Proteomics Analysis of the Tombusvirus Replicase: Hsp70 Molecular Chaperone Is Associated with the Replicase and Enhances Viral RNA Replication

Abstract

Viral replicases are the key enzymes in the replication of plus-stranded RNA viruses (1, 7). The viral replicase complexes, which are assembled on intracellular membranes, consist of virus-encoded proteins, such as the RNA-dependent RNA polymerase (RdRp) and auxiliary viral protein(s), as well as host-derived proteins and the viral RNA template (2, 19). Studies with a small number of plant RNA viruses identified several host factors which are likely involved in the targeting of the viral replication proteins to intracellular compartments and replicase assembly and/or function. For example, subunit 45 of eukaryotic initiation factor 3 was found in association with brome mosaic virus (BMV) RdRp (35), while subunit 56 of eukaryotic initiation factor 3 was detected in a tobacco mosaic virus replicase preparation (25). Moreover, a molecular chaperone (Ydj1p) is known to be essential for the activation of the BMV replicase (45). TOM1 and TOM3 integral membrane proteins of Arabidopsis were found to interact with the tobacco mosaic virus replicase proteins and cofractionated with the RdRp activity (46, 53).Host factors interacting with and/or affecting viral replicase activities have also been identified in animal plus-stranded RNA viruses. For example, hepatitis C virus (HCV) RdRp is known to bind to nucleolin (an RNA binding protein) and initiation factor 4A (11,15,20). The HCV NS5A and NS5B replication proteins interacted with hVAP-33, a SNARE-like protein bound to cellular membranes (47). hVAP-33 has been suggested to serve as a docking site to anchor the HCV RdRp to the membrane during assembly of the replicase complex (12, 47). Also, host chaperones are involved in proteolytic processing of the HCV nonstructural proteins (44). The Hsp90 molecular chaperone has been shown to enhance Flock House virus replication (18). Poly(C) and poly(A) binding proteins were found to interact with both the poliovirus RNA and the viral polymerase precursor 3CD (14, 49). Nucleolin and Sam68, which are involved in virus replication, are relocalized in the cell during poliovirus infection (21, 48). In addition, the cellular COPII proteins are involved in the assembly of the poliovirus replication complex (39). hnRNP A1 was found to be part of the mouse hepatitis virus transcription/replication complex, and it is involved in subgenomic RNA synthesis and genome replication (42,50). Overall, the above examples illustrate that host factors play complex and significant roles in the replication of many plus-stranded RNA viruses.Tomato bushy stunt virus (TBSV) and the closely related cucumber necrosis virus (CNV) are nonsegmented plusstranded viruses of plants. Among the five TBSV-encoded proteins, only p33 and p92 are required for replication (26,30,40,52), whereas the other proteins are involved in cell-to-cell movement, encapsidation, and suppression of gene silencing (52). The p92 replication protein has the RdRp signature motifs in its unique C terminus, whereas the auxiliary p33, which overlaps with the N-terminal sequence of p92, play...

Membrane-Bound Tomato Mosaic Virus Replication Proteins Participate in RNA Synthesis and Are Associated with Host Proteins in a Pattern Distinct from Those That Are Not Membrane Bound

“…These features resemble those reported for the replication complexes of eukaryotic (ϩ)RNA viruses including Brome mosaic virus (19,23,25). Replication complexes of Brome mosaic virus are formed in spherular invaginations of the ER membranes or between appressed double membranes (26), which is consistent with the detergent-dependent nuclease sensitivity of the (Ϫ)RNA. The morphology of tobamovirus replication complexes remains to be determined.…”

“…One of the possible interpretations of these observations is that the membrane-bound replication proteins have conformational and other physical properties that are distinct from those of the non-membrane-bound replication proteins. The putative conformational differentiation of ToMV (15,26,27,30) and suggests that the above process is mediated by chaperones.…”

Inhibition of Sterol Biosynthesis Reduces Tombusvirus Replication in Yeast and Plants

“…Total RNA isolation from yeast and Northern blot analyses of the accumulation of TBSV repRNA were performed as described previously (37,41). Western blotting for measuring p33/p92 levels was performed using anti-His antibody, whereas the secondary antibody was alkaline phosphatase-conjugated anti-mouse immunoglobulin G (Sigma), as described previously (49).…”

Identification of Essential Host Factors Affecting Tombusvirus RNA Replication Based on the Yeast Tet Promoters Hughes Collection

“…The identified host genes among the essential genes represent a higher ratio (3.75%) than among the nonessential genes (2%), suggesting that tombusviruses might have adapted to use and/or depend on essential genes to higher extent than nonessential genes. Altogether, this number of host genes is probably an underestimation, because genetic screens frequently overlook redundant genes (such as gene families) with similar functions whose deletion/down-regulation could be compensated for by other genes (39). Nevertheless, the identified host genes represent a diverse set of host genes shown to affect viral RNA replication, and thus they should be valuable for studies on the mechanism of tombusvirus replication.…”

“…Earlier work defined that p33 is involved in template selection and recruitment of viral RNA into replication (18,25,32). These proteins interact with each other, the viral RNA, and the host proteins in cells (25,34,35,39), which leads to the assembly of RC on peroxisomal membranes (22, 25). The CNV replication proteins can support the replication of TBSV defective interfering (DI) RNA, a small deletion derivative of the genomic RNA, as efficiently as TBSV replication proteins can (19,24).…”