RNA polymerase of vesicular stomatitis virus specifically associates with translation elongation factor-1 αβγ for its activity

Abstract

An RNA-dependent RNA polymerase is packaged within the virions of purified vesicular stomatitis virus, a nonsegmented negative-strand RNA virus, which carries out transcription of the genome RNA into mRNAs both in vitro and in vivo. The RNA polymerase is composed of two virally encoded polypeptides: a large protein L (240 kDa) and a phosphoprotein P (29 kDa). Recently, we obtained biologically active L protein from insect cells following infection by a recombinant baculovirus expressing L gene. During purification of the L protein from Sf21 cells, we obtained in addition to an active L fraction an inactive fraction that required uninfected insect cell extract to restore its activity. The cellular factors have now been purified, characterized, and shown to be ␤ and ␥ subunits of the protein synthesis elongation factor EF-1. We also demonstrate that the ␣ subunit of EF-1 remains tightly bound to the L protein in the inactive fraction and ␤␥ subunits associate with the L(␣) complex. Further purification of L(␣) from the inactive fraction revealed that the complex is partially active and is significantly stimulated by the addition of ␤␥ subunits purified from Sf21 cells. A putative inhibitor(s) appears to co-elute in the inactive fraction that blocked the L(␣) activity. The purified virions also package all three subunits of EF-1. These findings have a striking similarity with Q␤ RNA phage, which also associates with the bacterial homologue of EF-1 for its replicase function, implicating a possible evolutionary relationship between these host proteins and the RNA-dependent RNA polymerase of RNA viruses.Vesicular stomatitis virus (VSV), a prototype of nonsegmented negative-strand RNA viruses, has long been a paradigm for studying gene expression of this class of RNA viruses that infect vertebrates, invertebrates, and plants (1). Some of the most common human pathogens that belong to this category are rabies, measles, mumps, and human parainfluenza. A hallmark of all negative strand RNA viruses is the obligate packaging of an RNA-dependent RNA polymerase within the mature virions (2) that transcribes the genome RNA into mRNAs both in vitro and in vivo (3). For VSV, the virion-associated RNA polymerase is generally thought to consist of two virally encoded protein subunits, L (240 kDa) and P (29 kDa), which remain tightly complexed within the virion (3). Studies on the structure and function of VSV RNA polymerase have been greatly aided by the ability to isolate the polymerase subunits from the virions in a relatively pure form (4, 5). Active reconstitution of transcription is achieved by mixing the genome RNA enwrapped with the nucleocapsid protein (N) (referred to as N-RNA template) and purified L and P proteins (4, 5). From a large body of evidence, it appears that the L protein possesses the catalytic activity for RNA synthesis and the P protein is a transcription factor essential for L function (3); no cellular protein(s) has so far been shown to be required for the RNA polymerase activity. Only recently, ...

Rabies virus matrix protein regulates the balance of virus transcription and replication

“…Association of VSV L with different host cell or virus factors might affect L polymerase activity (Barik & Banerjee, 1992;Gupta & Banerjee, 1997;Das et al, 1998). Indeed, VSV P phosphorylation mutants able to support either replication or transcription of defective interfering particles (Pattnaik et al, 1997;Hwang et al, 1999) and P antibodies inhibiting replication but not transcription have been described (Richardson & Peluso, 1996).…”

“…This strongly argues in favour of a situation where the availability of N is just a prerequisite for replication and that other factors determine whether an RNP is a template for transcription or replication. These may include modifications of P and L proteins (Das et al, 1998;Pattnaik et al, 1997;Hwang et al, 1999) or involvement of other viral and cellular factors. Indeed, in the more complex negative-strand RNA virus (NSV) respiratory syncytial virus (RSV) the absence of the viral M2-2 gene product resulted in enhanced transcription and reduced replication (Bermingham & Collins, 1999).…”

Yeast genome-wide screen reveals dissimilar sets of host genes affecting replication of RNA viruses

“…2 and 3). An interesting gene in this group is TEF4, which codes for translation-elongation factor EF-1␥, which binds to the L replicase protein of vesicular stomatitis virus (21). In addition, the replicase proteins are expressed constitutively from separate plasmids by using the ADH1 promoter (PADH1), whereas the precursor for DI-72 RNA is under the control of inducible GAL1 promoter (PGAL1).…”

Immuno-characterization of the switch of peptide elongation factors eEF1A-1/EF-1α and eEF1A-2/S1 in the central nervous system during mouse development

“…For instance, eEF1A-1/EF-1α exerts pro-apoptotic functions, accelerating the cell death rate, whereas eEF1A-2/S1 protects differentiated myotubes from apoptotic cell death [3,5,15]. Among other non-canonical functions, eEF1A-1/EF-1α cleaves actin cytoskeleton and microtubules [14,16], and interacts with two nuclear proteins, RNA polymerase and zinc finger protein ZPR1, thus enabling cells to successfully cycle through the G 2 /M phase transition [4,6].…”

“…The results presented herein show definitely that eEF1A-2/S1 in vivo is only expressed in long-lived, terminally differentiated cells; similar results were obtained in skeletal muscle and heart (data not shown). The switching event is important during development, to maintain long-lived terminally differentiated neurons, cardiomyocytes, and myofibers in a non-replicating stage, protect them from apoptosis, and possibly also from debilitating effects of microtubule severing and myofiber bundling [3][4][5][6][14][15][16].…”