Increasing the Capping Efficiency of the Sindbis Virus nsP1 Protein Negatively Affects Viral Infection

Abstract

25Alphaviruses are arthropod-borne RNA viruses that are capable of causing severe 26 disease and are a significant burden to public health. Alphaviral replication results in the 27 production of both capped and noncapped viral genomic RNAs, which are packaged 28 into virions during the infections of vertebrate and invertebrate cells. However, the roles 29 that the noncapped genomic RNAs (ncgRNAs) play during alphaviral infection have yet 30 to be exhaustively characterized. Here, the importance of the ncgRNAs to alphaviral 31 infection was assessed by using mutants of the nsP1 protein of Sindbis virus (SINV), 32 which altered the synthesis of the ncgRNAs during infection by modulating the protein's 33 capping efficiency. Specifically, point mutants at residues Y286A and N376A decreased 34 capping efficiency, while a point mutant at D355A increased the capping efficiency of 35 the SINV genomic RNA during genuine viral infection. Viral growth kinetics were 36 significantly reduced for the D355A mutant relative to wild type infection, whereas the 37 Y286A and N376A mutants showed modest decreases in growth kinetics. Overall 38 genomic translation and nonstructural protein accumulation was found to correlate with 39 increases and decreases in capping efficiency. However, genomic, minus strand, and 40 subgenomic viral RNA synthesis was largely unaffected by the modulation of alphaviral 41 capping activity. In addition, translation of the subgenomic vRNA was found to be 42 unimpacted by changes in capping efficiency. The mechanism by which decreased 43 presence of ncgRNAs reduced viral growth kinetics was through the impaired 44 production of viral particles. Collectively, these data illustrate the importance of 45 ncgRNAs to viral infection and suggests that they play in integral role in the production 46 of viral progeny. 47 94 5' termini of the alphaviral genomic and subgenomic vRNAs, which provided the first 95 evidence for independent promoter initiation for the two positive-sense vRNA species 96 (9). While these studies were able to identify the presence of the 5' type-0 cap structure, 97 they were, by the nature of their design and technological limitations, unable to 98 determine the relative frequency with which the positive sense vRNAs were capped. 99Recently, we reported findings that indicated that the alphaviral genomic vRNAs 100 are not ubiquitously capped, and that a significant proportion of the genomic vRNAs 101 produced during SINV and RRV infection lack the 5' type-0 cap structure (18). 102 Furthermore, analyses of infectious and noninfectious viral particles demonstrated that 103 both the capped and noncapped genomic vRNAs are packaged into viral particles 104 throughout the course of infection. Through the use of tissue culture models of 105 alphaviral infection, the presence of the noncapped vRNAs were found to correlate with 106 the activation of a type-I IFN response. Moreover, an attenuated RRV mutant was found 107to produce fewer noncapped genomic RNAs relative to wild type virulent RRV (18...

Bortezomib inhibits chikungunya virus replication by interfering with viral protein synthesis

“…The alterations in ns and structural protein levels upon bortezomib treatment also reflect a similar phenotype recently described for a SINV mutant with increased nsP1 capping activity [55]. This mutant over-produced ns proteins and had somewhat elevated levels of viral RNA synthesis.…”

“…This mutant over-produced ns proteins and had somewhat elevated levels of viral RNA synthesis. Importantly, it also had slightly reduced synthesis of structural proteins and strongly reduced infectious virion production [55]. These similarities indicate that disrupting the balance between ns and structural protein levels is detrimental for alphavirus replication, perhaps by altering the cellular environment in a way that is unfavorable for virion formation and/or by directly interfering with virion formation.…”

Mature rotavirus particles contain equivalent amounts of7meGpppGcap and noncapped viral positive-sense RNAs

“…Recently, it was reported that noncapped RNAs have an important relevance during SINV infection in an in vitro and in vivo model, since when the viral protein involved in cap addition (nsP1) was mutated to decrease the amount of cap on viral RNAs a slight decrease in mortality of infected mice was observed. Conversely, an increased addition of cap resulted in an almost complete abrogation of mortality and morbidity compared to mice infected with a wild type virus (34,35). Thus, in SINV noncapped RNAs play a critical role in determining whether the virus is neurovirulent or not, however if the cap structure has a role in genome encapsidation is not known.…”

Non-catastrophic collapse of VEEV by lethal mutagenesis

“…This finding implies that the virus population is maintained by constituents with a small number of mutations. In other words, progeny virus with a high number of mutations is not capable of replicating and this could be a reason for low specific infectivity (pfu/copy number of viral genome) in addition to other observations, such as the absence of RNA capping in the viral genome(LaPointe et al, 2018). The replication incompetence of the high-mutation-containing population might interfere with the replication-competent members by competing for viral receptors or inducing innate immune responses.…”