Sindbis Macrodomain Poly-ADP-Ribose Hydrolase Activity Is Important for Viral RNA Synthesis.

ADP-ribosylation is a highly dynamic posttranslational modification frequently studied in stress response pathways with recent attention given to its role in response to viral infection. Notably, the alphaviruses encode catalytically active macrodomains capable of ADP-ribosylhydrolase (ARH) activities, implying a role in remodeling the cellular ADP-ribosylome. This report decouples mono- and poly-ARH contributions to macrodomain function using a newly engineered Sindbis virus (SINV) mutant with attenuated poly-ARH activity. Our findings indicate that viral poly-ARH activity is uniquely required for high titer replication in mammalian systems. Despite translating incoming genomic RNA as efficiently as WT virus, mutant viruses have a reduced capacity to establish productive infection, offering a more complete understanding of the kinetics and role of the alphavirus macrodomain with important implications for broader ADP-ribosyltransferase biology. IMPORTANCE Viral macrodomains have drawn attention in recent years due to their high degree of conservation in several virus families (e.g., coronaviruses and alphaviruses) and their potential druggability. These domains erase mono- or poly-ADP-ribose, posttranslational modifications written by host poly-ADP-ribose polymerase (PARP) proteins, from undetermined host or viral proteins to enhance replication. Prior work determined that efficient alphavirus replication requires catalytically active macrodomains; however, which form of the modification requires removal and from which protein(s) had not been determined. Here, we present evidence for the specific requirement of poly-ARH activity to ensure efficient productive infection and virus replication.

Increasing the Capping Efficiency of the Sindbis Virus nsP1 Protein Negatively Affects Viral Infection.

Alphaviruses are arthropod-borne RNA viruses that are capable of causing severe disease and are a significant burden to public health. Alphaviral replication results in the production of both capped and noncapped viral genomic RNAs (ncgRNAs), which are packaged into virions during infections of vertebrate and invertebrate cells. However, the roles that the ncgRNAs play during alphaviral infection have yet to be exhaustively characterized. Here, the importance of the ncgRNAs to alphaviral infection was assessed by using mutations of the nsP1 protein of Sindbis virus (SINV), which altered the synthesis of the ncgRNAs during infection by modulating the protein's capping efficiency. Specifically, point mutations at residues Y286A and N376A decreased capping efficiency whereas a point mutation at D355A increased the capping efficiency of the SINV genomic RNA during genuine viral infection. Viral growth kinetics levels were significantly reduced for the D355A mutant relative to wild-type infection, whereas the Y286A and N376A mutants showed modest decreases in growth kinetics. Overall genomic translation and nonstructural protein accumulation were found to correlate with increases and decreases in capping efficiency. However, genomic, minus-strand, and subgenomic viral RNA synthesis were largely unaffected by the modulation of alphaviral capping activity. In addition, translation of the subgenomic alphaviral RNA (vRNA) was found not to be impacted by changes in capping efficiency. The mechanism by which the decreased presence of ncgRNAs reduced viral growth kinetics levels operated through the impaired production of viral particles. Collectively, these data illustrate the importance of ncgRNAs to viral infection and suggest that they play an integral role in the production of viral progeny.IMPORTANCE Alphaviruses have been the cause of both localized outbreaks and large epidemics of severe disease. Currently, there are no strategies or vaccines which are either safe or effective for preventing alphaviral infection or treating alphaviral disease. This deficit of viable therapeutics highlights the need to better understand the mechanisms behind alphaviral infection in order to develop novel antiviral strategies for treatment of alphaviral disease. In particular, this report details a previously uncharacterized aspect of the alphaviral life cycle: the importance of noncapped genomic viral RNAs for alphaviral infection. This offers new insights into the mechanisms of alphaviral replication and the impact of the noncapped genomic RNAs on viral packaging.

Low-Fidelity Polymerases of Alphaviruses Recombine at Higher Rates To Overproduce Defective Interfering Particles.

UNLABELLED: Low-fidelity RNA-dependent RNA polymerases for many RNA virus mutators have been shown to confer attenuated phenotypes, presumably due to increased mutation rates. Additionally, for many RNA viruses, replication to high titers results in the production of defective interfering particles (DIs) that also attenuate infection. We hypothesized that fidelity, recombination, and DI production are tightly linked. We show that a Sindbis virus mutator replicating at a high multiplicity of infection manifests an earlier and greater accumulation of DIs than its wild-type counterpart. The isolated DIs interfere with the replication of full-length virus in a dose-dependent manner. Importantly, the ability of the mutator virus to overproduce DIs could be linked to an increased recombination frequency. These data confirm that RNA-dependent RNA polymerase fidelity and recombination are inversely correlated for this mutator. Our findings suggest that defective interference resulting from higher recombination rates may be more detrimental to RNA virus mutators than the increase in mutational burden. IMPORTANCE: Replication, adaptation, and evolution of RNA viruses rely in large part on their low-fidelity RNA-dependent RNA polymerase. Viruses artificially modified in their polymerases to decrease fidelity (mutator viruses) are attenuated in vivo, demonstrating the important role of fidelity in viral fitness. However, attenuation was attributed solely to the modification of the viral mutation rate and the accumulation of detrimental point mutations. In this work, we described an additional phenotype of mutator viruses: an increased recombination rate leading to defective interfering particle (DI) overproduction. Because DIs are known for their inhibitory effect on viral replication, our work suggests that fidelity variants may be attenuated in vivo via several mechanisms. This has important implications in the development of fidelity variants as live attenuated vaccine strains.

Alphavirus mutator variants present host-specific defects and attenuation in mammalian and insect models.

Arboviruses cycle through both vertebrates and invertebrates, which requires them to adapt to disparate hosts while maintaining genetic integrity during genome replication. To study the genetic mechanisms and determinants of these processes, we use chikungunya virus (CHIKV), a re-emerging human pathogen transmitted by the Aedes mosquito. We previously isolated a high fidelity (or antimutator) polymerase variant, C483Y, which had decreased fitness in both mammalian and mosquito hosts, suggesting this residue may be a key molecular determinant. To further investigate effects of position 483 on RNA-dependent RNA-polymerase (RdRp) fidelity, we substituted every amino acid at this position. We isolated novel mutators with decreased replication fidelity and higher mutation frequencies, allowing us to examine the fitness of error-prone arbovirus variants. Although CHIKV mutators displayed no major replication defects in mammalian cell culture, they had reduced specific infectivity and were attenuated in vivo. Unexpectedly, mutator phenotypes were suppressed in mosquito cells and the variants exhibited significant defects in RNA synthesis. Consequently, these replication defects resulted in strong selection for reversion during infection of mosquitoes. Since residue 483 is conserved among alphaviruses, we examined the analogous mutations in Sindbis virus (SINV), which also reduced polymerase fidelity and generated replication defects in mosquito cells. However, replication defects were mosquito cell-specific and were not observed in Drosophila S2 cells, allowing us to evaluate the potential attenuation of mutators in insect models where pressure for reversion was absent. Indeed, the SINV mutator variant was attenuated in fruit flies. These findings confirm that residue 483 is a determinant regulating alphavirus polymerase fidelity and demonstrate proof of principle that arboviruses can be attenuated in mammalian and insect hosts by reducing fidelity.

Evasion of the innate immune response: the Old World alphavirus nsP2 protein induces rapid degradation of Rpb1, a catalytic subunit of RNA polymerase II.

The Old World alphaviruses are emerging human pathogens with an ability to cause widespread epidemics. The latest epidemic of Chikungunya virus, from 2005 to 2007, affected over 40 countries in Africa, Asia, and Europe. The Old World alphaviruses are highly cytopathic and known to evade the cellular antiviral response by inducing global inhibition of transcription in vertebrate cells. This function was shown to be mediated by their nonstructural nsP2 protein; however, the detailed mechanism of this phenomenon has remained unknown. Here, we report that nsP2 proteins of Sindbis, Semliki Forest, and Chikungunya viruses inhibit cellular transcription by inducing rapid degradation of Rpb1, a catalytic subunit of the RNAPII complex. This degradation of Rpb1 is independent of the nsP2-associated protease activity, but, instead, it proceeds through nsP2-mediated Rpb1 ubiquitination. This function of nsP2 depends on the integrity of the helicase and S-adenosylmethionine (SAM)-dependent methyltransferase-like domains, and point mutations in either of these domains abolish Rpb1 degradation. We go on to show that complete degradation of Rpb1 in alphavirus-infected cells occurs within 6 h postinfection, before other previously described virus-induced changes in cell physiology, such as apoptosis, autophagy, and inhibition of STAT1 phosphorylation, are detected. Since Rpb1 is a subunit that catalyzes the polymerase reaction during RNA transcription, degradation of Rpb1 plays an indispensable role in blocking the activation of cellular genes and downregulating cellular antiviral response. This indicates that the nsP2-induced degradation of Rpb1 is a critical mechanism utilized by the Old World alphaviruses to subvert the cellular antiviral response.

Heterologous production, purification and characterization of enzymatically active Sindbis virus nonstructural protein nsP1.

Alphavirus nonstructural protein nsP1 possesses distinct methyltransferase (MTase) and guanylyltransferase (GTase) activities involved in the capping of viral RNAs. In alphaviruses, the methylation of GTP occurs before RNA transguanylation and nsP1 forms a covalent complex with m(7)GMP unlike the host mRNA guanylyltransferase which forms GMP-enzyme complex. In this study, full length SINV nsP1 was expressed in a soluble form with an N-terminal histidine tag in Escherichia coli and purified to homogeneity. The purified protein is enzymatically active and contains both MTase and GTase activity indicating that SINV nsP1 does not require membrane association for its enzymatic function. Biochemical analysis shows that detergents abolish nsP1 GTase activity, whereas nonionic detergents do not affect MTase activity. Furthermore, SINV nsP1 contains the metal-ion dependent GTase, whereas MTase does not require a metal ion. Circular dichroism spectroscopic analysis of purified protein indicate that nsP1 has a mixed α/ß structure and is in the folded native conformation.

An arthropod enzyme, Dfurin1, and a vertebrate furin homolog display distinct cleavage site sequence preferences for a shared viral proprotein substrate.

Alphaviruses replicate in vertebrate and arthropod cells and utilize a cellular enzyme called furin to process the PE2 glycoprotein precursor during virus replication in both cell types. Furin cleaves PE2 at a site immediately following a highly conserved four residue cleavage signal. Prior studies demonstrated that the amino acid immediately adjacent to the cleavage site influenced PE2 cleavage differently in vertebrate and mosquito cells (HW Heidner et al. 1996 . Journal of Virology 70: 2069-2073.). This finding was tentatively attributed to potential differences in the substrate specificities of the vertebrate and arthropod furin enzymes or to differences in the carbohydrate processing phenotypes of arthropod and vertebrate cells. To further address this issue, we evaluated Sindbis virus replication and PE2 cleavage in the Chinese hamster, Cricetulus griseus Milne-Edwards (Rodentia: Cricetidae) ovary cells (CHO-K1) and in a CHO-K1-derived furin-negative cell line (RPE.40) engineered to stably express the Dfurin1 enzyme of Drosophila melanogaster Meigen (Diptera: Drosophilidae). Expression of Dfurin1 enhanced Sindbis virus titers in RPE.40 cells by a factor of 10(2)-10(3), and this increase correlated with efficient cleavage of PE2. The PE2-cleavage phenotypes of viruses containing different amino acid substitutions adjacent to the furin cleavage site were compared in mosquito (C6/36), CHO-K1, and Dfurin1-expressing RPE.40 cells. This analysis confirmed that the substrate specificities of Dfurin1 and the putative mosquito furin homolog present in C6/36 cells are similar and suggested that the alternative PE2 cleavage phenotypes observed in vertebrate and arthropod cells were due to differences in substrate specificity between the arthropod and vertebrate furin enzymes and not to differences in host cell glycoprotein processing pathways.

Characterization of purified Sindbis virus nsP4 RNA-dependent RNA polymerase activity in vitro.

The Sindbis virus RNA-dependent RNA polymerase (nsP4) is responsible for the replication of the viral RNA genome. In infected cells, nsP4 is localized in a replication complex along with the other viral non-structural proteins. nsP4 has been difficult to homogenously purify from infected cells due to its interactions with the other replication proteins and the fact that its N-terminal residue, a tyrosine, causes the protein to be rapidly turned over in cells. We report the successful expression and purification of Sindbis nsP4 in a bacterial system, in which nsP4 is expressed as an N-terminal SUMO fusion protein. After purification the SUMO tag is removed, resulting in the isolation of full-length nsP4 possessing the authentic N-terminal tyrosine. This purified enzyme is able to produce minus-strand RNA de novo from plus-strand templates, as well as terminally add adenosine residues to the 3' end of an RNA substrate. In the presence of the partially processed viral replicase polyprotein, P123, purified nsP4 is able to synthesize discrete template length minus-strand RNA products. Mutations in the 3' CSE or poly(A) tail of viral template RNA prevent RNA synthesis by the replicase complex containing purified nsP4, consistent with previously reported template requirements for minus-strand RNA synthesis. Optimal reaction conditions were determined by investigating the effects of time, pH, and the concentrations of nsP4, P123 and magnesium on the synthesis of RNA.

Role for conserved residues of sindbis virus nonstructural protein 2 methyltransferase-like domain in regulation of minus-strand synthesis and development of cytopathic infection.

The plus-strand RNA genome of Sindbis virus (SINV) encodes four nonstructural proteins (nsP1 to nsP4) that are involved in the replication of the viral RNA. The approximately 800-amino-acid nsP2 consists of an N-terminal domain with nucleoside triphosphatase and helicase activities and a C-terminal protease domain. Recently, the structure determined for Venezuelan equine encephalitis virus nsP2 indicated the presence of a previously unrecognized methyltransferase (MTase)-like domain within the C-terminal approximately 200 residues and raised a question about its functional importance. To assess the role of this MTase-like region in viral replication, highly conserved arginine and lysine residues were mutated to alanine. The plaque phenotypes of these mutants ranged from large/wild-type to small plaques with selected mutations demonstrating temperature sensitive lethality. The proteolytic polyprotein processing activity of nsP2 was unaffected in most of the mutants. Some of the temperature-sensitive mutants showed reduction in the minus-strand RNA synthesis, a function that has not yet been ascribed to nsP2. Mutation of SINV residue R615 rendered the virus noncytopathic and incapable of inhibiting the host cell translation but with no effects on the transcriptional inhibition. This property differentiated the mutation at R615 from previously described noncytopathic mutations. These results implicate nsP2 in regulation of minus-strand synthesis and suggest that different regions of the nsP2 MTase-like domain differentially modulate host defense mechanisms, independent of its role as the viral protease.

Ovarian tumor domain-containing viral proteases evade ubiquitin- and ISG15-dependent innate immune responses.

Ubiquitin (Ub) and interferon-stimulated gene product 15 (ISG15) reversibly conjugate to proteins and mediate important innate antiviral responses. The ovarian tumor (OTU) domain represents a superfamily of predicted proteases found in eukaryotic, bacterial, and viral proteins, some of which have Ub-deconjugating activity. We show that the OTU domain-containing proteases from nairoviruses and arteriviruses, two unrelated groups of RNA viruses, hydrolyze Ub and ISG15 from cellular target proteins. This broad activity contrasts with the target specificity of known mammalian OTU domain-containing proteins. Expression of a viral OTU domain-containing protein antagonizes the antiviral effects of ISG15 and enhances susceptibility to Sindbis virus infection in vivo. We also show that viral OTU domain-containing proteases inhibit NF-kappaB-dependent signaling. Thus, the deconjugating activity of viral OTU proteases represents a unique viral strategy to inhibit Ub- and ISG15-dependent antiviral pathways.

Immunization with a low-dose replicon DNA vaccine encoding Phl p 5 effectively prevents allergic sensitization.

BACKGROUND: Replicase-based DNA vaccines stimulate T(H)1-biased immune responses at ultralow doses and induce self-removal of transfected cells through apoptosis. Both aspects are important requirements for efficient and safe DNA-based immunotherapy of type I allergies. OBJECTIVE: A Sindbis virus replicon-based DNA vaccine encoding the major timothy grass pollen allergen Phl p 5 was evaluated for its antiallergic potential compared with a conventional DNA vaccine in a BALB/c mouse model of allergy. METHODS: Mice were intradermally prevaccinated with plasmid DNA, followed by sensitization and intranasal allergen provocation with recombinant Phl p 5. In vitro proliferation and cytokine secretion was measured in splenocyte cultures. Distribution of IgG1, IgG2a, and IgE antibody subclasses was determined by means of ELISA. IgE-mediated degranulation was measured with the basophil release assay. Bronchoalveolar lavage fluid was analyzed for eosinophils, IL-4, IL-5, IL-13, and IFN-gamma. Mucus production, inflammatory infiltrates, and epithelial damage were determined in lung sections. RESULTS: Both vaccines induced T(H)1-biased immune responses, resulting in suppression of functional IgE, reduction of eosinophilia in bronchoalveolar lavage fluid, and alleviation of lung pathology. However, immunization with the replicon DNA vaccine elicited these effects at a 100-fold lower dose compared with the conventional DNA vaccine. CONCLUSIONS: The increased immunogenicity of replicon-based DNA vaccines allows for application of extremely low doses, thereby eliminating the concerns associated with conventional DNA vaccines, which have to be administered at milligram amounts to induce immune reactions in human subjects. CLINICAL IMPLICATIONS: Their high safety profile makes replicon-based DNA vaccines promising candidates for treatment of type I allergies in the clinic.

Sindbis virus nonstructural protein nsP2 is cytotoxic and inhibits cellular transcription.

Replication of alphaviruses in vertebrate cells strongly affects cell physiology and ultimately leads to development of a cytopathic effect (CPE) and cell death. Sindbis virus (SIN) replication causes major changes in cellular macromolecular synthesis, in which the strong downregulation of transcription of cellular mRNAs and rRNAs plays a critical role. SIN nonstructural protein nsP2 was previously proposed as one of the main regulators of virus-host cell interactions, because point mutations in the carboxy-terminal part of nsP2 could make SIN and other alphaviruses and replicons less cytopathic and capable of persisting in some vertebrate cell lines. These mutants were incapable of inhibiting transcription and downregulating a viral stress-induced cell response. In the present work, we demonstrate that (i) SIN nsP2 is critically involved in CPE development, not only during the replication of SIN-specific RNAs, but also when this protein is expressed alone from different expression cassettes; (ii) the cytotoxic effect of SIN nsP2 appears to be at least partially determined by its ability to cause transcriptional shutoff; (iii) these functions of SIN nsP2 are determined by the integrity of the carboxy-terminal peptide of this protein located outside its helicase and protease domains, rather than by its protease activity; and (iv) the cytotoxic activity of SIN nsP2 depends on the presence of this protein in a free form, and alterations in P123 processing abolish the ability of nsP2 to cause CPE.

RNA encoding the MPT83 antigen induces protective immune responses against Mycobacterium tuberculosis infection.

We have previously demonstrated that vaccination of mice with plasmid DNA vectors expressing immunodominant mycobacterial genes induced cellular immune responses and significant protection against challenge with Mycobacterium tuberculosis. We demonstrate here, using in vitro-synthesized RNA, that vaccination with DNA or RNA constructs expressing the M. tuberculosis MPT83 antigen are capable of inducing specific humoral and T-cell immune responses and confer modest but significant protection against M. tuberculosis challenge in mice. This is the first report of protective immunity conferred against intracellular bacteria by an RNA vaccine. This novel approach avoids some of the drawbacks of DNA vaccines and illustrates the potential for developing new antimycobacterial immunization strategies.

Identification of the amino acid sequence in Sindbis virus nsP4 that binds to the promoter for the synthesis of the subgenomic RNA.

A gel mobility-shift assay was used to demonstrate the binding of the Sindbis virus transcriptase to the promoter for the synthesis of subgenomic (SG) RNA. The assay made use of a P15 fraction (the cell fraction that is pelleted at 15,000 x g) from cells infected with recombinant vaccinia virions expressing various Sindbis virus nonstructural proteins (nsPs) and a (32)P-labeled 24-mer oligoribonucleotide representing the minimal sequence with SG promoter activity. By itself, nsP4, the viral RNA-dependent RNA polymerase, did not bind to the SG promoter; rather, all four nsPs were required for the binding of the transcriptase to the promoter. UV crosslinking of the transcriptase to a thiouridine-containing SG promoter, followed by V8 protease digestion of the complex, generated a peptide fragment that was bound to the SG promoter. This peptide fragment contained a sequence that corresponded to residues 329-334 of nsP4. This peptide may be in the fingers domain of nsP4. The peptide that was identified contained Arg residues at positions 331 and 332. Another Arg is present at position 327. By changing each of the Arg residues to Ala, we demonstrated that only the Arg residues at positions 331 and 332 were required for binding nsP4 to the SG promoter.

Apoptosis is essential for the increased efficacy of alphaviral replicase-based DNA vaccines.

Alphaviral replicons can increase the efficacy and immunogenicity of naked nucleic acid vaccines. To study the impact of apoptosis on this increased effectiveness, we co-delivered an anti-apoptotic gene (Bcl-X(L)) with the melanocyte/melanoma differentiation antigen TRP-1. Although cells co-transfected with Bcl-X(L) lived longer, produced more antigen and elicited increased antibody production in vivo, co-delivery of pro-survival Bcl-X(L) with antigen significantly reduced the ability of the replicase-based vaccine to protect against an aggressive tumor challenge. These data show for the first time that the induction of apoptotic cell death of transfected cells in vivo is required for the increased effectiveness of replicase-based vaccines. Our findings also provide an explanation for the paradoxical observation that replicase-based DNA vaccines are much more immunogenic than conventional constructs despite reduced antigen production.

Suppressor mutations that allow sindbis virus RNA polymerase to function with nonaromatic amino acids at the N-terminus: evidence for interaction between nsP1 and nsP4 in minus-strand RNA synthesis.

The alphavirus RNA polymerase, nsP4, invariably has a Tyr residue at the N-terminus. Previously we reported that the N-terminal Tyr residue of nsP4 of Sindbis virus, the type species of the genus Alphavirus, can be substituted with Phe, Trp, or His without altering the wild-type phenotype in cultured cells but that other substitutions tested, except for Met, were lethal or quasilethal. Here we report the identification of two suppressor mutations in nsP4 (Glu-191 to Leu and Glu-315 to Gly, Val, or Lys) and one in nsP1 (Thr-349 to Lys) that allow nsP4 with nonaromatic amino acids at the N-terminus to function at 30 degrees C. The suppressor mutation at nsP4 Glu-315 occurred most frequently. All three suppressor mutations suppressed the effects of Ala, Arg, or Leu at the N-terminus of nsP4 with almost equal efficiency and thus the effect of the suppressing mutation is independent of the nsP4 N-terminal residue. Reconstructed mutants containing nsP1-T349K or nsP4-E315G combined with Ala-nsP4 had a defect in minus-strand RNA synthesis at 40 degrees C. A double mutant containing nsP4-Q191L combined with Ala-nsP4 was unstable and could not be tested for RNA synthesis because it reverted to temperature-independence too rapidly. Combinations of nsP1-T349K or nsP4-E315G with Leu, Arg, His, or any aromatic amino acid at the N-terminus of nsP4 also made the mutant viruses temperature sensitive. The results from this study and from a previous report on the shutoff of minus-strand RNA synthesis at 40 degrees C with the nsP1-A348T mutation in ts11 suggests that the N-terminus nsP4 interacts with nsP1 during initiation of minus-strand RNA synthesis.

A mutant of Sindbis virus that is resistant to pyrazofurin encodes an altered RNA polymerase.

Pyrazofurin (PZF), a cytidine analog and an inhibitor of orotate monophosphate decarboxylase, has been shown to decrease the levels of UTP and CTP in treated cells. When Sindbis virus (SV)-infected Aedes albopictus cells were treated with PZF, the yield of virus was reduced 100- to 1000-fold. By serial passage of our standard SV(STD) in Ae. albopictus cells in the presence of increasing concentrations of PZF, a mutant, SV(PZF), was derived, which was not inhibited by PZF. SV(PZF) is also resistant to adenosine, guanosine, and phosphono-acetyl-N-aspartate, all of which have been shown to decrease levels of UTP and CTP. Analysis of chimeric viruses containing sequences from the SV(PZF) and parental genomes showed that the sequence between nt 5262 and 7999 conferred the PZF-resistant phenotype. Sequencing of this region identified four mutations (nt 5750, 6627, 7543, and 7593), which are predicted to lead to amino acid changes: opal550L in nsP3 and M287L, K592I, and P609T in nsP4. Characterization of viruses containing one or more of these mutations demonstrated that all three mutations in the nsP4 coding region are required to produce full resistance to PZF. Using a molecular model of nsP4 based on the structure of HIV reverse transcriptase, we located amino acid change M287L at the tip of the fingers domain and K592I and P609T at the base of the thumb domain of the viral RNA polymerase. We suggest that these three amino acid changes in nsP4 alter the geometry of the NTP binding pocket so as to increase the affinity of the enzyme for CTP and UTP.

The African swine fever virus prenyltransferase is an integral membrane trans-geranylgeranyl-diphosphate synthase.

In a previous study, it was shown that the protein encoded by the gene B318L of African swine fever virus (ASFV) is a trans-prenyltransferase that catalyzes in vitro the condensation of farnesyl diphosphate and isopentenyl diphosphate to synthesize geranylgeranyl diphosphate and longer chain prenyl diphosphates (Alejo, A., Yáñez, R. J., Rodríguez, J. M., Viñuela, E., and Salas, M. L. (1997) J. Biol. Chem. 272, 9417-9423). To investigate the in vivo function of the viral enzyme, we have determined, in this work, its subcellular localization and activity in cell extracts. Two systems were used in these studies: cells infected with ASFV and cells infected with a recombinant pseudo-Sindbis virus carrying the complete B318L gene. In this latter system, the trans-prenyltransferase was found to colocalize with the endoplasmic reticulum marker protein-disulfide isomerase, whereas in cells infected with ASFV, the viral enzyme was present in cytoplasmic viral assembly sites, associated with precursor viral membranes derived from the endoplasmic reticulum. In addition, after subcellular fractionation, the viral enzyme partitioned into the membrane fraction. Extraction of membrane proteins with alkaline carbonate and Triton X-114 indicated that the ASFV enzyme behaved as an integral membrane protein. The membrane enzyme synthesized predominantly all-trans-geranylgeranyl diphosphate from farnesyl diphosphate and isopentenyl diphosphate. These results indicate that the viral B318L protein is a trans-geranylgeranyl-diphosphate synthase, being the only enzyme of this type that is known to have a membrane localization.

Requirement for an aromatic amino acid or histidine at the N terminus of Sindbis virus RNA polymerase.

The N terminal amino acid of nonstructural protein nsP4, the viral RNA polymerase, is a tyrosine in all sequenced alphaviruses; this is a destabilizing amino acid for the N-end rule pathway and results in rapid degradation of nsP4 produced in infected cells or in reticulocyte lysates. We have constructed 11 mutants of Sindbis virus bearing Phe, Ala, Thr, Cys, Leu, Met, Asn, Gln, Glu, Arg, or Pro at the N terminus of nsP4. Translation of RNAs in reticulocyte lysates showed that cleavage at the nsP3/nsP4 site occurred efficiently for all mutants except for Glu-nsP4, which was cleaved inefficiently, and Pro-nsP4, which was not detectably cleaved, and that Tyr, Cys, Leu, Arg, and Phe destabilized nsP4 but Ala, Met, Thr, Asn, Gln, and Glu stabilized nsP4 to various extents. The viability of the mutants was examined by transfection of chicken cells at 30 or 40 degrees C. The Phe-nsP4 mutant formed large plaques at both temperatures. The Met-nsP4 mutant was also viable but formed small plaques at 30 degrees C and minute plaques at 40 degrees C. The remaining mutants did not form plaques at either temperature. However, after prolonged incubation at 30 degrees C, all the mutants except Glu-nsP4 and Pro-nsP4 produced viable viruses. In the case of Cys-, Leu-, Asn-, Gln-, or Arg-nsP4, revertants that were indistinguishable in plaque phenotype from the wild-type virus arose by same-site reversion to Tyr, Trp, Phe, or His by a single nucleotide substitution in the original mutant codon. Viable viruses also arose from the Ala-, Leu-, Cys-, Thr-, Asn-, Gln-, and Arg-nsP4 mutants that retained the original mutations at the N terminus of nsP4, but these viruses formed smaller plaques than the wild-type virus and many were temperature sensitive. Our results indicate that only nsP4s bearing N-terminal Tyr, Phe, Trp, or His have wild-type or near-wild-type activity for RNA replication and that rapid degradation of nsP4 is not a prerequisite for its function. nsP4s bearing other N-terminal residues, with the exception of Met-nsP4, have only very low or negligible activity, so that no detectable infectious virus can be produced. However, suppressor mutations can arise that enable most such nsP4s to regain significant but still suboptimal activity.

Construction of hepatitis C-SIN virus recombinants with replicative dependency on hepatitis C virus serine protease activity.

An in vivo assay system was developed for the serine protease of hepatitis C virus (HCV) using the sindbis (SIN) viral replication system in which HCV serine protease activity is essential for the replication of the HCV-SIN chimeric virus. Two chimeric viral cDNA clones were constructed by inserting the NS3/4A region and NS3/4A region with the putative helicase deleted, into the N-terminal region of SIN core protein. The constructs were named Tpro CT and Tpro T, respectively. BHK-21 cells transfected with the in vitro transcribed RNAs from Tpro CT and Tpro T showed specific cytopathic morphology and produced chimeric viruses, Vpro CT and Vpro T. In contrast, in vitro transcribed RNAs from Tpro CTI and Tpro TI, in which serine of catalytic triad of HCV protease was changed to alanine, were not infectious. When the chimeric viruses were passaged in BHK-21 cells at about 0.1 multiplicity of infection (MOI), Vpro T, but not Vpro CT, stably expressed HCV protease for up to five passages. Surprisingly, the cell culture media of BHK-21 cells infected with Vpro T, compared to wild-type sindbis virus, showed rapid pH changes by more than 0.8 pH degree at 72 h post-infection. HCV-SIN hybrid viruses could be used in screening the HCV protease-inhibitor in cell culture systems.