Function of Rotavirus VP3 Polypeptide in Viral Morphogenesis

Vásquez, Mónica; Sandino, Ana María; Pizarro, Javiera; Fernández, J; Valenzuela, Sofía; Spencer, Eugenio

doi:10.1099/0022-1317-74-5-937

Cited by 13 publications

(13 citation statements)

References 26 publications

(15 reference statements)

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“…The observation that these types of particles can be formed when either VP1 or VP3 is knocked down suggests that their assembly can proceed in the absence of either protein. These results are in agreement with those reported by Vasquez et al (39). Taken together, these results suggest that the decrease in viral replication observed when VP3 was silenced is most probably due to an RNA replication defect rather than to an assembly problem, and they suggest that VP3 may have a novel and thus far undetermined role during in vivo replication of the virus.…”

supporting

confidence: 83%

“…However, the newly synthesized dsRNA was not packaged, even when VP3 was added to the assay, so it was concluded that, in vivo, some other viral proteins, such as NSP2 and NSP5, should be needed for the encapsidation process and/or for the selection of the RNAs to be replicated. Using a temperature-sensitive virus with a lesion in VP3, Vásquez et al (39) demonstrated that this protein plays an important role during the RNA replication process since only empty, singleshelled particles were assembled at the nonpermissive temperature, suggesting that VP3 might have another function besides adding the cap structure to mRNAs. Additionally, Mansell and Patton (17) used a temperature-sensitive mutant with a lesion in VP6 to gain insight into the role of this protein in the replication of the virus.…”

mentioning

confidence: 99%

“…It has been proposed that this process leads to the production of new transcriptionally active, dsRNA-containing double-layered RI particles, which are thought to be responsible for an enhanced second round of transcription, resulting in a second wave of assembly of doublelayered RI particles (4). The occurrence of this second round, however, has not been directly demonstrated.The participation of several viral proteins in the replication process of rotavirus has been studied using different approaches; the characterization of temperature-sensitive mutants underlined the importance of VP1, VP2, and VP3 in the replication process (17,39). In a different approach, an in vitro replication assay was developed (2); in this system it has been observed that VP1 and VP2 are necessary and sufficient to replicate the genome and that no other structural or nonstructural protein was necessary although the lack of genome encapsidation in this system indicated the possible role of other viral proteins…”

mentioning

confidence: 99%

“…The participation of several viral proteins in the replication process of rotavirus has been studied using different approaches; the characterization of temperature-sensitive mutants underlined the importance of VP1, VP2, and VP3 in the replication process (17,39). In a different approach, an in vitro replication assay was developed (2); in this system it has been observed that VP1 and VP2 are necessary and sufficient to replicate the genome and that no other structural or nonstructural protein was necessary although the lack of genome encapsidation in this system indicated the possible role of other viral proteins in coordinating the process of replication and sorting of the RNA segments in equimolar amounts during an infection (24).…”

mentioning

confidence: 99%

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Analysis of the Kinetics of Transcription and Replication of the Rotavirus Genome by RNA Interference

Ayala-Bretón¹,

Arias²,

Espinosa³

et al. 2009

J Virol

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Rotaviruses have a genome composed of 11 segments of double-stranded RNA (dsRNA) surrounded by three protein layers. The virus contains an RNA-dependent RNA polymerase that synthesizes RNA transcripts corresponding to all segments of the viral genome. These transcripts direct the synthesis of the viral proteins and also serve as templates for the synthesis of the complementary strand to form the dsRNA genome. In this work, we analyzed the kinetics of transcription and replication of the viral genome throughout the replication cycle of the virus using quantitative reverse transcription-PCR. The role of the proteins that form doublelayered particles ([DLPs] VP1, VP2, VP3, and VP6) in replication and transcription of the viral genome was analyzed by silencing their expression in rotavirus-infected cells. All of them were shown to be essential for the replication of the dsRNA genome since in their absence there was little synthesis of viral mRNA and dsRNA. The characterization of the kinetics of RNA transcription and replication of the viral genome under conditions where these proteins were silenced provided direct evidence for a second round of transcription during the replication of the virus. Interestingly, despite the decrease in mRNA accumulation when any of the four proteins was silenced, the synthesis of viral proteins decreased when VP2 and VP6 were knocked down, whereas the absence of VP1 and VP3 did not have a severe impact on viral protein synthesis. Characterization of viral particle assembly in the absence of VP1 and VP3 showed that while the formation of triple-layered particles and DLPs was decreased, the amount of assembled lower-density particles, often referred to as empty particles, was not different from the amount in control-infected cells, suggesting that viral particles can assemble in the absence of either VP1 or VP3.The family Reoviridae includes viruses that have a genome composed of 9 to 12 segments of double-stranded RNA (dsRNA). Rotaviruses belong to the most medically significant genus of the family since they are the main cause of infantile gastroenteritis, causing approximately 500,000 deaths per year in children less than 5 years of age (23). These viruses have a genome composed of 11 segments of dsRNA, which is enclosed in a capsid formed by three concentric layers of protein (33). The innermost layer, formed by VP2, contains the viral genome and 12 copies each of the virus RNA-dependent RNA polymerase (RdRP; VP1) and the guanylyltransferase and methylase enzyme (VP3); these viral elements constitute the core of the virus. The addition of VP6 on top of the VP2 layer produces double-layered particles (DLPs). The outermost layer, characteristic of infectious, triple-layered particles (TLPs), is composed of two proteins, VP4 and VP7.During or shortly after cell entry, the infecting TLP uncoats, loosing the two proteins of the outer layer and yielding a DLP, which is transcriptionally active (14). The nascent transcripts are extruded into the cell's cytoplasm through channels located ne...

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supporting

confidence: 83%

mentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

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Analysis of the Kinetics of Transcription and Replication of the Rotavirus Genome by RNA Interference

Ayala-Bretón¹,

Arias²,

Espinosa³

et al. 2009

J Virol

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“…Notably, this enzyme does have an important role in genome replication in vivo, as small interfering RNA-mediated knockdown of VP3 expression in infected cells yields a reduction in progeny particles containing dsRNA (41,44). In contrast, both in vitro and in vivo experiments have shown that dsRNA synthesis by VP1 is strictly dependent upon the presence of the core shell protein VP2 (41,(45)(46)(47).…”

Section: Protein Requirements For Rv Rna Synthesismentioning

confidence: 99%

Mechanism of Intraparticle Synthesis of the Rotavirus Double-stranded RNA Genome

Guglielmi

McDonald

Patton

2010

Journal of Biological Chemistry

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Rotaviruses perform the remarkable tasks of transcribing and replicating 11 distinct double-stranded RNA genome segments within the confines of a subviral particle. Multiple viral polymerases are tethered to the interior of a particle, each dedicated to a solitary genome segment but acting in synchrony to synthesize RNA. Although the rotavirus polymerase specifically recognizes RNA templates in the absence of other proteins, its enzymatic activity is contingent upon interaction with the viral capsid. This intraparticle strategy of RNA synthesis helps orchestrate the concerted packaging and replication of the viral genome. Here, we review our current understanding of rotavirus RNA synthetic mechanisms.A distinguishing feature of viruses with segmented doublestranded RNA (dsRNA) 2 genomes is that they perform all stages of RNA synthesis inside icosahedral subviral particles (1-4). Each genome segment is dedicated to a single polymerase complex (PC) anchored to the interior of the particle. Particle-associated PCs act in synchrony to transcribe and replicate the viral dsRNA segments. Thus, subviral particles of dsRNA viruses can be viewed as nanoscale factories containing multiple highly coordinated PC machines. During transcription, each genome segment serves as a template for the generation of plus-strand RNAs (ϩRNAs). In addition to directing viral protein expression, ϩRNAs also serve as templates for minus-strand synthesis to recreate dsRNA duplexes. The intraparticle mechanism of RNA synthesis not only protects the genome from recognition by antiviral dsRNA sensors but also provides a mechanism for coordinating ϩRNA packaging with genome replication (4).As the primary cause of life-threatening gastroenteritis in young children, rotaviruses (RVs) have long been the subject of basic research (5). Specifically, biochemical studies have probed the functions of viral proteins involved in RNA synthesis and have elucidated critical features of viral RNA templates. Moreover, the recent determination of a high resolution structure of the RV RNA-dependent RNA polymerase (RdRp) in complex with RNA has greatly enhanced our understanding of intraparticle RNA synthesis (6).

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