Disease relevance of nsP4

• The RNA-dependent RNA polymerase nsP4 is an integral part of the alphavirus replication complex [1].
• Modification of the 5' terminus of Sindbis virus genomic RNA allows nsP4 RNA polymerases with nonaromatic amino acids at the N terminus to function in RNA replication [2].
• Two regions of the genome were examined: sequences of 477 nucleotides from the C terminus of the E1 envelope glycoprotein gene which in WEE virus was derived from the Sindbis-like virus parent, and 517 nucleotide sequences at the C terminus of the nsP4 gene which in WEE virus was derived from the EEE-like virus parent [3].
• To define the role of nsP4 in viral RNA replication and for a structure-function analysis, we expressed Sindbis virus nsP4 in Escherichia coli [1].
• The 26S mRNA and most of the nsP4 encoding regions of the eastern equine encephalomyelitis (EEE) viral genome have been cloned [4].

High impact information on nsP4

• This sequence of delayed processing of P1234 would explain the accumulation of P123 plus nsP4, the early short-lived minus-strand replicase [5].
• These results suggest that while selection of ONNV variants is occurring, de novo mutation at the position between nsP3 and nsP4 does not readily occur in the mosquito [6].
• Effects of an Opal Termination Codon Preceding the nsP4 Gene Sequence in the O'Nyong-Nyong Virus Genome on Anopheles gambiae Infectivity [6].
• The presence of an opal stop codon upstream of nsP4 nearly doubled (75.5%) the infectivity of ONNV over that of virus possessing a codon for the amino acid arginine at the corresponding position (39.8%) [6].
• Catalytic Core of Alphavirus Nonstructural Protein nsP4 Possesses Terminal Adenylyltransferase Activity [1].

Chemical compound and disease context of nsP4

• Recent insights into the early events in Sindbis virus RNA replication suggest a requirement for either the P123 or P23 polyprotein, as well as mature nsP4, the RNA-dependent RNA polymerase, for initiation of minus-strand RNA synthesis [7].
• We found that Ross River virus possesses an in-phase opal termination codon between nsP3 and nsP4, whereas in O'Nyong-nyong virus this termination codon is replaced by an arginine codon [8].
• Previously we reported that the N-terminal Tyr residue of nsP4 of Sindbis virus, the type species of the genus Alphavirus, can be substituted with Phe, Trp, or His without altering the wild-type phenotype in cultured cells but that other substitutions tested, except for Met, were lethal or quasilethal [9].

Biological context of nsP4

• In chicken cells, the nsP4 Arg183 mutants had a nonconditionally lethal, temperature-sensitive (ts) growth phenotype caused by a ts defect in minus-strand synthesis whose extent varied with the particular amino acid substituted (Ser>Ala>Lys) [10].
• Previous studies (D.L. Sawicki, D. B. Barkhimer, S. G. Sawicki, C. M. Rice, and S. Schlesinger, Virology 174:43-52, 1990) identified a temperature-sensitive (ts) defect in Sindbis virus nonstructural protein 4 (nsP4) that reactivated negative-strand synthesis after its normal cessation at the end of the early phase of replication [11].
• The core catalytic domain of nsP4 (Delta97nsP4, a deletion of the N-terminal 97 amino acids), which consists of the predicted polymerase domain containing the GDD amino acid motif required for viral RNA synthesis, was stable against proteolytic degradation during expression [1].
• All three suppressor mutations suppressed the effects of Ala, Arg, or Leu at the N-terminus of nsP4 with almost equal efficiency and thus the effect of the suppressing mutation is independent of the nsP4 N-terminal residue [9].

Anatomical context of nsP4

• In alphavirus-infected cells the four virus-specific nonstructural proteins (nsP1-nsP4) are located on modified endosomes and lysosomes known as type I cytopathic vacuoles [12].

Associations of nsP4 with chemical compounds

• These mutations resulted in the following amino acid changes in nsP4: leucine to valine at residue 48, aspartate to glycine at residue 142, and proline to arginine at residue 187 [13].
• (4) SFV mutants that produce nsP1, P23, nsP4, as well as the precursor P123 are viable but produce an order of magnitude less virus than wild type at 30 degrees C and two orders of magnitude less virus at 39 degrees C. The ratio of subgenomic mRNA to genomic RNA is much reduced in these mutants relative to the parental viruses [14].
• A unique base change of an A for a C residue at nt 6339, predicting a change from glutamine to lysine at amino acid 195 in nsP4, was found in genomes of ts24, ts24R1, and ts24R2 [15].

Physical interactions of nsP4

• However, the nsP3 of both ts4 and ts7 allowed reactivation of negative-strand synthesis by stable replication complexes containing nsP4 from ts24 [16].
• The results from this study and from a previous report on the shutoff of minus-strand RNA synthesis at 40 degrees C with the nsP1-A348T mutation in ts11 suggests that the N-terminus nsP4 interacts with nsP1 during initiation of minus-strand RNA synthesis [9].

Other interactions of nsP4

• The membrane-associated alphavirus RNA replication complex contains four virus-encoded subunits, the nonstructural proteins nsP1 to nsP4 [17].
• Reactivation of negative-strand synthesis by mutations in nsP2 resembled that in nsP4: it was a reversible property of stable replication complexes and did not require continuation of viral protein synthesis [11].
• We designed 4 primer pairs to amplify conserved regions of the E1 or nsP4 genes of salmonid alphavirus (SAV) and evaluated their performance in optimized 1-step SYBR green real-time RT-PCR (RRT-PCR) assays [18].

Analytical, diagnostic and therapeutic context of nsP4

• Functional mapping and sequence analysis of the mutant cDNAs revealed several mutations which mapped to the amino terminus of nsP4, the putative polymerase subunit of the viral RNA replicase [13].

References