Role of the inner protein capsid on in vitro human rotavirus transcription

Sandino, Ana María; Jashés, M; Faundez, Gustavo; Spencer, Eugenio

doi:10.1128/jvi.60.2.797-802.1986

Cited by 59 publications

(23 citation statements)

References 25 publications

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“…In addition, the replicase reactions were programmed with twice the mass of particles as was used for double-layered VLPs and made less product. Finally, VP6 may play a role in maintaining the optimal conformation of the replicase, as it apparently does in the case of the transcriptase for which the addition of VP6 to single-layered (core) particles activates the transcriptase through what is thought to be a conformational effect (35). A combination of these factors may provide the explanation.…”

Section: Discussionmentioning

confidence: 99%

Characterization and replicase activity of double-layered and single-layered rotavirus-like particles expressed from baculovirus recombinants

Zeng

Wentz

Cohen

et al. 1996

J Virol

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Rotavirus has a capsid composed of three concentric protein layers. We coexpressed various combinations of the rotavirus structural proteins of single-layered (core) and double-layered (single-shelled) capsids from baculovirus vectors in insect cells and determined the ability of the various combinations to assemble into viruslike particles (VLPs). VLPs were purified by centrifugation, their structure was examined by negativestain electron microscopy, their protein content was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and GTP binding assays, and their ability to support synthesis of negative-strand RNAs on positive-sense template RNAs was determined in an in vitro replication system. Coexpression of all possible combinations of VP1, VP2, VP3, and VP6, the proteins of double-layered capsids, resulted in the formation of VP1/2/3/6, VP1/2/6, VP2/3/6, and VP2/6 double-layered VLPs. These VLPs had the structural characteristics of empty rotavirus double-layered particles and contained the indicated protein species. Only VP1/2/3/6 and VP1/2/6 particles supported RNA replication. Coexpression of all possible combinations of VP1, VP2, and VP3, the proteins of single-layered capsids, resulted in the formation of VP1/2/3, VP1/2, VP2/3, and VP2 singlelayered VLPs. These VLPs had the structural characteristics of empty single-layered rotavirus particles and contained the indicated protein species. Only VP1/2/3 and VP1/2 VLPs supported RNA replication. We conclude that (i) the assembly of VP1 and VP3 into VLPs requires the presence of VP2, (ii) the role of VP2 in the assembly of VP1 and VP3 and in replicase activity is most likely structural, (iii) VP1 is required and VP3 is not required for replicase activity of VLPs, and (iv) VP1/2 VLPs constitute the minimal replicase particle in the in vitro replication system.

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Section: Discussionmentioning

confidence: 99%

Characterization and replicase activity of double-layered and single-layered rotavirus-like particles expressed from baculovirus recombinants

Zeng

Wentz

Cohen

et al. 1996

J Virol

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“…The idea that the replication of the rotavirus genome involves two unique polymerase activities is not novel, given earlier experiments which revealed that the minimum protein components of particles with transcriptase and replicase activity differ from one another (1,24,46). More precisely, although cores surrounded by a layer of VP6 have transcriptase activity, cores alone do not, thus indicating that VP6 is an essential component of functional transcriptase particles (42). In addition to VP6, the polymerase core VP1, the guanylyltransferase VP3, and the major core protein VP2 are probably also essential components of particles with transcriptase activity (32).…”

Section: Discussionmentioning

confidence: 99%

“…Cores surrounded by VP6 (double-shelled particles) have an associated transcriptase activity able to synthesize viral mRNA in vitro (1,7). The ability of double-shelled particles to carry out transcription is dependent on VP6, as removal of this protein produces cores that are transcriptionally inactive (42).…”

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confidence: 99%

cis-Acting signals that promote genome replication in rotavirus mRNA

Patton

Wentz

Xiaobo

et al. 1996

J Virol

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A previous study has shown that rotavirus cores have an associated replicase activity which can direct the synthesis of double-stranded RNA from viral mRNA in a cell-free system (. 68:7030-7039, 1994). To define the cis-acting signals in rotavirus mRNA that are important for RNA replication, gene 8 transcripts which contained internal and terminal deletions and chimeric transcripts which linked gene 8-specific 3-terminal sequences to the ends of nonviral sequences were generated. Analysis of these RNAs in the cell-free system led to the identification of a cis-acting signal in the gene 8 mRNA which is essential for RNA replication and two cis-acting signals which, while not essential for replication, serve to enhance the process. The sequence of the essential replication signal is located at the extreme 3 end of the gene 8 mRNA and, because of its highly conserved nature, is probably a common feature of all 11 viral mRNAs. By site-specific mutagenesis of the gene 8 mRNA, residues at positions ؊1, ؊2, ؊5, ؊6, and ؊7 of the 3 essential signal were found to be particularly important for promoting RNA replication. One of the cis-acting signals shown to enhance the replication in the cell-free system was located near the 5 end of the 3 untranslated region (UTR) of the gene 8 mRNA, while remarkably the other was located in the 5 UTR of the message. The existence of an enhancement signal in the 5 UTR raises the possibility that the 5 and 3 ends of the rotavirus mRNA may interact with each other and/or with the viral replicase during genome replication.

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“…In rotavirus, the transcriptionally competent form of the virus has a double-layered capsid consisting of the structural proteins VP2 and VP6 surrounding the dsRNA genome segments and the enzymatic machinery in the core. Not only is the RNA-dependent RNA polymerase unable to synthesize RNA efficiently unless associated with the inner capsid protein VP2 (Patton et al, 1997), but the intermediate VP6 capsid layer must also be present (Bican et al, 1982;Kohli et al, 1993;Sandino et al, 1986) and the outermost VP7 capsid layer absent (Cohen et al, 1979;Chen and Ramig, 1993;Lawton et al, 1999). These observations have led to the proposal that the functional endogenous transcription apparatus contains three components: (1) the enzymes required for RNA synthesis and capping, (2) a scaffold consisting of the intact VP2 capsid layer functionally enhanced by the presence of VP6 on the exterior surface, and (3) the viral nucleic acid .…”

Section: Components Of Endogenous Transcription Apparatusmentioning

confidence: 99%

Mechanism of genome transcription in segmented dsRNA viruses

Lawton

Estes

Prasad

2000

Advances in Virus Research

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Genome transcription is a critical stage in the life cycle of a virus, as this is the process by which the viral genetic information is presented to the host cell protein synthesis machinery for the production of the viral proteins needed for genome replication and progeny virion assembly. Viruses with dsRNA genomes face a particular challenge in that host cells do not produce proteins which can transcribe from a dsRNA template. Therefore, dsRNA viruses contain all of the necessary enzymatic machinery to synthesize complete mRNA transcripts within the core without the need for disassembly. Indeed one of the more striking observations about genome transcription in dsRNA viruses is that this process occurs efficiently only when the transcriptionally competent particle is fully intact. This observation suggests that all of the components of the TCP, including the viral genome, the transcription enzymes, and the viral capsid, function together to produce and release mRNA transcripts and that each component has a specific and critical role to play in promoting the efficiency of this process. This review has examined the process of genome transcription in dsRNA viruses from the perspective of rotavirus as a model system. However, despite numerous architectural and organizational differences among the families of dsRNA viruses, numerous studies suggest that the basic mechanism of mRNA production may be similar in most, if not all, viruses having dsRNA genomes. Important functional similarities include (1) the presence of a capsid-bound RNA-dependent RNA polymerase, which produces single-stranded mRNA transcripts from the dsRNA genome and regenerates the dsRNA genome from single-stranded RNA templates; (2) in viruses infecting eukaryotic hosts, the presence of all the enzymatic activities needed to generate the 5' cap required by the eukaryotic translation machinery; (3) the high degree of structural order present in the packaged genome, suggesting the requirement for organization in the viral core; (4) the role of the innermost capsid protein as a scaffold on which the core components of the transcription apparatus are assembled; and (5) the release of nascent mRNA transcripts through channels at the icosahedral vertices. The process of genome transcription in dsRNA viruses will become better understood as structural studies progress to higher resolution and as more viruses become amenable to study using site-directed mutagenesis coupled with viral reconstitution to generate recombinant particles having precise functional and structural changes. Future studies will dissect important intermolecular interactions required for efficient mRNA synthesis and will shed further light on the reasons for which the viral core must be structurally intact in order for transcription to occur efficiently. Structural studies of the capping enzymes at atomic resolution will reveal how multiple enzyme activities reside within a single polypeptide and how they act in concert to synthesize the 5' cap on the end of each mature transcript. Pe...

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Role of the inner protein capsid on in vitro human rotavirus transcription

Cited by 59 publications

References 25 publications

Characterization and replicase activity of double-layered and single-layered rotavirus-like particles expressed from baculovirus recombinants

Characterization and replicase activity of double-layered and single-layered rotavirus-like particles expressed from baculovirus recombinants

cis-Acting signals that promote genome replication in rotavirus mRNA

Mechanism of genome transcription in segmented dsRNA viruses

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