De novo initiation of RNA synthesis by the RNA-dependent RNA polymerase (NS5B) of hepatitis C virus.

Hepatitis C virus (HCV) NS5B protein possesses an RNA-dependent RNA polymerase (RdRp) activity, a major function responsible for replication of the viral RNA genome. To further characterize the RdRp activity, NS5B proteins were expressed from recombinant baculoviruses, purified to near homogeneity, and examined for their ability to synthesize RNA in vitro. As a result, a highly active NS5B RdRp (1b-42), which contains an 18-amino acid C-terminal truncation resulting from a newly created stop codon, was identified among a number of independent isolates. The RdRp activity of the truncated NS5B is comparable to the activity of the full-length protein and is 20 times higher in the presence of Mn(2+) than in the presence of Mg(2+). When a 384-nucleotide RNA was used as the template, two major RNA products were synthesized by 1b-42. One is a complementary RNA identical in size to the input RNA template (monomer), while the other is a hairpin dimer RNA synthesized by a "copy-back" mechanism. Substantial evidence derived from several experiments demonstrated that the RNA monomer was synthesized through de novo initiation by NS5B rather than by a terminal transferase activity. Synthesis of the RNA monomer requires all four ribonucleotides. The RNA monomer product was verified to be the result of de novo RNA synthesis, as two expected RNA products were generated from monomer RNA by RNase H digestion. In addition, modification of the RNA template by the addition of the chain terminator cordycepin at the 3' end did not affect synthesis of the RNA monomer but eliminated synthesis of the self-priming hairpin dimer RNA. Moreover, synthesis of RNA on poly(C) and poly(U) homopolymer templates by 1b-42 NS5B did not require the oligonucleotide primer at high concentrations (>/=50 microM) of GTP and ATP, further supporting a de novo initiation mechanism. These findings suggest that HCV NS5B is able to initiate RNA synthesis de novo.

Template-dependent initiation of Sindbis virus RNA replication in vitro.

Recent insights into the early events in Sindbis virus RNA replication suggest a requirement for either the P123 or P23 polyprotein, as well as mature nsP4, the RNA-dependent RNA polymerase, for initiation of minus-strand RNA synthesis. Based on this observation, we have succeeded in reconstituting an in vitro system for template-dependent initiation of SIN RNA replication. Extracts were isolated from cells infected with vaccinia virus recombinants expressing various SIN proteins and assayed by the addition of exogenous template RNAs. Extracts from cells expressing P123C>S, a protease-defective P123 polyprotein, and nsP4 synthesized a genome-length minus-sense RNA product. Replicase activity was dependent upon addition of exogenous RNA and was specific for alphavirus plus-strand RNA templates. RNA synthesis was also obtained by coexpression of nsP1, P23C>S, and nsP4. However, extracts from cells expressing nsP4 and P123, a cleavage-competent P123 polyprotein, had much less replicase activity. In addition, a P123 polyprotein containing a mutation in the nsP2 protease which increased the efficiency of processing exhibited very little, if any, replicase activity. These results provide further evidence that processing of the polyprotein inactivates the minus-strand initiation complex. Finally, RNA synthesis was detected when soluble nsP4 was added to a membrane fraction containing P123C>S, thus providing a functional assay for purification of the nsP4 RNA polymerase.

Immunization with nonstructural proteins promotes functional recovery of alphavirus-infected neurons.

The encephalitic alphaviruses are useful models for understanding virus-neuron interactions. A neurovirulent strain of Sindbis virus (NSV) causes fatal paralysis in mice by infecting motor neurons and inducing apoptosis of these nonrenewable cells. Antibodies to the surface glycoproteins suppress virus replication, but other recovery-promoting components of the immune response have not been recognized. We assessed the effect on the outcome of NSV-induced encephalomyelitis of immunization of mice with nonstructural proteins (nsPs) by using recombinant vaccinia viruses. Mice immunized with vaccinia virus expressing nsPs and challenged with NSV initially developed paralysis similar to unimmunized mice but then recovered neurologic function. Mice preimmunized with vaccinia virus expressing structural proteins were completely protected from paralysis. Mice immunized with vaccinia virus alone showed paralysis with little evidence of recovery. Vaccinia virus expressing only nsP2 was as effective as vaccinia virus expressing all the nsPs. Protection provided by immunity to nsPs was not associated with a reduction in virus replication or with improved antibody responses to structural proteins. Protection could not be passively transferred with nsP immune serum. The depletion of T cells at the time of NSV infection decreased protection. The data show that antiviral immune responses can improve the ability of neurons to survive infection and to recover function without altering virus replication.

Genetic analysis of the nsP3 region of Sindbis virus: evidence for roles in minus-strand and subgenomic RNA synthesis.

Sindbis virus nonstructural polyproteins and their cleavage products are believed to be essential components of viral RNA replication and transcription complexes. Although numerous studies have investigated the effect of mutations in nsP1-, nsP2-, and nsP4-coding regions on Sindbis virus-specific RNA synthesis, relatively little is known about the function of the region encoding nsP3. nsP3 is a phosphoprotein comprising two regions: an N-terminal portion which is highly conserved among alphaviruses and a C-terminal portion which is not conserved, varying both in sequence and in length. We have constructed a library of random linker insertion mutations in the nsP3-coding region and characterized selected viable mutants. Initially, 126 mutants containing insertions in the conserved region and 23 with insertions in the nonconserved region were screened for temperature-sensitive (ts) plaque formation or for significant differences in plaque morphology. All nonconserved-region mutants were similar to the parental virus, whereas 13 of those in the conserved region were either ts or exhibited altered plaque phenotypes. Ten of these 13 mutants were ts for plaque formation as well as RNA accumulation at 40 degrees C. Highly ts mutants CR3.36 and CR3.39 were defective in their ability to synthesize minus-strand RNAs at the nonpermissive temperature. The CR3.36 and CR3.39 insertion mutations localized to different regions near nsP3 residues 58 and 226, respectively. CR3.39 was able to complement ts mutants from Sindbis virus complementation groups A, B, F, and G. Another mutant isolated from the library, CR3.34, while not ts for plaque formation or RNA synthesis, formed smaller plaques and was defective in subgenomic RNA synthesis at all temperatures examined. These results suggest a role for nsP3 or nsP3-containing polyproteins in the synthesis of viral minus-strand and subgenomic RNAs.

Polypeptide requirements for assembly of functional Sindbis virus replication complexes: a model for the temporal regulation of minus- and plus-strand RNA synthesis.

Proteolytic processing of the Sindbis virus non-structural polyproteins (P123 and P1234) and synthesis of minus- and plus-strand RNAs are highly regulated during virus infection. Although their precise roles have not been defined, these polyproteins, processing intermediates or mature cleavage products (nsP1-4) are believed to be essential components of viral replication and transcription complexes. In this study, we have shown that nsP4 can function as the polymerase for both minus- and plus-strand RNA synthesis. Mutations inactivating the nsP2 proteinase, resulting in uncleaved P123, led to enhanced accumulation of minus-strand RNAs and reduced accumulation of genomic and subgenomic plus-strand RNAs. In contrast, no RNA synthesis was observed with a mutation which increased the efficiency of P123 processing. Inclusion of this mutation in a P123 polyprotein with cleavage sites 1/2 and 2/3 blocked allowed synthesis of both minus- and plus-strand RNAs. We conclude that nsP4 and uncleaved P123 normally function as the minus-strand replication complex, and propose that processing of P123 switches the template preference of the complex to minus-strands, resulting in efficient synthesis of plus-strand genomic and subgenomic RNAs and shut-off of minus-strand RNA synthesis.

Assembly of functional Sindbis virus RNA replication complexes: requirement for coexpression of P123 and P34.

A vaccinia virus transient expression system was used to determine which of the Sindbis virus (SIN) proteins and/or polyproteins are necessary for the formation of active replication complexes and, in particular, to analyze the role of nsP4, the putative polymerase, versus P34 in RNA replication. We generated vaccinia virus recombinants in which the cDNA for the entire SIN nonstructural coding region as well as cDNA copies of the individual nonstructural proteins (nsPs) and several intermediate polyproteins were placed downstream of the promoter for T7 RNA polymerase and the encephalomyocarditis virus 5' untranslated region. The proteins expressed by the vaccinia virus recombinants comigrate with authentic proteins synthesized in SIN-infected cells, and the polyproteins appear to be processed to the individual proteins of the correct size. To examine the replication efficiencies of different protein combinations, a vaccinia virus recombinant was designed to express an engineered substrate RNA which could serve as a template for replication and subgenomic mRNA transcription by the SIN nsPs. Expression of the entire SIN nonstructural coding region resulted in the synthesis of high levels of both genomic and subgenomic RNAs derived from the engineered template. No RNA replication could be detected during coexpression of the four individual nsPs, although the proteins were indistinguishable, in terms of electrophoretic mobility, from those synthesized in SIN-infected cells. Coexpression of polyproteins P12, P23, and/or P34 with the individual nsPs also did not result in detectable levels of RNA replication. However, when P123 and P34 were coexpressed, efficient RNA replication and subgenomic mRNA transcription of the substrate RNA was observed. Coexpression of nsP4 with P123 resulted in the synthesis of only minus-strand RNAs. These studies show that expression of both P123 and P34 is necessary for establishment of functional RNA replication and transcription complexes and raise the possibility that the polyproteins themselves may be functional components of these complexes. In addition, these data indicate that an nsP4 moiety expressed independently with an additional N-terminal methionine is capable of functioning in minus-strand but not plus-strand RNA synthesis.

Roles of nonstructural polyproteins and cleavage products in regulating Sindbis virus RNA replication and transcription.

Using vaccinia virus to express Sindbis virus (SIN) nonstructural proteins (nsPs) and template RNAs, we showed previously that synthesis of all three viral RNAs occurred only during expression of either the entire nonstructural coding region or the polyprotein precursors P123 and P34. In this report, the vaccinia virus system was used to express cleavage-defective polyproteins and nsP4 proteins containing various N-terminal extensions to directly examine the roles of the P123 and P34 polyproteins in RNA replication. Replication and subgenomic mRNA transcription occurred during coexpression of P34 and P123 polyproteins in which cleavage was blocked at either or both of the 1/2 and 2/3 sites. For all cleavage-defective P123 polyproteins, however, the ratio of subgenomic to genomic RNA was decreased, suggesting that both the 1/2 and 2/3 cleavages are required for efficient subgenomic RNA transcription. These studies indicate that the uncleaved P123 polyprotein can function as a component of the viral replicase capable of synthesizing both plus- and minus-strand RNAs. In contrast, cleavage-defective P34 was unable to function in RNA replication, even in complementation experiments in which minus-strand RNAs were provided by nsP4. A P34 polyprotein whose cleavage site was not altered could only function in RNA replication in the presence of an active nsP2 protease. Although nsP4, the putative RNA polymerase, was capable of synthesizing only minus-strand RNAs during coexpression with P123, the addition of only 22 upstream residues to nsP4 allowed both replication and transcription of subgenomic RNA to occur. These data show that the conserved domains of both nsP3 and the nsP4 polymerase do not need to be present in a P34 polyprotein to form a functional plus-strand replicase-transcriptase and suggest that the presence of an active nsP2 protease and a cleavable 3/4 site correlates with synthesis of all virus-specific RNA species.

Mutations which alter the level or structure of nsP4 can affect the efficiency of Sindbis virus replication in a host-dependent manner.

Two mutants of Sindbis virus have been isolated which grow inefficiently at 34.5 degrees C in mosquito cells yet replicate normally in chicken embryo fibroblast cells at the same temperature. In addition, these mutants exhibit temperature-sensitive growth in both cell types and are RNA- at the nonpermissive temperatures (K.J. Kowal and V. Stollar, Virology 114:140-148, 1981). To clarify the basis of this host restriction, we have mapped the causal mutations for these temperature-dependent, host-restricted mutants. Functional mapping and sequence analysis of the mutant cDNAs revealed several mutations which mapped to the amino terminus of nsP4, the putative polymerase subunit of the viral RNA replicase. These mutations resulted in the following amino acid changes in nsP4: leucine to valine at residue 48, aspartate to glycine at residue 142, and proline to arginine at residue 187. Virus containing any of these mutations was restricted in its ability to replicate in mosquito but not chicken embryo fibroblast cells at 34.5 degrees C. In addition to its temperature-dependent, host-restricted phenotype, virus derived from one cDNA clone also exhibited decreased levels of nsP34 and nsP4 yet contained only a silent change in its genome. This C-to-U mutation occurred at nucleotide 5751, the first nucleotide after the opal termination codon separating nsP3 and nsP4. Our results suggest that this substitution decreases readthrough of the opal codon and diminishes production of nsP34 and nsP4. Such a decrease in synthesis rates might lead to levels of these products which are insufficient for viral RNA replication in mosquito cells at the higher temperature. This work provides the first evidence that nsP4 function can be strongly influenced by the host environment.