Characterization of the Nucleoside Triphosphate Phosphohydrolase and Helicase Activities of the Reovirus λ1 Protein

Abstract

Previous studies have shown that the reovirus 1 core protein harbors a putative nucleotide-binding motif and exhibits an affinity for nucleic acids. In addition, a nucleoside triphosphate phosphohydrolase activity present in reovirus cores has been recently assigned to 1 using gene reassortment analysis. In this study, it was demonstrated that the recombinant 1 protein, expressed in the yeast Pichia pastoris, is able to hydrolyze nucleoside 5-triphosphates or deoxynucleoside 5-triphosphates. This activity was absolutely dependent on the presence of a divalent cation, Mg 2؉ or Mn 2؉. The protein can also unwind double-stranded nucleic acid molecules in the presence of a nucleoside 5-triphosphate or deoxynucleoside 5-triphosphate. These results provide the first biochemical evidence that the reovirus 1 protein is a nucleoside triphosphate phosphohydrolase/helicase and strongly support the idea that 1 participates in transcription of the viral genome.Mammalian reoviruses are members of the Reoviridae family, and since their genome is made up of 10 segments of double-stranded RNA (dsRNA) 1 and replicates in the cytoplasm, they must encode their own transcriptional and replicative enzymes (1). During reovirus infection, the viral genome remains in the inner capsid (core) of the virus, composed of two major (1 and 2) and two minor (3 and 2) proteins. Gene reassortment experiments have resulted in the assignment of transcriptase activity to 3 (2). Although functions of other core proteins have not been firmly established, it is suspected that additional enzymatic functions are needed to achieve transcription and replication of the viral genome. For example, it has been postulated, by analogy with other viruses, that a helicase function could be present in the viral core (1).Nucleic acid helicases unwind double-stranded DNA and/or RNA, a process energetically coupled to the hydrolysis of nucleoside 5Ј-triphosphates (NTPs) or deoxynucleoside 5Ј-triphosphates (dNTPs) (3, 4). Helicases play a key role in nucleic acid replication, transcription, splicing, translocation, recombination, and repair (5-8). Helicases of prokaryotic, eukaryotic, and viral origins have been isolated and classified into defined superfamilies (9 -14). These proteins are characterized by seven conserved motifs designated I, Ia, and II-VI (15). Motifs I and II are very well conserved and correspond to the A and B consensus sequences of a nucleotide-binding domain (16). Superfamily II includes an expanding group of DNA and RNA helicases that harbor a DEA(D/H) sequence in motif II (17). The sequences present in motifs III-V are less strictly conserved, and their roles are not clearly defined, whereas motif VI is supposed to be involved in nucleic acid binding given its high content of positively charged amino acids (13).The 1 protein, a major component of the reovirus core, exhibits an affinity for dsRNA and dsDNA in filter binding assays and can also bind single-stranded RNA in gel retardation assays (18).2 Furthermore, analysis of gene reassortment ha...

Reovirus polymerase λ3 localized by cryo-electron microscopy of virions at a resolution of 7.6 Å

“…[18][19][20][21][22]. As suggested for bluetongue virus 49 , the reovirus core may also require a diffusion-enhanced mechanism to pump NTPs into the particle interior for maintaining the observed rate of transcript elongation.…”

“…Parker and M.L.N., unpublished data). Shell protein λ1 (142 kDa) also has a genetic influence on the NTPase activities of cores 21 and mediates NTPase, RNA 5′-triphosphatase and RNA and DNA helicase activities in vitro 22,23 . Despite these in vitro findings, the specific roles of μ2 and λ1 in viral mRNA synthesis remain unclear.…”

Structural Comparisons of Empty and Full Cytoplasmic Polyhedrosis Virus

“…Nascent mRNA is capped before being released from the intact capsid. The mechanism by which this characteristic endogenous transcription takes place inside intact dsRNA viruses has been studied extensively (3)(4)(5)(6)(7), and it is generally believed that the RNA polymerase complex is structurally anchored to the core during transcription (7)(8)(9). The dsRNA template must be flexible enough to move freely within the densely packed core so that it can slide through the RNAdependent RNA polymerase (RDRP) and the capping enzyme complex to ensure efficient transcription (10).…”

Atomic model of a cypovirus built from cryo-EM structure provides insight into the mechanism of mRNA capping

“…Previous studies have shown that the inner shell protein λ1 of orthoreovirus exhibits an affinity for both dsRNA and single-stranded RNA binding (26) and participates as a helicase during mRNA transcription (27). Sequence comparisons of our CPV with other cypoviruses and previous studies have shown that the N-terminal segment of the inner shell protein of the reovirus is the most divergent segment in the whole sequence (28,29).…”