Distinct sites on the Sindbis virus RNA-dependent RNA polymerase for binding to the promoters for the synthesis of genomic and subgenomic RNA

Sindbis virus-infected cells make two positive-strand RNAs, a genomic (G) RNA and a subgenomic (SG) RNA. Here we report the amino acid sequence in nonstructural protein 4 (nsP4), the viral RNA-dependent RNA polymerase, that binds to the promoter for the synthesis of G RNA. In addition, using a cell-free system that makes both G and SG RNA, we show that specific amino acid changes in nsP4 that abolish the synthesis of SG RNA have no effect on the synthesis of G RNA. Our findings indicate that nsP4 has distinct sites for the recognition of the G and SG promoters.     

Separate pyrimidine-nucleotide pools for messenger-RNA and ribosomal-RNA synthesis in HeLa S3 cells.

Kinetic analyses of mRNA and 28-S RNA labeling [3H]uridine revealed distinctly different steady-state specific radioactivities finally reached for uridine in mRNA and 28-S RNA when exogenous [3H]uridine was kept constant for several cell doubling times. While the steady-state label of (total) UTP and of uridine in mRNA responded to the same extent to a suppression of pyrimidine synthesis de novo by high uridine concentrations in the culture medium, uridine in 28-S RNA was scarcely influenced. Similar findings were obtained with respect to labeling of cytidine in the various RNA species due to an equilibration of UTP with CTP [5-3H]Uridine is also incorporated into deoxycytidine of DNA, presumably via dCTP. The specific radioactivity of this nucleosidase attained the same steady-state value as UTP, uridine in mRNA and cytidine in mRNA. The data indicate the existence of two pyrimidine nucleotide pools. One is a large, general UTP pool comprising the bulk of the cellular UTP and serving nucleoplasmic nucleic acid formation (uridine and cytidine in mRNA, deoxycytidine in DNA). Its replenishment by de novo synthesis can be suppressed completely by exogenous uridine above 100 muM concentrations. A second, very small UTP (and CTP) pool with a high turnover provides most of the precursors for nucleolar RNA formation (rRNA). This pool is not subject to feedback inhibition by extracellular uridine to an appreciable extent. Determinations of (total) UTP turnover also show that the bulk of cellular RNA (rRNA) cannot be derived from the large UTP pool.     

Utilization of L-cell nucleoside triphosphates by Chlamydia psittaci for ribonucleic acid synthesis.

Long-term, 32-P-labeled L cells were infected with the obligately intracellular parasite Chlamydia psittaci (strain 6 BC). At 20 h postinfection, [3-H]uridine was added, and the infected cells were sampled at intervals for incorporation of the labels into the uridine triphosphate (UTP) and cytidine triphosphate (CTP) pools of the host L cell and the uridine monophosphate (UMP) and cytidine monophosphate (CMP) in 16S ribosomal ribonucleic acid (RNA) of the parasite. The specific activity of the nucleotides was calculated from the ratio of 3-H to 32-P counts in the nucleotides. The rate of approach to equilibrium labeling of UTP and CTP in L-cell pools and UMP and CMP in 16S RNA from the exogenous uridine label was determined from the increase in the ratios of the specific activities of CTP to UTP and CMP to UMP with time. The rate of approach to equilibrium CMP:UMP labeling of the 16S RNA of C. psittaci was consistent with the rate predicted from the kinetics of labeling of the CTP and UTP pools of the host L cell. In analogous experiments, the rate of approach to equilibrium guanosine monophosphate:adenosine monophosphate labeling of 16S RNA from an exogenous [14-C]adenine label was consistent with the rate predicted from the kinetics of labeling of the purine nucleoside triphosphate pool of the host cell. These results support the concept that members of the genus Chlamydia owe their obligate intracellular mode of reproduction to a requirement for energy intermediates which is fulfilled by the host cell. In addition, evidence was obtained that the total acid-soluble purine nucleoside triphosphate pool of L cells accurately represents the precursors of L-cell 18S ribosomal RNA.     

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.     

Requirement for the amino-terminal domain of sindbis virus nsP4 during virus infection

The Sindbis virus RNA-dependent RNA polymerase nsP4 possesses an amino-terminal region that is unique to alphaviruses and is predicted to be disordered. To determine the importance of this region during alphavirus replication, 29 mutations were introduced, and resultant viruses were assessed for growth defects. Three small plaque mutants, D41A, G83L, and the triple mutant GPG((8-10))VAV, had defects in subgenome synthesis, minus-strand synthesis, and overall levels of viral RNA synthesis, respectively. Large plaque viruses were selected following passage in BHK-21 cells, and the genomes of these were sequenced. Suppressor mutations in nsP1, nsP2, and nsP3 that restored viral RNA synthesis were identified. An nsP2 change from M282 to L and an nsP3 change from H99 to N corrected the D41A-induced defect in subgenomic RNA synthesis. Three changes in nsP1, I351 to V, I388 to V, or the previously identified change, N374 to H (C. L. Fata, S. G. Sawicki, and D. L. Sawicki, J. Virol. 76:8641-8649, 2002), suppressed the minus-strand synthetic defect. A direct reversion back to G at position 8 reduced the RNA synthesis defect of the GPG((8-10))VAV virus. These results imply that nsP4's amino-terminal domain participates in distinct interactions with other nsPs in the context of differentially functioning RNA synthetic complexes, and flexibility in this domain is important for viral RNA synthesis. Additionally, the inability of the mutant viruses to efficiently inhibit host protein synthesis suggests a role for nsP4 in the regulation of host cell gene expression.     

Tracking and elucidating alphavirus-host protein interactions

Viral infections cause profound alterations in host cells. Here, we explore the interactions between proteins of the Alphavirus Sindbis and host factors during the course of mammalian cell infection. Using a mutant virus expressing the viral nsP3 protein tagged with green fluorescent protein (GFP) we directly observed nsP3 localization and isolated nsP3-interacting proteins at various times after infection. These results revealed that host factor recruitment to nsP3-containing complexes was time dependent, with a specific early and persistent recruitment of G3BP and a later recruitment of 14-3-3 proteins. Expression of GFP-tagged G3BP allowed reciprocal isolation of nsP3 in Sindbis infected cells, as well as the identification of novel G3BP-interacting proteins in both uninfected and infected cells. Note-worthy interactions include nuclear pore complex components whose interactions with G3BP were reduced upon Sindbis infection. This suggests that G3BP is a nuclear transport factor, as hypothesized previously, and that viral infection may alter RNA transport. Immunoelectron microscopy showed that a portion of Sindbis nsP3 is localized at the nuclear envelope, suggesting a possible site of G3BP recruitment to nsP3-containing complexes. Our results demonstrate the utility of using a standard GFP tag to both track viral protein localization and elucidate specific viral-host interactions over time in infected mammalian cells.     

Catalytic core of alphavirus nonstructural protein nsP4 possesses terminal adenylyltransferase activity

The RNA-dependent RNA polymerase nsP4 is an integral part of the alphavirus replication complex. To define the role of nsP4 in viral RNA replication and for a structure-function analysis, we expressed Sindbis virus nsP4 in Escherichia coli. The core catalytic domain of nsP4 (Delta97nsP4, a deletion of the N-terminal 97 amino acids), which consists of the predicted polymerase domain containing the GDD amino acid motif required for viral RNA synthesis, was stable against proteolytic degradation during expression. Therefore, the recombinant core domain and selected mutants were expressed and purified to homogeneity. We determined that Delta97nsP4 possesses terminal adenylyltransferase (TATase) activity, as it specifically catalyzed the addition of adenine to the 3' end of an acceptor RNA in the presence of divalent cations. Furthermore, Delta97nsP4 is unable to transfer other nucleotides (UTP, CTP, GTP, and dATP) to the acceptor RNA in the absence or presence of other nucleotides. Delta97nsP4 possessing a GDD-to-GAA mutation completely inactivates the enzymatic activity. However, a GDD-to-SNN mutation did not inactivate the enzyme but reduced its activity to approximately 45% of that of the wild type in the presence of Mg(2+). Investigation of the TATase of the GDD-to-SNN mutant revealed that it had TATase equivalent to that of the wild type in the presence of Mn(2+). Identification of Delta97nsP4 TATase activity suggests a novel function of the alphavirus RNA-dependent RNA polymerase in the maintenance and repair of the poly(A) tail, an element required for replication of the viral genome.     

Inhibition of host protein synthesis by Sindbis virus: correlation with viral RNA replication and release of nuclear proteins to the cytoplasm

Infection of mammalian cells by Sindbis virus (SINV) profoundly blocks cellular mRNA translation. Experimental evidence points to viral non‐structural proteins (nsPs), in particular nsP2, as the mediator of this inhibition. However, individual expression of nsP1, nsP2, nsP3 or nsP1‐4 does not block cellular protein synthesis in BHK cells. Trans‐complementation of a defective SINV replicon lacking most of the coding region for nsPs by the co‐expression of nsP1‐4 propitiates viral RNA replication at low levels, and inhibition of cellular translation is not observed. Exit of nuclear proteins including T‐cell intracellular antigen and polypyrimidine tract‐binding protein is clearly detected in SINV‐infected cells, but not upon the expression of nsPs, even when the defective replicon was complemented. Analysis of a SINV variant with a point mutation in nsP2, exhibiting defects in the shut‐off of host protein synthesis, indicates that both viral RNA replication and the release of nuclear proteins to the cytoplasm are greatly inhibited. Furthermore, nucleoside analogues that inhibit cellular and viral RNA synthesis impede the blockade of host mRNA translation, in addition to the release of nuclear proteins. Prevention of the shut‐off of host mRNA translation by nucleoside analogues is not due to the inhibition of eIF2α phosphorylation, as this prevention is also observed in PKR^?/? mouse embryonic fibroblasts that do not phosphorylate eIF2α after SINV infection. Collectively, our observations are consistent with the concept that for the inhibition of cellular protein synthesis to occur, viral RNA replication must take place at control levels, leading to the release of nuclear proteins to the cytoplasm.     

Sequestration of G3BP coupled with efficient translation inhibits stress granules in Semliki Forest virus infection

Dynamic, mRNA-containing stress granules (SGs) form in the cytoplasm of cells under environmental stresses, including viral infection. Many viruses appear to employ mechanisms to disrupt the formation of SGs on their mRNAs, suggesting that they represent a cellular defense against infection. Here, we report that early in Semliki Forest virus infection, the C-terminal domain of the viral nonstructural protein 3 (nsP3) forms a complex with Ras-GAP SH3-domain–binding protein (G3BP) and sequesters it into viral RNA replication complexes in a manner that inhibits the formation of SGs on viral mRNAs. A viral mutant carrying a C-terminal truncation of nsP3 induces more persistent SGs and is attenuated for propagation in cell culture. Of importance, we also show that the efficient translation of viral mRNAs containing a translation enhancer sequence also contributes to the disassembly of SGs in infected cells. Furthermore, we show that the nsP3/G3BP interaction also blocks SGs induced by other stresses than virus infection. This is one of few described viral mechanisms for SG disruption and underlines the role of SGs in antiviral defense.     

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.     

Pyrimidine Nucleotide Metabolism and Pathways of Thymidine Triphosphate Biosynthesis in Salmonella typhimurium

The nucleoside triphosphate pools of two cytidine auxotrophic mutants of Salmonella typhimurium LT-2 were studied under different conditions of pyrimidine starvation. Both mutants, DP-45 and DP-55, are defective in cytidine deaminase and cytidine triphosphate (CTP) synthase. In addition, DP-55 has a requirement for uracil (uridine). Cytidine starvation of the mutants results in accumulation of high concentrations of uridine triphosphate (UTP) in the cells, while the pools of CTP and deoxy-CTP drop to undetectable levels within a few minutes. Addition of deoxycytidine to such cells does not restore the dCTP pool, indicating that S. typhimurium has no deoxycytidine kinase. From the kinetics of UTP accumulation during cytidine starvation, it is concluded that only cytidine nucleotides participate in the feedback regulation of de novo synthesis of UTP; both uridine and cytidine nucleotides participate in the regulation of UTP synthesis from exogenously supplied uracil or uridine. Uracil starvation of DP-55 in presence of cytidine results in extensive accumulation of CTP, suggesting that CTP does not regulate its own synthesis from exogenous cytidine. Analysis of the thymidine triphosphate (dTTP) pool of DP-55 labeled for several generations with (32)P-orthophosphate and (3)H-uracil in presence of (12)C-cytidine shows that only 20% of the dTTP pool is derived from uracil (via the methylation of deoxyuridine monophosphate); 80% is apparently synthesized from a cytidine nucleotide.     

An attenuating mutation in nsP1 of the Sindbis-group virus S.A.AR86 accelerates nonstructural protein processing and up-regulates viral 26S RNA synthesis

The Sindbis-group alphavirus S.A.AR86 encodes a threonine at nonstructural protein 1 (nsP1) 538 that is associated with neurovirulence in adult mice. Mutation of the nsP1 538 Thr to the consensus Ile found in nonneurovirulent Sindbis-group alphaviruses attenuates S.A.AR86 for adult mouse neurovirulence, while introduction of Thr at position 538 in a nonneurovirulent Sindbis virus background confers increased neurovirulence (M. T. Heise et al., J. Virol. 74:4207-4213, 2000). Since changes in the viral nonstructural region are likely to affect viral replication, studies were performed to evaluate the effect of Thr or Ile at nsP1 538 on viral growth, nonstructural protein processing, and RNA synthesis. Multistep growth curves in Neuro2A and BHK-21 cells revealed that the attenuated s51 (nsP1 538 Ile) virus had a slight, but reproducible growth advantage over the wild-type s55 (nsP1 538 Thr) virus. nsP1 538 lies within the cleavage recognition domain between nsP1 and nsP2, and the presence of the attenuating Ile at nsP1 538 accelerated the processing of S.A.AR86 nonstructural proteins both in vitro and in infected cells. Since nonstructural protein processing is known to regulate alphavirus RNA synthesis, experiments were performed to evaluate the effect of Ile or Thr at nsP1 538 on viral RNA synthesis. A combination of S.A.AR86-derived reporter assays and RNase protection assays determined that the presence of Ile at nsP1 538 led to earlier expression from the viral 26S promoter without affecting viral minus- or plus-strand synthesis. These results suggest that slower nonstructural protein processing and delayed 26S RNA synthesis in wild-type S.A.AR86 infections may contribute to the adult mouse neurovirulence phenotype of S.A.AR86.     

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 P123_C>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, P23_C>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 P123_C>S, thus providing a functional assay for purification of the nsP4 RNA polymerase.     

Key role of uridine kinase and uridine phosphorylase in the homeostatic regulation of purine and pyrimidine salvage in brain

Uridine, the major circulating pyrimidine nucleoside, participating in the regulation of a number of physiological processes, is readily uptaken into mammalian cells. The balance between anabolism and catabolism of intracellular uridine is maintained by uridine kinase, catalyzing the first step of UTP and CTP salvage synthesis, and uridine phosphorylase, catalyzing the first step of uridine degradation to β-alanine in liver. In the present study we report that the two enzymes have an additional role in the homeostatic regulation of purine and pyrimidine metabolism in brain, which relies on the salvage synthesis of nucleotides from preformed nucleosides and nucleobases, rather than on the de novo synthesis from simple precursors. The experiments were performed in rat brain extracts and cultured human astrocytoma cells. The rationale of the reciprocal regulation of purine and pyrimidine salvage synthesis in brain stands (i) on the inhibition exerted by UTP and CTP, the final products of the pyrimidine salvage pathway, on uridine kinase and (ii) on the widely accepted idea that pyrimidine salvage occurs at the nucleoside level (mostly uridine), while purine salvage is a 5-phosphoribosyl-1-pyrophosphate (PRPP)-mediated process, occurring at the nucleobase level. Thus, at relatively low UTP and CTP level, uptaken uridine is mainly anabolized to uridine nucleotides. On the contrary, at relatively high UTP and CTP levels the inhibition of uridine kinase channels uridine towards phosphorolysis. The ribose-1-phosphate is then transformed into PRPP, which is used for purine salvage synthesis.