Role for nsP2 Proteins in the Cessation of Alphavirus Minus-Strand Synthesis by Host Cells

Abstract

In order to establish nonlytic persistent infections (PI) of BHK cells, replicons derived from Sindbis (SIN) and Semliki Forest (SFV) viruses have mutations in nsP2. Five different nsP2 PI replicons were compared to wild-type (wt) SIN, SFV, and wt nsPs SIN replicons. Replicon PI BHK21 cells had viral RNA synthesis rates that were less than 5% of those of the wt virus and ϳ10% or less of those of SIN wt replicon-infected cells, and, in contrast to wt virus and replicons containing wt nsP2, all showed a phenotype of continuous minus-strand synthesis and of unstable, mature replication/transcription complexes (RC؉) that are active in plus-strand synthesis. Minus-strand synthesis and incorporation of [ 3 H]uridine into replicative intermediates differed among PI replicons, depending on the location of the mutation in nsP2. Minus-strand synthesis by PI cells appeared normal; it was dependent on continuous P123 and P1234 polyprotein synthesis and ceased when protein synthesis was inhibited. The failure by the PI replicons to shut off minus-strand synthesis was not due to some defect in the PI cells but rather was due to the loss of some function in the mutated nsP2. This was demonstrated by showing that superinfection of PI cells with wt SFV triggered the shutdown of minus-strand synthesis, which we believe is a host response to infection with alphaviruses. Together, the results indicate alphavirus nsP2 functions to engage the host response to infection and activate a switch from the early-to-late phase. The loss of this function leads to continuous viral minus-strand synthesis and the production of unstable RC؉.The alphaviruses Sindbis (SIN) and Semliki Forest (SFV) are plus-stranded RNA viruses whose greater than 40S (11.7 kb) genomes encode four nonstructural proteins (nsP1-4), numbered according to their gene order (reviewed in reference 55), that are the essential components of the viral replicases and transcriptase. They are synthesized initially as polyproteins P1234 and P123, the former by readthrough of an opal termination codon between the nsP3 and nsP4 genes (54). The replicases involved in minus-strand (P123, P23) and genome (P23 or fully cleaved nsPs) synthesis contain uncleaved nsP2-containing polyproteins (32,33,52,59). The transcriptase makes a subgenomic 26S mRNA that encodes the viral structural proteins. Capping of viral genome and 26S plus strands employs methyltransferase and guanylyltransferase activities present within the nsP1 protein (2-4, 23, 35, 51, 58) and 5Ј-triphosphatase activity resident in the N domain of nsP2 (57). The nsP1 protein also is involved in the initiation of minusstrand synthesis (21, 50, 53, 60) and interacts with nsP4 (12, 53), which is the RNA-dependent RNA polymerase and also affects host cell-dependent replication (11,33). The N half to two-thirds of the nsP3 phosphoprotein is conserved among alphaviruses and provides essential functions for minus-strand and 26S mRNA syntheses (5,26,29,30,38,59); it also includes a conserved macrohistone 2A-like sequence predicted to b...

A New Role for ns Polyprotein Cleavage in Sindbis Virus Replication

“…It has been found that nsP2 expresses functions that are involved in the regulation of the host response to infection (6,9,11,26,32). One of its regulatory activities might be an overall cessation of negative-strand synthesis that is observed at 4 to 8 h p.i., depending on the cell type (33,35).…”

“…Moreover, like the nsP2 proteins of other alphaviruses (25), SINV nsP2 is coisolated with ribosomes from infected cells (1), but its functional role in these complexes is unknown. Specific amino acid substitutions in SINV and SFV nsP2 strongly affected the ability of the protein to modify the intracellular environment (6,26,32); the mutations altering the carboxy-terminal fragment of nsP2 made viruses attenuated in vivo and incapable of inhibiting host cell transcription and/or translation (9,13,14). Thus, accumulated data suggest that the processing of SINV and SFV nsP precursors plays a role not only in the temporal regulation of negative-and positive-strand viral RNA syntheses by the RC but also in the modification of the cellular response to infection.…”

Genome-Scale Phylogeny of the Alphavirus Genus Suggests a Marine Origin

“…The four nonstructural proteins produced, nsP1 to nsP4, are involved in RNA replication and modification and in proteolytic cleavage. A leaky opal stop codon near the 3= end of the nsP3 gene is present in the genomes of most but not all alphaviruses (42,51), such that two products, P123 and P1234, are produced during translation (63,71). The second polyprotein encodes the structural proteins, including the capsid protein, two major envelope proteins (E2 and E1), and two smaller structural proteins not usually found in virions (23,71).…”

Mobilization of Full-Length Semliki Forest Virus Replicon by Retrovirus Particles

“…This is useful in eliminating a target cell or in triggering an immune response (11,18). The use of noncytotoxic SFV vectors would lead to long-lasting but weak expression (39). Alternatively, cells that do not support SFV replication exist, and provided that they allow a high retroviral budding, the use of a Pol II-dependent expression system for a ⌿ MLV SFV vector (5,18,34) should promote the efficient formation of retrovirus particles containing the full-length vector.…”