RNA replication

Characterization of subviral particles purified from infected cells has indicated that the synthesis of dsRNA occurs simultaneously with the packaging of mRNA

Rotavirus Panhandle Rna

FIG. 1. Model for assembly of core pentamers with replicase activity. The RNA polymerase, VP1, and capping enzyme, VP3, interact with viral mRNA to form a pre-core complex that lacks replicase activity. In the viroplasm, the complex then interacts with VP2 dimers, NSP2 octamers, and NSP5 multimers producing a structure that represents one of the vertices (pentamers) of the core. In core replication intermediates, the pentamer structures have replicase activity that catalyses the synthesis of dsRNA. The properties of NSP2 and NSP5 suggest that they facilitate the packaging of viral mRNA into cores during RNA replication.

FIG. 1. Model for assembly of core pentamers with replicase activity. The RNA polymerase, VP1, and capping enzyme, VP3, interact with viral mRNA to form a pre-core complex that lacks replicase activity. In the viroplasm, the complex then interacts with VP2 dimers, NSP2 octamers, and NSP5 multimers producing a structure that represents one of the vertices (pentamers) of the core. In core replication intermediates, the pentamer structures have replicase activity that catalyses the synthesis of dsRNA. The properties of NSP2 and NSP5 suggest that they facilitate the packaging of viral mRNA into cores during RNA replication.

templates into core RIs (Gallegos & Patton 1989). RNase treatment of core RIs with replicase activity prevents further dsRNA synthesis and reduces the size of the intermediates, results that indicate that the mRNAs pass from the exterior to the interior of the RI as dsRNA synthesis takes place (Fig. 2) (Patton & Gallegos 1990). On the basis of rotavirus structure predictions, the mRNA templates may be predicted to pass into cores through channels that exist at the fivefold axes of the VP2 shell (Prasad et al 1996). VP1 is situated at the interior base of these fivefold channels (Lawton et al 1997), which presumably would assure that each mRNA template encounters the RNA polymerase and is replicated as it is packaged.

Plication Rotavirus

FIG. 2. A possible structure of a core-like replication intermediate. During the synthesis of dsRNA, the viral mRNA is proposed to move through channels present at the 5'-fold axes of the VP2. Whether packaging of the 11 mRNAs proceeds through a common site or eleven separate sites of the core is not known. It is also not resolved whether core assembly occurs before or after the assortment of the 11 viral mRNAs.

FIG. 2. A possible structure of a core-like replication intermediate. During the synthesis of dsRNA, the viral mRNA is proposed to move through channels present at the 5'-fold axes of the VP2. Whether packaging of the 11 mRNAs proceeds through a common site or eleven separate sites of the core is not known. It is also not resolved whether core assembly occurs before or after the assortment of the 11 viral mRNAs.

Considerable insight into the process of dsRNA synthesis has been gained using the template-dependent cell-free replication system developed by Chen et al (1994). The source of replicase activity in the system consists of cores which have been purified from virions and disrupted ('opened') by incubation in hypotonic buffer. The replicase activity of open cores is specific, catalysing the de novo synthesis of minus-strand RNA from exogenous viral mRNA to yield dsRNA.

Initiation complexfor minus-strand RNA

The level of dsRNA synthesis in the open core replication system decreases as the concentration of monovalent salt, e.g. NaCl, in the reaction mixtures increases (Chen & Patton 1999). Recent studies have shown that the salt inhibition can be overcome if open cores, the mRNA template and GTP are pre-incubated prior to the addition of salt and the other 3 ribonucleotides (Chen & Patton 1999). Notably, pre-incubation will not overcome salt inhibition if any of these three components are left out or if GTP is replaced with ATP or CTP. When GTP is included in the pre-incubation mixture, the dinucleotides pGpG and ppGpG are made, possibly by a template-independent process (Chen & Patton 2000). When included in replication assays, pGpG can serve as a primer for minus-strand synthesis. Together, the data indicate that during rotavirus genome replication, an initiation complex is formed by the interaction of the viral RNA polymerase, the mRNA template and GTP or (p)pGpG and that this event is salt sensitive. Once formed, the initiation complex is salt resistant, and GTP or a derivative of it, i.e. pGpG or ppGpG, serves as the primer for elongation of the minus-strand.

mRNA structure anddsRNA synthesis

The open core replication system has been used to identify features of viral mRNAs that contribute to the synthesis of dsRNA. By adding viral mRNAs containing deletions of the 3'-consensus sequence, 5'-UGUGACC-3', to the replication system, it was shown that this sequence contains a cis-acting signal that is essential for minus-strand synthesis (Patton et al 1996, Wentz et al 1996). Indeed, placement of the 3'-consensus sequence onto the 3'-end of foreign RNAs is sufficient to allow them to serve as templates for the synthesis of dsRNA by open cores. By site-specific mutagenesis of viral mRNA templates, it has also been possible to assess the importance of the individual residues of the 3'-consensus sequence on the synthesis of dsRNA. The results have indicated that the mRNA template must terminate with one or two C residues for efficient RNA replication to occur (Patton et al 1996, Wentz et al 1996).

While nearly all viral mRNAs possess the same 3'-terminal sequence, there are some important exceptions. For instance, instead of the consensus sequence, the gene 5 RNAs of the SA11 and SA11-4F strains of rotavirus terminate with the sequence, 5'-UGUGAACC-3', and therefore contain an insertion of an A residue (underlined) (Mitchell & Both 1990a, Patton et al 2000). More remarkably, a triple-plaque purified variant of a RRVxDS1 mono-reassortant has been isolated whose gene 5 RNA has the 3'-terminal sequence, 5'-UGUUUCC-3' and therefore differs at both the -3 and -4 position (underlined) from the 3'-consensus sequence (K. Kearney, D. Chen & J. Patton, unpublished results). On the basis of sequence comparisons of all group A rotavirus RNAs, the only strictly conserved sequences at the 5'- and 3'-termini are 5'-GGC-3' and 5'-CC-3', respectively. Given the sequence variations that have been detected in the 3'-consensus sequence, only the 3'-terminal CC residues can be considered as potentially required for packaging or replication of rotavirus mRNAs in vivo.

Several studies using the open core replication system have identified cis-acting signals in viral mRNAs that will enhance the synthesis of dsRNA, but unlike the cis-acting signal of the 3'-consensus sequence, are not essential for the synthesis of dsRNA. Most notably, analysis of viral mRNAs containing deletions of all or parts of their 5'-untranslated region (UTR) revealed that this region contains sequences that stimulate RNA replication (Patton et al 1996, 1999, Wentz et al 1996). Similarly, deletion mutagenesis showed that sequences upstream of the 3'-consensus sequence in the 3'-UTR contribute to efficient synthesis ofdsRNA by open cores. Based on the location of cis-acting signals and computer modelling, the ends of the viral mRNAs have been proposed to interact in cis via complementary terminal sequences to form panhandle structures that promote the synthesis of dsRNA (Fig. 3) (Patton et al 1996, Chen & Patton 1998).

Besides the 5'- and 3'-termini, replacement of the open reading frame (ORF) of rotavirus mRNAs with foreign sequences of equivalent size has also been shown to decrease the efficiency of replication of the mRNA template in vitro (Patton et al 1999). However, more recently, an exhaustive analysis performed by deletion mutagenesis of short and overlapping sequences spanning the entire gene 11 mRNA of porcine CN86 rotavirus has determined that not all regions of the ORF play a role in enhancing dsRNA synthesis (J. Patton & M. Jones, unpublished results). Those regions that do play a role are involved in base-pairing between the termini of the mRNA and, thereby, have an impact on the formation and stability of the panhandle structure. From these analyses, it is clear that sequences that contribute to replication can be located both in the UTRs and the ORF of viral mRNAs and that RNA folding is a critical element affecting the ability of the mRNAs to promote minus-strand synthesis.

An important feature of the predicted secondary structures of the rotaviral mRNAs is that within the panhandle structure, the 3'-consensus sequence is either not base-paired or only partially base-paired to the 5'-terminus (Chen & Patton 1998). Mutations introduced into the RNA which increase the extent of complementarity between the 3'-consensus sequence and the 5'-terminus inhibit dsRNA synthesis in the open core replication system. In particular, when the 5'-end of the mRNA is changed so that it is fully complementary to the 3'-consensus sequence, replication of the mRNA in vitro is reduced by more than 100-fold. Hence, the single-stranded nature of the 3'-consensus sequence of the mRNA panhandle is essential for efficient dsRNA synthesis.

Rearrangements and deletions within viral RNAs

Several rotavirus variants have been described with atypical genotypes stemming from sequence duplications or deletions occurring within genome segments encoding VP6, NSP1 (gene 5 product), NSP2, NSP4 or NSP5 (Desselberger 1996). The abnormal segments probably result from the viral RNA polymerase and nascent RNA detaching from the RNA template during plus-strand synthesis, then re-attaching at an upstream or a downstream site on the template where transcription re-initiates (Kojima et al 1996). For genes other than gene 5, sequence duplications begin downstream of the ORF and, hence, the genes still encode a wild-type protein. However, rearrangements occurring within gene 5 can alter the ORF such that the NSP1 product contains a C-terminal truncation, a duplication or a deletion (Fig. 4) (Tian et al 1993, Hua & Patton 1994). Such results indicate that NSP1 is not essential for virus replication (Okada et al 1999). The

Rotavirus Rna

FIG. 3. Predicted secondary structure of the gene 11 mRNA of porcine rotavirus CN86 illustrating the interaction of the 5'- and 3'-termini of the mRNA. The structure was generated with the mfold program (http:\\www.ibc.wustl.edu\ ~%uker). Deletion of residues 1—50, 50—100, 500—550, or 650—664 reduces the efficiency of replication of the mRNA in vitro by greater than twofold suggesting that sequences within the 5'-UTR, 3'-UTR and ORF of the mRNA all may promote dsRNA synthesis.

FIG. 3. Predicted secondary structure of the gene 11 mRNA of porcine rotavirus CN86 illustrating the interaction of the 5'- and 3'-termini of the mRNA. The structure was generated with the mfold program (http:\\www.ibc.wustl.edu\ ~%uker). Deletion of residues 1—50, 50—100, 500—550, or 650—664 reduces the efficiency of replication of the mRNA in vitro by greater than twofold suggesting that sequences within the 5'-UTR, 3'-UTR and ORF of the mRNA all may promote dsRNA synthesis.

Rotavirus Panhandle

FIG. 4. Schematic illustrating rearrangements and deletions of the gene 5 RNA. The size of each atypical RNA and the NSP1 protein encoded by it are given, and the size of its wild-type counterpart RNA and protein is given in parentheses. Sequence duplications are shaded; the sizes of the duplications and deletions are also shown. The A5-10 RNA does not contain a duplication or deletion, but encodes a truncated NSP1 protein due to a point mutation in the ORF.

FIG. 4. Schematic illustrating rearrangements and deletions of the gene 5 RNA. The size of each atypical RNA and the NSP1 protein encoded by it are given, and the size of its wild-type counterpart RNA and protein is given in parentheses. Sequence duplications are shaded; the sizes of the duplications and deletions are also shown. The A5-10 RNA does not contain a duplication or deletion, but encodes a truncated NSP1 protein due to a point mutation in the ORF.

largest of the sequence duplications and deletions that have been described occur in gene 5 and are 1112 and 500 nucleotides long, respectively (Hua & Patton 1994, Okada et al 1999). Since these results indicate that the amount of viral RNA present in virion cores can vary by as much as 1600 nucleotides, a headful mechanism may not operate during RNA packaging. Analysis of bovine rotavirus variants with deletions in the normally 1579 nucleotide gene 5 RNA provide evidence that a 600+ nucleotide stretch from residues 141 to 768 is not required for RNA packaging and replication (Okada et al 1999). Isolation and characterization of many such rearranged genes should provide further information about the location of cis-acting signals in rotavirus mRNAs that promote packaging and replication.

Viralproteinfunction in RNA synthesis

RIs that have replicase activity contain the structural proteins VP1, VP2 and VP3 and the non-structural proteins NSP2 and NSP5 (Fig. 1) (Gallegos & Patton 1989). As reviewed in Table 1, all these proteins have affinity for single-stranded RNA (Labbe et al 1994, Patton & Chen 1999, Taraporewala et al 1999). As core RIs synthesize dsRNA, they simultaneously undergo maturation into double-layered RIs through the acquisition of VP6 (Gallegos & Patton 1989), a protein that is not required for dsRNA synthesis and, based on in vitro assays, has no effect on the activity of the replicase.

Several lines of evidence suggest that VP1 is the viral RNA-dependent RNA polymerase. These include (i) sequence analysis demonstrating that VP1 contains motifs shared among RNA-dependent RNA polymerase (Mitchell & Both 1990b), (ii) experiments showing that VP1 has affinity for nucleotides and that cross-linking of photo-reactable azido-ATP to VP1 inhibits transcription (Valenzuela et al 1991), (iii) electrophoretic gel-shift assays indicating that VP1 has affinity for the 3'-end of viral mRNAs (Patton 1996), and (iv) in vitro replication assays showing that VP1 is a common component of RIs and recombinant viral particles with replicase activity (Gallegos & Patton 1989, Zeng et al 1996). By assaying recombinant proteins for enzymatic functions in vitro, we found that VP1 only exhibited replicase activity in the presence of VP2, the protein that forms the T = 1 shell of the core (Patton et al 1997). Studies with rotavirus ts mutants likewise indicate that VP2 plays an essential role in the formation of RIs with replicase activity (Mansell et al 1994). Deletion mutagenesis has shown that the N-terminus of VP2 is necessary for the interaction of the protein with RNA and VP1, and for replicase activity, but not for the assembly of VP2 into cores (Labbe et al 1994, Lawton et al 1997, Patton et al 1997, Zeng et al 1998). While the precise role of VP2 in dsRNA synthesis is not known, it is possible that pentamers formed by VP2 serve as platforms on which the mRNA template binds and VP1 catalyses minus-strand synthesis (Prasad et al 1996). Analysis of VP3 indicates that this protein contains at least two enzymatic activities, guanylyltransferase and methyltransferase, involved in the capping of rotavirus mRNAs (Pizzaro et al 1991, Chen et al 1999). Because VP3 has affinity for GTP, it may be speculated that the protein plays some role in the formation of the initiation complex for minus-strand synthesis.

The non-structural proteins, NSP2 and NSP5, both accumulate in viroplasms, and indeed, the co-expression of these viral proteins in uninfected cells generates viroplasm-like structures (Fabbretti et al 1999). Earlier studies provided evidence that NSP2 forms homo-multimeric complexes in vivo and interacts with VP1 (Kattoura et al 1994). More recent work with purified recombinant protein has shown that NSP2 self-assembles into stable barrel-shaped octamers of ~12S, consisting of two tetramers of ~7S (Schuck et al 2001). The NSP2 octamers have strong non-specific affinity for single-stranded RNA, and exhibit Mg2+-dependent NTPase activity (Taraporewala et al 1999) and Mg2+-independent helix-destabilizing activity (Taraporewala & Patton 2001). During hydrolysis of NTP by NSP2, the protein undergoes phosphorylation (Taraporewala et al 1999). Together, these properties suggest that NSP2 octamers may serve as molecular motors, catalyzing the packaging of the mRNA templates into cores as dsRNA synthesis occurs.

The recovery of NSP2—NSP5 complexes from infected cells raises the possibility that NSP5 may modulate the activity of NSP2. NSP5 is an O-glycosylated phosphoprotein that self-assembles into dimers and that has non-specific RNA-binding protein activity (Gonzalez & Burrone 1991, P. Vende, Z. Taraporewala & J. Patton, unpublished results). The protein has autokinase activity and exists in several phosphorylated isomers in infected cells (Afrikanova et al 1996, Blackhall et al 1997). When co-expressed in uninfected cells, NSP2 induces the hyperphosphorylation of NSP5 (Afrikanova et al 1998). The mechanism of NSP5 hyperphosphorylation is not known but may involve a cascade of events initiated by the NTPase activity of NSP2. Indeed, the interaction of NSP2 and NSP5 may result in the transfer of phosphate groups generated by the NTPase activity of NSP2 to NSP5, causing NSP5 to undergo hyperphosphorylation. In a recent study, NSP5 was shown to form complexes with NSP6, which like NSP5 is a product of gene 11 and accumulates in viroplasms (Torres-Vega et al 2000).

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    How does rotavirus replicate?
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