Nonstructural proteins nsP3 and nsP4 of Ross River and O'Nyong-nyong viruses: Sequence and comparison with those of other alphaviruses

Strauss, Ellen G.; Levinson, Randy; Rice, Charles M.; Dalrymple, Joel M.; Strauss, James H.

doi:10.1016/0042-6822(88)90644-7

Cited by 74 publications

(63 citation statements)

References 30 publications

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“…In contrast to that work, our study showed that the SFV nsP3 C-terminal L/ITFGDFD repeat motifs at positions 449 to 455 and 466 to 472, both well conserved in the Old World alphaviruses (11), were necessary and sufficient for formation of the nsP3/G3BP complex in infected cells (8). Both these regions, although situated within the hypervariable domain (HVD) of nsP3 (12)(13)(14)(15), are highly conserved between both CHIKV and SFV, and it was therefore surprising that the viruses would differ in the region used to bind and recruit G3BP.…”

contrasting

confidence: 50%

The C-Terminal Repeat Domains of nsP3 from the Old World Alphaviruses Bind Directly to G3BP

Panas

Ahola

McInerney

2014

J Virol

129

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bThe Old World alphaviruses block stress granule assembly by sequestration of RasGAP SH3-domain binding protein (G3BP). Here, we show that the proline-rich sequences in the hypervariable domain of nonstructural protein 3 (nsP3) of both Semliki Forest virus and Chikungunya virus were dispensable for binding to G3BP. nsP3 variants with or without this domain colocalized with G3BP. Furthermore, we show that the C-terminal repeat motifs of nsP3 were sufficient for G3BP binding. Stress granules (SGs) are foci of accumulation of translationally silent mRNP complexes, which are induced during cellular stress (1). Their assembly is dependent on the proteins TIA-1/R (2) and G3BP (3). Many diverse virus infections employ mechanisms to restrict the formation of SGs (4, 5), which are induced by early events in the replication of many viruses, including the alphaviruses (6, 7). We recently showed that in cells infected with the alphavirus Semliki Forest virus (SFV), nonstructural protein 3 (nsP3) binds RasGAP SH3-domain binding proteins 1 and 2 (G3BP-1 and G3BP-2, respectively), recruits them to foci containing other viral proteins and often double-stranded RNA (dsRNA), and, in doing so, inhibits SG assembly (8). Fros and colleagues (9) also showed that the expression of the closely related Chikungunya virus (CHIKV) nsP3 blocks SG assembly by recruitment of G3BP-1 into cytoplasmic foci. These reports therefore describe a function for Old World alphavirus nsP3 in inhibition of the stress response. A surprising difference in the findings of the two groups was that the work of Fros and colleagues suggested that the CHIKV nsP3 sequence binding the SH3 domain of amphiphysins ) was essential for colocalization of nsP3 and G3BP (9). In contrast to that work, our study showed that the SFV nsP3 C-terminal L/ITFGDFD repeat motifs at positions 449 to 455 and 466 to 472, both well conserved in the Old World alphaviruses (11), were necessary and sufficient for formation of the nsP3/G3BP complex in infected cells (8). Both these regions, although situated within the hypervariable domain (HVD) of nsP3 (12-15), are highly conserved between both CHIKV and SFV, and it was therefore surprising that the viruses would differ in the region used to bind and recruit G3BP.Recently, a variant of SFV (SFV-⌬P1ϩ2) was described, lacking the proline-rich SH3-domain binding sequences of nsP3 (10). It was shown that the deletion impairs the recruitment of amphiphysin proteins to foci of nsP3 accumulation and delays replication complex formation (10). To determine if this domain of SFV nsP3 was important for the recruitment of G3BP-1, we infected mouse embryonic fibroblasts (MEFs) maintained as described previously (16) with SFV wild type (wt) or SFV-⌬P1ϩ2 at a multiplicity of infection (MOI) of 1, fixed the cells at 8 h postinfection (hpi), and stained them for nsP3 and G3BP-1. Single-plane images were captured using a supercontinuum confocal TCS SP5 X microscope (Leica, Wetzlar, Germany) with a pulsed white light laser. No deficiency in G3BP-1 recruitment...

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contrasting

confidence: 50%

The C-Terminal Repeat Domains of nsP3 from the Old World Alphaviruses Bind Directly to G3BP

Panas

Ahola

McInerney

2014

J Virol

129

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“…Similar to several other alphaviruses, rA774 has an opal termination codon at position 469 of the 39 end of the nsP3 gene, while an arginine residue is at this position in both the SFV prototype and SFV4 (Takkinen, 1986;Tuittila et al, 2000). Similar strain-specific opal/sense codon variation is found also in Sindbis and o'nyong-nyong viruses (Simpson et al, 1996;Strauss et al, 1988). Although mutation of the arginine residue to an opal codon dramatically attenuates SFV4, an opal to arginine mutation in rA774 does not reconstitute full neurovirulence, even though it clearly increases pathogenicity .…”

Section: Introductionmentioning

confidence: 78%

Amino acid mutations in the replicase protein nsP3 of Semliki Forest virus cumulatively affect neurovirulence

Tuittila

Hinkkanen

2003

Journal of General Virology

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It has been shown previously that an avirulent Semliki Forest virus (SFV) clone, rA774, engineered to carry the nsP3 gene of the virulent clone SFV4 becomes highly neurovirulent and is lethal for adult BALB/c mice. rA774, like several other alphaviruses, has an opal termination codon close to the 59 end of nsP3 (aa 469), while SFV4 has an arginine residue at this position. Mutation of the opal codon to an arginine residue increases the virulence of rA774 but does not reconstruct the severe neurovirulence of SFV4. Additionally, nsP3 amino acid sequences differ between these two strains by eight amino acids and by a deletion of seven amino acids in the C-terminal third of rA774 nsP3. This study shows that neurovirulence can be reconstituted gradually by exchanging individual amino acids and is fully retained when combinations of two nsP3 mutations, V 11 RI into a moderately virulent rA774 recombinant carrying the SFV4 nsP1 and nsP2 genes. In conclusion, virulence determinants in SFV are distributed over a wide region of the nonstructural genes.

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“…For these viruses, it was presumed that only P1234 was generated during replication. However, passaging of ONNV prior to sequencing may have determined the codon at this particular locus (29). Consensus sequences obtained from later, lower passage, isolates indicate that ONNVs possess both sense and stop codons at this position (18).…”

mentioning

confidence: 99%

“…Sequence information initially indicated that the termination codon before nsP4 had been replaced with an arginine codon (CGA) in at least two alphaviruses, ONNV and Semliki Forest virus (SFV) (29,32). For these viruses, it was presumed that only P1234 was generated during replication.…”

mentioning

confidence: 99%

Effects of an Opal Termination Codon Preceding the nsP4 Gene Sequence in the O'Nyong-Nyong Virus Genome on Anopheles gambiae Infectivity

Myles

Kelly

Ledermann

et al. 2006

J Virol

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The genomic RNA of an alphavirus encodes four different nonstructural proteins, nsP1, nsP2, nsP3, and nsP4. The polyprotein P123 is produced when translation terminates at an opal termination codon between nsP3 and nsP4. The polyprotein P1234 is produced when translational readthrough occurs or when the opal termination codon has been replaced by a sense codon in the alphavirus genome. Evolutionary pressures appear to have maintained genomic sequences encoding both a stop codon (opal) and an open reading frame (arginine) as a general feature of the O'nyong-nyong virus (ONNV) genome, indicating that both are required at some point. Alternate replication of ONNVs in both vertebrate and invertebrate hosts may determine predominance of a particular codon at this locus in the viral quasispecies. However, no systematic study has previously tested this hypothesis in whole animals. We report here the results of the first study to investigate in a natural mosquito host the functional significance of the opal stop codon in an alphavirus genome. We used a full-length cDNA clone of ONNV to construct a series of mutants in which the arginine between nsP3 and nsP4 was replaced with an opal, ochre, or amber stop codon. 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%). Although the frequency with which the opal virus disseminated from the mosquito midgut did not differ significantly from that of the arginine virus on days 8 and 10, dissemination did began earlier in mosquitoes infected with the opal virus. Although a clear fitness advantage is provided to ONNV by the presence of an opal codon between nsP3 and nsP4 in Anopheles gambiae, sequence analysis of ONNV RNA extracted from mosquito bodies and heads indicated codon usage at this position corresponded with that of the virus administered in the blood meal. 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. Taken together, these results suggest that the primary fitness advantage provided to ONNV by the presence of an opal codon between nsP3 and nsP4 is related to mosquito infectivity.

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Nonstructural proteins nsP3 and nsP4 of Ross River and O'Nyong-nyong viruses: Sequence and comparison with those of other alphaviruses

Cited by 74 publications

References 30 publications

The C-Terminal Repeat Domains of nsP3 from the Old World Alphaviruses Bind Directly to G3BP

The C-Terminal Repeat Domains of nsP3 from the Old World Alphaviruses Bind Directly to G3BP

Amino acid mutations in the replicase protein nsP3 of Semliki Forest virus cumulatively affect neurovirulence

Effects of an Opal Termination Codon Preceding the nsP4 Gene Sequence in the O'Nyong-Nyong Virus Genome on Anopheles gambiae Infectivity

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