Tracking and Elucidating Alphavirus -Host Protein Interactions

Laboratory of Virology and Infectious Disease, The Rockefeller University, New York, New York, United States
Journal of Biological Chemistry (Impact Factor: 4.57). 11/2006; 281(40):30269-78. DOI: 10.1074/jbc.M603980200
Source: PubMed


Viral infections cause profound alterations in host cells. Here, we explore the interactions between proteins of the Alphavirus Sindbis and host factors during the course of mammalian cell infection. Using a mutant virus expressing the viral nsP3 protein
tagged with green fluorescent protein (GFP) we directly observed nsP3 localization and isolated nsP3-interacting proteins
at various times after infection. These results revealed that host factor recruitment to nsP3-containing complexes was time
dependent, with a specific early and persistent recruitment of G3BP and a later recruitment of 14-3-3 proteins. Expression
of GFP-tagged G3BP allowed reciprocal isolation of nsP3 in Sindbis infected cells, as well as the identification of novel
G3BP-interacting proteins in both uninfected and infected cells. Note-worthy interactions include nuclear pore complex components
whose interactions with G3BP were reduced upon Sindbis infection. This suggests that G3BP is a nuclear transport factor, as
hypothesized previously, and that viral infection may alter RNA transport. Immunoelectron microscopy showed that a portion
of Sindbis nsP3 is localized at the nuclear envelope, suggesting a possible site of G3BP recruitment to nsP3-containing complexes.
Our results demonstrate the utility of using a standard GFP tag to both track viral protein localization and elucidate specific
viral-host interactions over time in infected mammalian cells.

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    • "To determine the susceptibility of L. dispar cells to RNA virus infection, we challenged LD652 cells with recombinant strains of VSV and SINV that express either green fluorescent protein (GFP) or luciferase (LUC) from viral promoters. In single infections with VSV-GFP (Kato et al., 2005) or SINV-GFP (Cristea et al., 2006), we found that, even at a high multiplicity of infection (MOI) of 10, <4% of LD652 cells exhibited GFP fluorescence by 96 hr post-infection (hpi) (Figure 1A,B). A previous report indicated that VACV enters LD652 cells and reaches the stage of late gene expression but ultimately fails to complete virion morphogenesis (Li et al., 1998). "
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    • "However, since we have also found that the efficient translation of viral RNAs in the normally restrictive milieu of a cell containing high levels of phospho-eIF2α can itself contribute to the disruption of SG, it might be worth considering that such a passive mechanism might operate in other viral infections also. G3BP-1 has been shown to form a complex with alphaviral nsP3 proteins (Cristea et al., 2006; Frolova et al., 2006; Gorchakov et al., 2008; Fros et al., 2012) but also with nsP2 (Atasheva et al., 2007) and nsP4 (Cristea et al., 2010), whereas other studies of the host protein components of the alphaviral RCs failed to detect this (Bourai et al., 2012). Our results show that the formation of a complex with G3BP is dependent only on the C-terminal seven–amino acid repeat motifs of nsP3. "
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    ABSTRACT: Dynamic, mRNA-containing stress granules (SGs) form in the cytoplasm of cells under environmental stresses including viral infection. Many viruses appear to employ mechanisms to disrupt the formation of SGs on their mRNAs, suggesting that they represent a cellular defence against infection. Here, we report that early in Semliki Forest virus infection, the C-terminal domain of the viral non-structural protein 3 (nsP3) forms a complex with Ras-GAP SH3-domain binding protein (G3BP) and sequesters it into viral RNA replication complexes in a manner that inhibits the formation of SGs on viral mRNAs. A viral mutant carrying a C-terminal truncation of nsP3 induces more persistent SGs and is attenuated for propagation in cell culture. Importantly, we also show that the efficient translation of viral mRNAs containing a translation enhancer sequence also contributes to the disassembly of SGs in infected cells. Further, we show that the nsP3/G3BP interaction also blocks SGs induced by other stresses than virus infection. This is one of few described viral mechanisms for SG disruption and underlines the role of SGs in anti-viral defence.
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    • "Recent work by Burnham et al. [27] has demonstrated that the host protein hnRNP K interacts with nsP2 and SG RNA in SV-infected cells. Several reports [17,28,29] have identified other cellular proteins associated with the SV replicase/transcriptase. These include cytoskeleton proteins, ribosomal subunits, chaperones, and hnRNPs. "
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    ABSTRACT: Sindbis virus (SV) is the prototype of alphaviruses which are a group of widely distributed human and animal pathogens. Heterogeneous nuclear ribonucleoprotein (hnRNP) A1 is an RNA-binding protein that shuttles between the nucleus and the cytoplasm. Our recent studies found that hnRNP A1 relocates from nucleus to cytoplasm in Sindbis virus (SV)-infected cells. hnRNP A1 binds to the 5' UTR of SV RNA and facilitates the viral RNA replication and translation. Making use of standard molecular techniques, virology methods and an in vitro system developed by our lab to assess the role of hnRNP A1 in SV positive strand RNA synthesis. hnRNP A1 interacted with the genomic (G) and subgenomic (SG) RNA promoters. Knockdown of hnRNP A1 resulted in markedly decrease in the synthesis of G and SG RNA both in infected cells and in vitro. Our study provides the first direct evidence that hnRNP A1 actively participates in viral RNA replication and is required for the synthesis of G and SG RNA.
    Full-text · Article · Jul 2010 · Journal of Biomedical Science
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