Characterization of rotavirus replication intermediates: A model for the assembly of single-shelled particles

Gallegos, Claudia O.; Patton, John T.

doi:10.1016/0042-6822(89)90204-3

Cited by 136 publications

(152 citation statements)

References 30 publications

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“…The mechanism by which salt prevents the formation of a functional (Ϫ) strand initiation complex in the open core replication system is not known+ One possibility is that salt increases the stability of the 59-39 panhandle or causes other structural changes in the (ϩ) strand RNA template, making it more difficult for the viral singlestranded RNA-binding proteins to interact to form a ternary initiation complex+ Indeed, neither VP1 nor VP3 has affinity for dsRNA and the affinity of VP2 for dsRNA is less than that for single-stranded RNA ; thus any changes in the stability or the extent of secondary structure in the template RNA could significantly impact the ability of these proteins to become part of an initiation complex+ In contrast to what is observed in vitro, salt may not have any real impact on the formation of initiation complexes in vivo because the nonstructural proteins, NSP2 and NSP5, are components of replication intermediates with replicase activity in the infected cells (Gallegos & Patton, 1989)+ In particular, NSP2 is an NTPase with helix destabilizing activity and the protein may assist in the formation of initiation complexes by disrupting base pairing that impedes the necessary binding of VP1, VP2, and/or VP3 to the (ϩ) RNA template (Taraporewala et al+, 1999;Taraporewala & Patton, unpubl+ results)+…”

Section: Discussionmentioning

confidence: 99%

De novo synthesis of minus strand RNA by the rotavirus RNA polymerase in a cell-free system involves a novel mechanism of initiation

Chen

Patton

2000

RNA

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The replicase activity of rotavirus open cores has been used to study the synthesis of (-) strand RNA from viral (1) strand RNA in a cell-free replication system. The last 7 nt of the (1) strand RNA, 59-UGUGACC-39, are highly conserved and are necessary for efficient (-) strand synthesis in vitro. Characterization of the cell-free replication system revealed that the addition of NaCl inhibited (-) strand synthesis. By preincubating open cores with (1) strand RNA and ATP, CTP, and GTP prior to the addition of NaCl and UTP, the salt-sensitive step was overcome. Thus, (-) strand initiation, but not elongation, was a salt-sensitive process in the cell-free system. Further analysis of the requirements for initiation showed that preincubating open cores and the (1) strand RNA with GTP or UTP, but not with ATP or CTP, allowed (-) strand synthesis to occur in the presence of NaCl. Mutagenesis suggested that in the presence of GTP, (-) strand synthesis initiated at the 39-terminal C residue of the (1) strand template, whereas in the absence of GTP, an aberrant initiation event occurred at the third residue upstream from the 39 end of the (1) strand RNA. During preincubation with GTP, formation of the dinucleotides pGpG and ppGpG was detected; however, no such products were made during preincubation with ATP, CTP, or UTP. Replication assays showed that pGpG, but not GpG, pApG, or ApG, served as a specific primer for (-) strand synthesis and that the synthesis of pGpG may occur by a templateindependent process. From these data, we conclude that initiation of rotavirus (-) strand synthesis involves the formation of a ternary complex consisting of the viral RNA-dependent RNA polymerase, viral (1) strand RNA, and possibly a 59-phosphorylated dinucleotide, that is, pGpG or ppGpG.

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Section: Discussionmentioning

confidence: 99%

De novo synthesis of minus strand RNA by the rotavirus RNA polymerase in a cell-free system involves a novel mechanism of initiation

Chen

Patton

2000

RNA

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“…Viroplasms are composed of viral RNA and the proteins VP1, VP2, VP3, VP6, NSP2, and NSP5 (Petrie et al 1982, Gallegos & Patton 1989, and while virion assembly occur in these structures (Petrie et al 1982, Gallegos & Patton 1989) the mechanism of viroplasms formation is unknown. Recently, it was reported that the number of rotavirus viroplasms decrease with post-infection time (Eichwald et al 2004), and while the diameter of single viroplasms increased with time, the total number of viroplasms per cell diminishes, suggesting that growth of these inclusion bodies occur by fusion and probably also by stepwise addition of viral components to the viroplasms surface (Eichwald et al 2004).…”

Section: Rotavirus Replication and Virus Assembly Take Place In Electmentioning

confidence: 99%

Association of rotavirus viroplasms with microtubules through NSP2 and NSP5

Cabral-Romero

Padilla-Noriega

2006

Mem. Inst. Oswaldo Cruz

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Rotavirus replication and virus assembly take place in electrodense spherical structures known as viroplasms whose main components are the viral proteins NSP2 and NSP5. The viroplasms are produced since early times after infection and seem to grow by stepwise addition of viral proteins and by fusion, however, the mechanism of viropIasms formation is unknown. In this study we found that the viroplasms surface colocalized with microtubules, and seem to be caged by a microtubule network. Moreover inhibition of microtubule assembly with nocodazole interfered with viroplasms growth in rotavirus infected cells. We searched for a physical link between viroplasms and microtubules by co-immunoprecipitation assays, and we found that the proteins NSP2 and NSP5 were co-immunoprecipitated with anti-tubulin in rotavirus infected cells and also when they were transiently co-expressed or individually expressed. These results indicate that a functional microtubule network is needed for viroplasm growth presumably due to the association of viroplasms with microtubules via NSP2 and NSP5.Key words: rotavirus -viroplasms -microtubules -NSP2 -NSP5Group A rotaviruses are the leading etiological agents of severe diarrheal disease in infants and young children worldwide (Parashar 2003). These viruses belong to the genus Rotavirus within the Reoviridae family and their genome consist of 11 double-stranded RNA segments that encode six structural proteins named VP1-VP4, VP6, and VP7, as well as six nonstructural proteins named NSP1 to NSP6 (Estes 2001).Rotavirus replication takes place in the cytoplasm of infected cells in electrodense spherical structures known as "viroplasms" that can be found as early as 4 h after infection . Viroplasms are composed of viral RNA and the proteins VP1, VP2, VP3, VP6, NSP2, and NSP5 (Petrie et al. 1982, Gallegos & Patton 1989, and while virion assembly occur in these structures (Petrie et al. 1982, Gallegos & Patton 1989) the mechanism of viroplasms formation is unknown. Recently, it was reported that the number of rotavirus viroplasms decrease with post-infection time (Eichwald et al. 2004), and while the diameter of single viroplasms increased with time, the total number of viroplasms per cell diminishes, suggesting that growth of these inclusion bodies occur by fusion and probably also by stepwise addition of viral components to the viroplasms surface (Eichwald et al. 2004).In the viroplasms the viral mRNAs are replicated to produce genomic double stranded RNAs (dsRNA) which are simultaneously encapsidated into double layerded viral particles (DLPs), that contain the 11 dsRNA at the core of the particle surrounded by the inner and intermediate protein layers of VP2 and VP6, respectively. The third layer formed by VP4 and VP7 is acquired by budding of the DLP particles into the endoplasmic reticulum (ER), a process that requires assistance of the viral non-structural protein 4 (NSP4), whereby a transient envelope is acquired, that is finally lost within the ER along with NSP4. The fundamental role of NSP...

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“…Infective particles replicate in the cytoplasm of infected cells. Although several reports have described the characterization of rotavirus replication intermediates (Gallegos & Patton, 1989 ;Patton & Gallegos, 1988), molecular details of the replication mechanism remain unclear. Partially purified intermediate complexes (or sub-viral particles) obtained from SA11-infected cells were able to catalyse run-off synthesis of endogenous dsRNAs (Patton, 1986), as well as complete replication of exogenously added viral ss (j)-RNAs (Chen et al, 1994 ;Wentz et al, 1996 ;Patton et al, 1996 ;E.…”

Section: Introductionmentioning

confidence: 99%

Rotavirus NSP5 phosphorylation is up-regulated by interaction with NSP2.

Afrikanova

Fabbretti

Miozzo

et al. 1998

Journal of General Virology

129

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We have previously shown that a number of isoforms of the non-structural rotavirus protein NSP5 are found in virus-infected cells. These isoforms differ in their level of phosphorylation which, at least in part, appears to occur through autophosphorylation. NSP5 co-localizes with another non-structural protein, NSP2, in the viroplasms of infected cells where virus replication takes place. We now show that NSP5 can be chemically cross-linked in living cells with the viral polymerase VP1 and NSP2. Interaction of NSP5 with NSP2 was also demonstrated by coimmunoprecipitation of NSP2 and NSP5 from extracts of UV-treated rotavirus-infected cells. In

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Characterization of rotavirus replication intermediates: A model for the assembly of single-shelled particles

Cited by 136 publications

References 30 publications

De novo synthesis of minus strand RNA by the rotavirus RNA polymerase in a cell-free system involves a novel mechanism of initiation

De novo synthesis of minus strand RNA by the rotavirus RNA polymerase in a cell-free system involves a novel mechanism of initiation

Association of rotavirus viroplasms with microtubules through NSP2 and NSP5

Rotavirus NSP5 phosphorylation is up-regulated by interaction with NSP2.

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