonucleoproteins. Finally, we demonstrate that ISG20 was associated with U1 and U2 snRNAs, and U3 snoRNA. The accumulation of ISG20 in the CBs after IFN treatment strongly suggests its involvement in a new route for IFN-mediated inhibition of protein synthesis by modulating snRNA and rRNA maturation.
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