Role of the nonstructural polyproteins in alphavirus RNA synthesis.
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Alphavirus↗
Biomedical subjects
D L Sawicki
Publications and source records attributed to D L Sawicki.
A new model for coronavirus transcription.
Coronaviruses contain an unusually long (27-32,000 ribonucleotide) positive sense RNA genome that is polyadenylated at the 3' end and capped at the 5' end. In addition to the genome, infected cells contain subgenomic mRNAs that form a 3' co-terminal nested set with the genome. In addition to their common 3' ends, the genome and the subgenomic mRNAs contain an identical 5' leader sequence. The transcription mechanism that coronaviruses use to produce subgenomic mRNA is not known and has been the subject of speculation since sequencing of the subgenomic mRNAs showed they must arise by discontinuous transcription. The current model called leader-primed transcription has subgenomic mRNAs transcribed directly from genome-length negative strands. It was based on the failure to find in coronavirus infected cells subgenome-length negative strands or replication intermediates containing subgenome-length negative strands. Clearly, these structures exist in infected cells and are transcriptionally active. We proposed a new model for coronavirus transcription which we called 3' discontinuous extension of negative strands. This model predicts that subgenome-length negative strands would be derived directly by transcription using the genome RNA as a template. The subgenome-length templates would contain the common 5' leader sequence and serve as templates for the production of subgenomic mRNAs. Our findings include showing that: 1. Replication intermediates (RIs) containing subgenome-length RNA exist in infected cells and are separable from RIs with genome-length templates. The RFs with subgenome-length templates are not derived by RNase treatment of RIs with genome-length templates. 2. The subgenome-length negative strands are formed early in infection when RIs are accumulating and the rate of viral RNA synthesis is increasing exponentially. 3. Subgenome-length negative strands contain at their 3' ends a complementary copy of the 72 nucleotide leader RNA that is found in the genome only at their 5' end. 4. RIs with subgenomic templates serve immediately as templates for transcription of subgenomic mRNAs. Because subgenomic mRNAs are not replicated, i.e., copied into negative strands that in turn are used as templates for subgenomic mRNA synthesis, we propose that the subgenome-length negative strands must arise directly by transcription of the genome and acquire their common 3' anti-leader sequence after polymerase jumping from the intergenic regions to the leader sequence at the 5' end of the genome. This would make negative strand synthesis discontinuous and subgenomic mRNA synthesis continuous, which is the opposite of what was proposed in the leader primed model.
Animals↗
Regulation of alphavirus 26S mRNA transcription by replicase component nsP2.
Semliki Forest virus (SFV) mutant ts4 has a reversible temperature-sensitive defect in the synthesis the subgenomic 26S mRNA. The viral nonstructural protein nsP2 was identified as a regulator of 26S synthesis by transferring nsP2 coding sequences from ts4 into the infectious SFV cDNA clone (SFoto) to create SFots4. Sequencing identified the causal mutation as C4038U, predicting the amino acid change M781T in nsP2. A revertant was isolated in which a back mutation of U to C restored the wild-type phenotype. Compared to Sindbis virus nsP2 mutants ts15, ts17, ts18, ts24 and ts133, which also exhibit temperature-sensitive 26S RNA synthesis, ts4 and SFots4 reduced 26S RNA synthesis faster and to lower levels after temperature shift. Under these conditions, ts4 and SFots4 also displayed complete conversion of RFII+RFIII into RFI and reactivated minus-strand synthesis. After shift to 39 degrees C, ts4 nsP2 was released from a crude RNA polymerase preparation consisting of membranes sedimenting at 15,000 g (P15) and the remaining, unreleased nsP2 was capable of being cross-linked in almost equimolar ratio with nsP1 and nsP3. This supports the hypothesis that nsP2 binds directly or undirectly to the promoter for 26S RNA and that it is also an essential component of the viral replicase synthesizing 42S RNA plus strands. Only the former activity is temperature-sensitive in ts4 mutant.
Alphavirus↗
Sindbis virus RNA-negative mutants that fail to convert from minus-strand to plus-strand synthesis: role of the nsP2 protein.
We identified mutations in the gene for nsP2, a nonstructural protein of the alphavirus Sindbis virus, that appear to block the conversion of the initial, short-lived minus-strand replicase complex (RCinitial) into mature, stable forms that are replicase and transcriptase complexes (RCstable), producing 49S genome or 26S mRNA. Base changes at nucleotide (nt) 2166 (G-->A, predicting a change of Glu-163-->Lys), at nt 2502 (G-->A, predicting a change of Val-275-->Ile), and at nt 2926 (C-->U, predicting a change of Leu-416-->Ser) in the nsP2 N domain were responsible for the phenotypes of ts14, ts16, and ts19 members of subgroup 11 (D.L. Sawicki and S.G. Sawicki, Virology 44:20-34, 1985) of the A complementation group of Sindbis virus RNA-negative mutants. Unlike subgroup I mutants, the RCstable formed at 30 degrees C transcribed 26S mRNA normally and did not synthesize minus strands in the absence of protein synthesis after temperature shift. The N-domain substitutions did not inactivate the thiol protease in the C domain of nsP2 and did not stop the proteolytic processing of the polyprotein containing the nonstructural proteins. The distinct phenotypes of subgroup I and 11 A complementation group mutants are evidence that the two domains of nsP2 are essential and functionally distinct. A detailed analysis of ts14 found that its nsPs were synthesized, processed, transported, and assembled at 40 degrees C into complexes with the properties of RCinitial and synthesized minus strands for a short time after shift to 40 degrees C. The block in the pathway to the formation of RCstable occurred after cleavage of the minus-strand replicase P123 or P23 polyprotein into mature nsP1, nsP2, nsP3, and nsP4, indicating that structures resembling RCstable, were formed at 40 degrees C. However, these RCstable or pre-RCstable structures were not capable of recovering activity at 30 degrees C. Therefore, failure to increase the rate of plus-strand synthesis after shift to 40 degrees C appears to result from failure to convert RCinitial to RCstable. We conclude that RCstable is derived from RCinitial by a conversion process and that ts14 is a conversion mutant. From their similar phenotypes, we predict that other nsP2 N-domain mutants are blocked also in the conversion of RCinitial to RCstable. Thus, the N domain of nsP2 plays an essential role in a folding pathway of the nsPs responsible for formation of the initial minus-strand replicase and for its conversion into stable plus-strand RNA-synthesizing enzymes.
Amino Acid Sequence↗
Coronaviruses use discontinuous extension for synthesis of subgenome-length negative strands.
We have developed a new model for coronavirus transcription, which we call discontinuous extension, to explain how subgenome-length negatives stands are derived directly from the genome. The current model called leader-primed transcription, which states that subgenomic mRNA is transcribed directly from genome-length negative-strands, cannot explain many of the recent experimental findings. For instance, subgenomic mRNAs are transcribed directly via transcription intermediates that contain subgenome-length negative-strand templates; however subgenomic mRNA does not appear to be copied directly into negative strands. In our model the subgenome-length negative strands would be derived using the genome as a template. After the polymerase had copied the 3'-end of the genome, it would detach at any one of the several intergenic sequences and reattach to the sequence immediately downstream of the leader sequence at the 5'-end of genome RNA. Base pairing between the 3'-end of the nascent subgenome-length negative strands, which would be complementary to the intergenic sequence at the end of the leader sequence at the 5'-end of genome, would serve to align the nascent negative strand to the genome and permit the completion of synthesis, i.e., discontinuous extension of the 3'-end of the negative strand. Thus, subgenome-length negative strands would arise by discontinuous synthesis, but of negative strands, not of positive strands as proposed originally by the leader-primed transcription model.
Animals↗
Alphavirus positive and negative strand RNA synthesis and the role of polyproteins in formation of viral replication complexes.
The genome of alphaviruses is translated into polyproteins that are processed into a viral replicase that produces both negative and positive strands. In infected cells, negative strand synthesis is short-lived and occurs only early, whereas positive strand synthesis is stable and occurs both early and late. Analysis of temperature sensitive mutants indicated: nsP1 functioned in the initiation of transcription; nsP3 acted to form initial transcription complexes; and nsP2 and nsP4 first recognized positive strands as templates and then made negative strands the preferred templates. While nsP4 and nsP1 individually rescued early defects in transcription, nsP2 and nsP3 acted initially in cis. We interpret our results to suggest nsP1234 was cleaved to nsP4, nsP1 and nsP23, bound a positive strand and synthesized a negative strand. Cleavage of P23 or other modifications to nsP2 and nsP4 convert the initial transcription complex to a stable complex that synthesizes positive strands. Negative strand synthesis is unstable because of the failure to form initial transcription complexes after host factors that are part of the replicase are depleted or the half-life of polyprotein precursors like P23 is shortened.
Alphavirus↗
Alphavirus nsP3 functions to form replication complexes transcribing negative-strand RNA.
The alphavirus mutant Sindbis virus HR ts4, which has been assigned to the A complementation group, possessed a selective defect in negative-strand synthesis that was similar although not identical to that observed for the B complementation group mutant ts11 (Y.-F. Wang, S. G. Sawicki, and D. L. Sawicki, J. Virol. 65:985-988, 1991). The causal mutation was identified as a change of a C to a U residue at nucleotide 4903 in the nsP3 open reading frame that predicted a change of Ala-268 to Val. Thus, both nsP3 and nsP1 play a role selectively in the transcription of negative strands early in infection. The assignment of the mutation carried by an A complementation group mutant of Sindbis virus HR to nsP3 was unexpected, as mutations in other A complementation group mutants studied to date mapped to nsP2. Another mutant with a conditionally lethal mutation, ts7 of the G complementation group, also possessed a causal mutation resulting from a single-residue change in nsP3. Negative-strand synthesis ceased more slowly after a shift to the nonpermissive temperature in ts7-than in ts4-infected cells, and ts7 complemented ts11, but ts4 did not. However, the nsP3 of both ts4 and ts7 allowed reactivation of negative-strand synthesis by stable replication complexes containing nsP4 from ts24. Therefore, mutations in nsP3 affected only early events in replication and probably prevent the formation and/or function of the initial replication complex that synthesizes its negative-strand template. Because neither ts4 nor ts7 complemented 10A complementation group mutants, the genes for nsP2 and nsP3 function initially as a single cistron. We interpret these findings and present a model to suggest that the initial alphavirus replication complex is formed from tightly associated nsP2 and nsP3, perhaps in the form of P23, and proteolytically processed and trans-active nsP4 and nsP1.
Alanine↗
A second nonstructural protein functions in the regulation of alphavirus negative-strand RNA synthesis.
Previous studies (D.L. Sawicki, D. B. Barkhimer, S. G. Sawicki, C. M. Rice, and S. Schlesinger, Virology 174:43-52, 1990) identified a temperature-sensitive (ts) defect in Sindbis virus nonstructural protein 4 (nsP4) that reactivated negative-strand synthesis after its normal cessation at the end of the early phase of replication. We now report identification of two different ts alterations in nsP2 of Ala-517 to Thr in ts17 or Asn-700 to Lys in ts133 that also reactivated negative-strand synthesis. These same mutations caused severely reduced protease processing by nsP2 and recognition of the internal promoter for subgenomic mRNA synthesis and were responsible for the conditional lethality and RNA negativity of these mutants. Reactivation of negative-strand synthesis by mutations in nsP2 resembled that in nsP4: it was a reversible property of stable replication complexes and did not require continuation of viral protein synthesis. Recombinant viruses expressing both mutant nsP2 and nsP4 reactivated negative-strand synthesis more efficiently than did either mutant protein alone, consistent with the hypothesis that both nsP2 and nsP4 participate in template recognition. We propose that these alterations cause nsP2 and nsP4 to switch from their normal preference to recognize negative strands as templates to recognize positive strands and thereby mimic the initial formation of a replication complex.
Alphavirus↗
Sindbis virus nsP1 functions in negative-strand RNA synthesis.
A mutation at nucleotide 1101 of Sindbis virus ts11 nsP1 caused temperature-sensitive negative-strand synthesis and suppressed the 24R phenotype, which is caused by a mutation in nsP4. Nonstructural proteins synthesized and accumulated by ts11 at 40 degrees C did not cause the reactivation of negative-strand synthesis upon return to 30 degrees C and did not prevent the formation of new replication complexes at 30 degrees C.
Animals↗
Solubilization and immunoprecipitation of alphavirus replication complexes.
Alphavirus replication complexes that are located in the mitochondrial fraction of infected cells which pellets at 15,000 x g (P15 fraction) were used for the in vitro synthesis of viral 49S genome RNA, subgenomic 26S mRNA, and replicative intermediates (RIs). Comparison of the polymerase activity in P15 fractions from Sindbis virus (SIN)- and Semliki Forest virus (SFV)-infected cells indicated that both had similar kinetics of viral RNA synthesis in vitro but the SFV fraction was twice as active and produced more labeled RIs than SIN. When assayed in vitro under conditions of high specific activity, which limits incorporation into RIs, at least 70% of the polymerase activity was recovered after detergent treatment. Treatment with Triton X-100 or with Triton X-100 plus deoxycholate (DOC) solubilized some prelabeled SFV RIs but little if any SFV or SIN RNA polymerase activity from large structures that also contained cytoskeletal components. Treatment with concentrations of DOC greater than 0.25% or with 1% Triton X-100-0.5% DOC in the presence of 0.5 M NaCl released the polymerase activity in a soluble form, i.e., it no longer pelleted at 15,000 x g. The DOC-solubilized replication complexes, identified by their polymerase activity in vitro and by the presence of prelabeled RI RNA, had a density of 1.25 g/ml, were 20S to 100S in size, and contained viral nsP1, nsP2, phosphorylated nsP3, nsP4, and possibly nsP34 proteins. Immunoprecipitation of the solubilized structures indicated that the nonstructural proteins were complexed together and that a presumed cellular protein of approximately 120 kDa may be part of the complex. Antibodies specific for nsP3, and to a lesser extent antibodies to nsP1, precipitated native replication complexes that retained prelabeled RIs and were active in vitro in viral RNA synthesis. Thus, antibodies to nsP3 bound but did not disrupt or inhibit the polymerase activity of replication complexes in vitro.
Alphavirus↗
Coronavirus transcription: subgenomic mouse hepatitis virus replicative intermediates function in RNA synthesis.
Both genomic and subgenomic replicative intermediates (RIs) and replicative-form (RF) structures were found in 17CL1 mouse cells that had been infected with the A59 strain of mouse hepatitis virus (MHV), a prototypic coronavirus. Seven species of RNase-resistant RF RNAs, whose sizes were consistent with the fact that each was derived from an RI that was engaged in the synthesis of one of the seven MHV positive-strand RNAs, were produced by treatment with RNase A. Because the radiolabeling of the seven RF RNAs was proportional to that of the corresponding seven positive-strand RNAs, the relative rate of synthesis of each of the MHV positive-strand RNAs may be controlled by the relative number of each of the size classes of RIs that are produced. In contrast to alphavirus, which produced its subgenome-length RF RNAs from genome-length RIs, MHV RF RNAs were derived from genome- and subgenome-length RIs. Only the three largest MHV RF RNAs (RFI, RFII, and RFIII) were derived from the RIs that migrated slowest on agarose gels. The four smallest RF RNAs (RFIV, RFV, RFVI, and RFVII) were derived from RIs that migrated in a broad region of the gel that extended from the position of 28S rRNA to the position of the viral single-stranded MHV mRNA-3. Because all seven RIs were labeled during very short pulses with [3H]uridine, we concluded that the subgenome-length RIs are transcriptionally active. These findings, with the recent report of the presence of subgenome-length negative-strand RNAs in cells infected with porcine transmissible gastroenteritis virus (P. B. Sethna, S.-L. Hung, and D. A. Brian, Proc. Natl. Acad. Sci. USA 86: 5626-5630, 1989), strongly suggest that coronaviruses utilize a novel replication strategy that employs the synthesis of subgenomic negative strands to produce subgenomic mRNAs.
Animals↗
Demonstration in vitro of temperature-sensitive elongation of RNA in Sindbis virus mutant ts6.
Characterization of conditionally lethal mutants of alphaviruses, Sindbis virus and Semliki Forest virus, has indicated that in almost all the RNA-negative mutants the temperature-sensitive (ts) defect prevents the formation of active transcription complexes at nonpermissive temperature (40 degrees C), but such complexes retain activity at 40 degrees C if formed first at permissive temperature (30 degrees C). Our recent results have extended the characterization of one exception to this finding: Sindbis ts6 transcription complexes, once formed at 30 degrees C, do not function at 40 degrees C. We used an in vitro assay for viral RNA synthesis to determine whether the ts defect was the result of dissociation of the complex or of a failure to elongate RNA chains in a stable complex. Our results indicated that the phenotype of ts6 observed in vivo was retained in vitro. In vivo incorporation into single-stranded 49S and 26S RNA was inhibited simultaneously with its incorporation into replicative intermediates upon shifting ts6-infected cells to 40 degrees C, which was compatible with a defect in elongation. Complexes formed at 30 degrees C and inactivated in vivo by shifting to 40 degrees C were reactivated by incubation in vitro at 30 degrees C but not at 40 degrees C. Thus, the transcription complexes were stable. Nascent RNA chains initiated in vivo and pulse-labeled in vitro were chased into single-stranded 49S and 26S RNA only when incubation was at 30 degrees C, indicating that the ts6 transcription complex was temperature sensitive in elongation. It should be possible to study in vitro other alphavirus RNA-negative mutants that demonstrate a change in viral RNA synthesis after shift to 40 degrees C. These would include ts mutants in the synthesis of subgenomic 26S mRNA and of minus-strand RNA.
Animals↗
The effect of overproduction of nonstructural proteins on alphavirus plus-strand and minus-strand RNA synthesis.
We determined the effect of the overproduction of viral nonstructural proteins on alphavirus plus-strand and minus-strand RNA synthesis. Because alphavirus minus-strand synthesis ceases normally at 3 to 4 hr postinfection and requires continuous protein synthesis [D. L. Sawicki and S. G. Sawicki, J. Virol. 34, 108-118 (1980); D. L. Sawicki, S. G. Sawicki, S. Keranen, and L. Kaariainen, J. Virol. 39, 348-358 (1981a)], we determined if the cessation of minus-strand synthesis was the result of the failure to continue synthesis of viral nonstructural proteins after 3-4 hr postinfection and if the overproduction of viral nonstructural proteins would increase the rate of plus-strand synthesis. Cells infected with ts1, an RNA-positive mutant of Semliki Forest virus (SFV) which overproduced the viral nonstructural proteins and underproduced the viral structural proteins at the nonpermissive temperature, did not cause the synthesis of increased amounts of viral minus strands relative to parental SFV and did not affect the time at which minus-strand synthesis ceased. All four viral nonstructural proteins were synthesized at early and late times after infection in the same relative proportions. The overproduction and the continued synthesis of nonstructural proteins late in infection did not increase the maximal rate of plus-strand synthesis above that in wild-type SFV-infected cells.
Alphavirus↗
The effect of loss of regulation of minus-strand RNA synthesis on Sindbis virus replication.
During the replication cycle of Sindbis virus minus-strand synthesis stops normally at the time that plus-strand synthesis reaches a maximum rate. We have isolated and characterized revertants of ts24, a member of the A complementation group of Sindbis HR mutants, that we had demonstrated previously to have a temperature-sensitive defect in the regulation of minus-strand synthesis. These revertants of ts24 replicated efficiently at 40 degrees but nevertheless retained the temperature sensitive defect in the regulation of minus-strand synthesis: they continued to synthesize minus strands late in the replication cycle at 40 degrees but not at 30 degrees and in the presence or absence of protein synthesis. Although failure to regulate the synthesis of minus strands resulted in continuous minus-strand synthesis and in the accumulation of minus strands, the rate of plus-strand synthesis was not increased concertedly. Minus strands synthesized after the rate of plus-strand synthesis had become constant were demonstrated to be utilized as templates for 26 S mRNA synthesis. Thus, the change from an increasing to a constant rate of plus-strand synthesis during the alphavirus replication cycle cannot be governed solely by the number of minus strands that accumulate in infected cells. We present a model for the preferential utilization of minus strands as a mechanism for the cessation of minus-strand synthesis that occurs normally during alphavirus replication.
Animals↗
Coronavirus minus-strand RNA synthesis and effect of cycloheximide on coronavirus RNA synthesis.
The temporal sequence of coronavirus plus-strand and minus-strand RNA synthesis was determined in 17CL1 cells infected with the A59 strain of mouse hepatitis virus (MHV). MHV-induced fusion was prevented by keeping the pH of the medium below pH 6.8. This had no effect on the MHV replication cycle, but gave 5- to 10-fold-greater titers of infectious virus and delayed the detachment of cells from the monolayer which permitted viral RNA synthesis to be studied conveniently until at least 10 h postinfection. Seven species of poly(A)-containing viral RNAs were synthesized at early and late times after infection, in nonequal but constant ratios. MHV minus-strand RNA synthesis was first detected at about 3 h after infection and was found exclusively in the viral replicative intermediates and was not detected in 60S single-stranded form in infected cells. Early in the replication cycle, from 45 to 65% of the [3H]uridine pulse-labeled RF core of purified MHV replicative intermediates was in minus-strand RNA. The rate of minus-strand synthesis peaked at 5 to 6 h postinfection and then declined to about 20% of the maximum rate. The addition of cycloheximide before 3 h postinfection prevented viral RNA synthesis, whereas the addition of cycloheximide after viral RNA synthesis had begun resulted in the inhibition of viral RNA synthesis. The synthesis of both genome and subgenomic mRNAs and of viral minus strands required continued protein synthesis, and minus-strand RNA synthesis was three- to fourfold more sensitive to inhibition by cycloheximide than was plus-strand synthesis.
Animals↗
Functional analysis of the A complementation group mutants of Sindbis HR virus.
The 10 members of the A complementation group of temperature-sensitive (ts) mutants of SIN HR, the heat-resistant strain of Sindbis virus, were divided into two phenotypic subgroups. Subgroup I mutants (ts15, ts17, ts21, ts24, and ts133) demonstrated temperature-sensitive 26 S mRNA synthesis, whereas subgroup II mutants (ts4, ts14, ts16, ts19, and ts138) did not; both ts4 and ts19 demonstrated defective 26 S mRNA synthesis at 30 degrees. None of the A group mutants demonstrated temperature-sensitive 49 S plus-strand synthesis. Temperature-sensitive cleavage of ns230 was demonstrated by subgroup I mutants, except ts21, but not by subgroup II mutants. A revertant of ts133 that grew at 40 degrees retained temperature-sensitive 26 S mRNA synthesis but lost temperature-sensitive cleavage of ns230 and the RNA-negative phenotype. Only ts4, like ts11 of the B complementation group, demonstrated temperature-sensitive minus-strand RNA synthesis. In addition to ts24, cells infected with ts17 or ts133 continued to synthesize minus strands after shiftup in the absence of continued protein synthesis, and resumed synthesis of minus strands if shifted to the nonpermissive temperature after minus-strand synthesis had ceased at the permissive temperature.
Animals↗
A Sindbis virus mutant temperature-sensitive in the regulation of minus-strand RNA synthesis.
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Cycloheximide↗
Specific Sindbis virus-coded function for minus-strand RNA synthesis.
The synthesis of minus-strand RNA was studied in cell cultures infected with the heat-resistant strain of Sindbis virus and with temperature-sensitive (ts) belonging to complementation groups A, B, F, and G, all of which exhibited an RNA-negative (RNA-) phenotype when infection was initiated and maintained at 39 degrees C, the nonpermissive temperature. When infected cultures were shifted from 28 degrees C (the permissive temperature) to 39 degrees C at 3 h postinfection, the synthesis of viral minus-strand RNA ceased in cultures infected with ts mutants of complementation groups B and F, but continued in cultures infected with the parental virus and mutans of complementation groups A and G. In cultures infected with ts11 of complementation group B, the synthesis of viral minus-strand RNA ceased, whereas the synthesis of 42S and 26S plus-strand RNAs continued for at least 5 h after the shift to 39 degrees C. However, when ts11-infected cultures were returned to 28 degrees C 1 h after the shift to 39 degrees C, the synthesis of viral minus-strand RNA resumed, and the rate of viral RNA synthesis increased. The recovery of minus-strand synthesis translation of new proteins. We conclude that at least one viral function is required for alphavirus minus-strand synthesis that is not required for plus-strand synthesis. In cultures infected with ts6 of complementation group F, the syntheses of both viral plus-strand and minus-strand RNAs were drastically reduced after the shift to 39 degrees C. Since ts6 failed to synthesize both plus-strand and minus-strand RNAs after the shift to 39 degrees C, at least one common viral component appears to be required for the synthesis of both minus-strand and plus-strand RNAs.
Animals↗