Dorothea L. Sawicki

A Contemporary View of Coronavirus Transcription

Coronaviruses are a family of enveloped, plus-stranded RNA viruses with helical nucleocapsids and extraordinarily large genomes. The hallmark of coronavirus transcription is the production of multiple subgenomic mRNAs that contain sequences corresponding to both ends of the genome. (Transcription is

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.

Coronavirus transcription: a perspective.

At the VIth International Symposium on Corona and Related Viruses held in Québec, Canada in 1994 we presented a new model for coronavirus transcription to explain how subgenome-length minus strands, which are used as templates for the synthesis of subgenomic mRNAs, might arise by a process involving discontinuous RNA synthesis. The old model explaining subgenomic mRNA synthesis, which was called leader-primed transcription, was based on erroneous evidence that only genome-length negative strands were present in replicative intermediates. To explain the discovery of subgenome-length minus strands, a related model, called the replicon model, was proposed: The subgenomic mRNAs would be produced initially by leader-primed transcription and then replicated into minus-strand templates that would in turn be transcribed into subgenomic mRNAs. We review the experimental evidence that led us to formulate a third model proposing that the discontinuous event in coronavirus RNA synthesis occurs during minus strand synthesis. With our model the genome is copied both continuously to produce minus-strand templates for genome RNA synthesis and discontinuously to produce minus-strand templates for subgenomic mRNA synthesis, and the subgenomic mRNAs do not function as templates for minus strand synthesis, only the genome does.

Role for nsP2 Proteins in the Cessation of Alphavirus Minus-Strand Synthesis by Host Cells

ABSTRACT In order to establish nonlytic persistent infections (PI) of BHK cells, replicons derived from Sindbis (SIN) and Semliki Forest (SFV) viruses have mutations in nsP2. Five different nsP2 PI replicons were compared to wild-type (wt) SIN, SFV, and wt nsPs SIN replicons. Replicon PI BHK21 cells had viral RNA synthesis rates that were less than 5% of those of the wt virus and ∼10% or less of those of SIN wt replicon-infected cells, and, in contrast to wt virus and replicons containing wt nsP2, all showed a phenotype of continuous minus-strand synthesis and of unstable, mature replication/transcription complexes (RC+) that are active in plus-strand synthesis. Minus-strand synthesis and incorporation of [3H]uridine into replicative intermediates differed among PI replicons, depending on the location of the mutation in nsP2. Minus-strand synthesis by PI cells appeared normal; it was dependent on continuous P123 and P1234 polyprotein synthesis and ceased when protein synthesis was inhibited. The failure by the PI replicons to shut off minus-strand synthesis was not due to some defect in the PI cells but rather was due to the loss of some function in the mutated nsP2. This was demonstrated by showing that superinfection of PI cells with wt SFV triggered the shutdown of minus-strand synthesis, which we believe is a host response to infection with alphaviruses. Together, the results indicate alphavirus nsP2 functions to engage the host response to infection and activate a switch from the early-to-late phase. The loss of this function leads to continuous viral minus-strand synthesis and the production of unstable RC+.

A New Role for ns Polyprotein Cleavage in Sindbis Virus Replication

ABSTRACT One of the distinguishing features of the alphaviruses is a sequential processing of the nonstructural polyproteins P1234 and P123. In the early stages of the infection, the complex of P123+nsP4 forms the primary replication complexes (RCs) that function in negative-strand RNA synthesis. The following processing steps make nsP1+P23+nsP4, and later nsP1+nsP2+nsP3+nsP4. The latter mature complex is active in positive-strand RNA synthesis but can no longer produce negative strands. However, the regulation of negative- and positive-strand RNA synthesis apparently is not the only function of ns polyprotein processing. In this study, we developed Sindbis virus mutants that were incapable of either P23 or P123 cleavage. Both mutants replicated in BHK-21 cells to levels comparable to those of the cleavage-competent virus. They continuously produced negative-strand RNA, but its synthesis was blocked by the translation inhibitor cycloheximide. Thus, after negative-strand synthesis, the ns proteins appeared to irreversibly change conformation and formed mature RCs, in spite of the lack of ns polyprotein cleavage. However, in the cells having no defects in α/β interferon (IFN-α/β) production and signaling, the cleavage-deficient viruses induced a high level of type I IFN and were incapable of causing the spread of infection. Moreover, the P123-cleavage-deficient virus was readily eliminated, even from the already infected cells. We speculate that this inability of the viruses with unprocessed polyprotein to productively replicate in the IFN-competent cells and in the cells of mosquito origin was an additional, important factor in ns polyprotein cleavage development. In the case of the Old World alphaviruses, it leads to the release of nsP2 protein, which plays a critical role in inhibiting the cellular antiviral response.

Functional Analysis of nsP3 Phosphoprotein Mutants of Sindbis Virus

ABSTRACT Alphavirus nsP3 phosphoprotein is essential for virus replication and functions initially within polyprotein P123 or P23 components of the short-lived minus-strand replicase, and upon polyprotein cleavage, mature nsP3 likely functions also in plus-strand synthesis. We report the identification of a second nsP3 mutant from among the A complementation group of Sindbis virus (SIN) heat-resistant strain, ts RNA-negative mutants. The ts138 mutant possessed a change of G4303 to C, predicting an Ala68-to-Gly alteration that altered a conserved His-Ala-Val tripeptide in the ancient (pre-eukaryotic),“ X” or histone 2A phosphoesterase-like macrodomain that in SIN encompasses nsP3 residues 1 to 161 and whose role is unknown. We undertook comparative analysis of three nsP3 N-terminal region mutants and observed (i) that nsP3 and nsP2 functioned initially as a single unit as deduced from complementation analysis and in agreement with our previous studies, (ii) that the degree of phosphorylation varied among the nsP3 mutants, and (iii) that reduced phosphorylation of nsP3 correlated with reduced minus-strand synthesis. The most striking phenotype was exhibited by ts4 (Ala268 to Val), which after shift to 40°C made significantly underphosphorylated P23/nsP3 and lost selectively the ability to make minus strands. After shift to 40°C, mutant ts7 (Phe312 to Ser) made phosphorylated P23/nsP3 and minus strands but failed to increase plus-strand synthesis. Macrodomain mutant ts138 was intermediate, making at 40°C partially phosphorylated P23/nsP3 and reduced amounts of minus strands. The mutants were able to assemble their nsPs at 40°C into complexes that were membrane associated. Our analyses argue that P23/P123 phosphorylation is affected by macrodomain and Ala268 region sequences and in turn affects the efficient transcription of the alphavirus genome.

A sindbis virus mutant temperature-sensitive in the regulation of minus-strand RNA synthesis

Abstract The synthesis of Sindbis virus (SIN) minus-strand RNA which is utilized as a template for the synthesis of 49 S plus-strand RNA and the 26 S mRNA is normally short-lived and regulated. Minus-strand RNA synthesis ceases 3–4 hr after infection or quickly after the inhibition of protein synthesis; this is in contrast to plus-strand RNA synthesis which continues throughout the infectious cycle and is resistant to inhibition of protein synthesis. In cells infected with SIN HR ts24, a member of the A complementation group, the synthesis of minus-strand RNA ceased normally at 6−8 hr p.i. when the infection was at 28°, the permissive temperature. However, shifting SIN HR ts24-infected cells to the nonpermissive temperature, 39°, during the early phase of the infectious cycle resulted in the failure to turn off minus-strand transcription, the accumulation of replicative intermediates, and the loss of sensitivity of minus-strand RNA synthesis to inhibition of protein synthesis. Furthermore, shifting SIN HR ts24-infected cells to the nonpermissive temperature late in the infectious cycle, after minus-strand synthesis had ceased normally at 28°, resulted in the resumption of minus-strand synthesis, even in the absence of protein synthesis. Thus, SIN HR ts24 appears to have a temperature-sensitive lesion in a viral polypeptide that acts to turn off minus-strand synthesis and that may be responsible for the normal short-lived nature of the minus-strand polymerase.

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.

Alphavirus Minus-Strand Synthesis and Persistence in Mouse Embryo Fibroblasts Derived from Mice Lacking RNase L and Protein Kinase R

ABSTRACT We report our studies to probe the possible role of the host response to double-stranded RNA in cessation of alphavirus minus-strand synthesis. Mouse embryo fibroblasts (MEF) from Mx1-deficient mice that also lack either the protein kinase R (PKR) or the latent RNase L or both PKR and RNase L were screened. In RNase L-deficient but not wild-type or PKR-deficient MEF, there was continuous synthesis of minus-strand templates and the formation of new replication complexes producing viral plus strands. Inhibiting translation caused minus-strand synthesis to stop and a loss of transcription activity of the mature replication complexes. This turnover of replication complexes that were stable in cells containing RNase L suggested that RNase L plays some role, albeit possibly indirect, in the formation of stable replication complexes during alphavirus infection. In addition, confluent monolayers of RNase L-deficient murine cells readily established persistent infections and were not killed. This phenotype is contrary to what has been observed for infection in vertebrate cells with a presumably functional RNase L gene and more resembled alphavirus replication in Aedes mosquito cells, in which the activity of replication complexes making plus stands was also found to decay with inhibition of translation.

Modification of Asn374 of nsP1 Suppresses a Sindbis Virus nsP4 Minus-Strand Polymerase Mutant

ABSTRACT Our recent study (C. L. Fata, S. G. Sawicki, and D. L. Sawicki, J. Virol. 76:8632-8640, 2002) found minus-strand synthesis to be temperature sensitive in vertebrate and invertebrate cells when the Arg183 residue of the Sindbis virus nsP4 polymerase was changed to Ser, Ala, or Lys. Here we report the results of studies identifying an interacting partner of the region of the viral polymerase containing Arg183 that suppresses the Ser183 codon mutation. Large-plaque revertants were observed readily following growth of the nsP4 Ser183 mutant at 40°C. Fifteen revertants were characterized, and all had a mutation in the Asn374 codon of nsP1 that changed it to either a His or an Ile codon. When combined with nsP4 Ser183, substitution of either His374 or Ile374 for Asn374 restored wild-type growth in chicken embryo fibroblast (CEF) cells at 40°C. In Aedes albopictus cells at 34.5°C, neither nsP1 substitution suppressed the nsP4 Ser183 defect in minus-strand synthesis. This argued that the nsP4 Arg183 residue itself is needed for minus-strand replicase assembly or function in the mosquito environment. The nsP1 His374 suppressor when combined with the wild-type nsP4 gave greater than wild-type levels of viral RNA synthesis in CEF cells at 40°C (∼140%) and in Aedes cells at 34.5°C (200%). Virus producing nsP1 His374 and wild-type nsP4 Arg183 made more minus strands during the early period of infection and before minus-strand synthesis ceased at about 4 h postinfection. Shirako et al. (Y. Shirako, E. G. Strauss, and J. H. Strauss, Virology 276:148-160, 2000) identified amino acid substitutions in nsP1 and nsP4 that suppressed mutations that changed the N-terminal Tyr of nsP4. The nsP4 N-terminal mutants were defective also in minus-strand synthesis. Our study implicates an interaction between another conserved nsP1 region and an internal region, predicted to be in the finger domain, of nsP4 for the formation or activity of the minus-strand polymerase. Finally, the observation that a single point mutation in nsP1 results in minus-strand synthesis at greater than wild-type levels supports the concept that the wild-type nsP sequences are evolutionary compromises.