Enzymatic defects of the nsP2 proteins of Semliki Forest virus temperature-sensitive mutants

We have analyzed the biochemical consequences of mutations that affect viral RNA synthesis in Semliki Forest virus temperature-sensitive (ts) mutants. Of the six mutations mapping in the multifunctional replicase protein nsP2, three were located in the N-terminal helicase region and three were in the C-terminal protease domain. Wild-type and mutant nsP2s were expressed, purified, and assayed for nucleotide triphosphatase (NTPase), RNA triphosphatase (RTPase), and protease activities in vitro at 24°C and 35°C. The protease domain mutants (ts4, ts6, and ts11) had reduced protease activity at 35°C but displayed normal NTPase and RTPase. The helicase domain mutation ts1 did not have enzymatic consequences, whereas ts13a and ts9 reduced both NTPase and protease activities but in different and mutant-specific ways. The effects of these helicase domain mutants on protease function suggest interdomain interactions within nsP2. NTPase activity was not directly required for protease activity. The similarities of the NTPase and RTPase results, as well as competition experiments, suggest that these two reactions utilize the same active site. The mutations were also studied in recombinant viruses first cultivated at the permissive temperature and then shifted up to the restrictive temperature. Processing of the nonstructural polyprotein was generally retarded in cells infected with viruses carrying the ts4, ts6, ts11, and ts13a mutations, and a specific defect appeared in ts9. All mutations except ts13a were associated with a large reduction in the production of the subgenomic 26S mRNA, indicating that both protease and helicase domains influence the recognition of the subgenomic promoter during virus replication.     

Identification of mutations causing temperature-sensitive defects in Semliki Forest virus RNA synthesis

We have sequenced the nonstructural protein coding region of Semliki Forest virus temperature-sensitive (ts) mutant strains ts1, ts6, ts9, ts10, ts11, ts13, and ts14. In each case, the individual amino acid changes uncovered were transferred to the prototype strain background and thereby identified as the underlying cause of the altered RNA synthesis phenotype. All mutations mapping to the protease domain of nonstructural protein nsP2 caused defects in nonstructural polyprotein processing and subgenomic RNA synthesis, and all mutations in the helicase domain of nsP2 affected subgenomic RNA production. These types of defects were not associated with mutations in other nonstructural proteins.     

Elimination of phosphorylation sites of Semliki Forest virus replicase protein nsP3

nsP3 is one of the four RNA replicase subunits encoded by alphaviruses. The specific essential functions of nsP3 remain unknown, but it is known to be phosphorylated on serine and threonine residues. Here we have completed mapping of the individual phosphorylation sites on Semliki Forest virus nsP3 (482 amino acids) by point mutational analysis of threonine residues. This showed that threonines 344 and 345 represented the major threonine phosphorylation sites in nsP3. Experiments with deletion variants suggested that nsP3 itself had no kinase activity; instead, it was likely to be phosphorylated by multiple cellular kinases. Phosphorylation was not necessary for the peripheral membrane association of nsP3, which was mediated by the N-terminal region preceding the phosphorylation sites. Two deletion variants of nsP3 with either reduced or undetectable phosphorylation were studied in the context of virus infection. Cells infected with mutant viruses produced close to wild type levels of infectious virions; however, the rate of viral RNA synthesis was significantly reduced in the mutants. A virus totally defective in nsP3 phosphorylation and exhibiting a decreased rate of RNA synthesis also exhibited greatly reduced pathogenicity in mice.     

Proteins synthesized by Semliki Forest virus and its 16 temperature-sensitive mutants.

The proteins synthesized in chicken embryo fibroblasts infected with wild-type Semliki Forest virus and 16 temperature-sensitive mutants derived from it were studied by polyacrylamide gel electrophoresis. In addition to the structural proteins, five nonvirion proteins (NVP) with molecular weight of 130,000, 97,000, 86,000, 78,000 and 62,000 were found varying amounts in cells infected with the different RNA+ mutants and also in the wild-type-infected cells. Pulse-chase experiments suggested that NVP 130, NVP 97, NVP 86, and NVP 62 are precursors presumably of the structural proteins. The amount of NVP 78 was not affected by the chase, and it may represent a translational product of the nonstructural part of the genome. The NVP 130 was shown to be a common precursor of the structural proteins by tryptic peptide mapping. Kinetic evidence from one of the mutants (ts-3) suggested that NVP 86 is one of the precursors of the capsid protein. A common feature of all the RNA+mutants was the inability to cleave the NVP 62 into E2 and E3, suggesting that this cleavage is a crucial reaction in the virus maturation.     

Expression of Semliki Forest virus nsP1-specific methyltransferase in insect cells and in Escherichia coli.

We have expressed the Semliki Forest virus (SFV)-specific nonstructural protein nsP1 both in insect cells and in Escherichia coli in the absence of other viral proteins. A substantial amount of nsP1 was synthesized in Sf9 cells infected with the recombinant Autographa californica nuclear polyhedrosis virus (AcNPV) AcNPV-nsP1. These cells had a high level of guanine-7-methyltransferase activity compared with that of wild-type AcNPV-infected cells. The methyltransferase activity and nsP1 were mostly in the mitochondrial pellet fraction (P15). The enzymatic activity was increased by treatment with deoxycholate (DOC), as in the case of SFV-infected BHK cells. The material released by DOC treatment from P15 of the AcNPV-nsP1-infected cells was analyzed by gel filtration and sucrose gradient centrifugation. Both the methyltransferase activity and nsP1 were in aggregates. nsP1 expressed in E. coli at 37 degrees C sedimented at 15,000 x g, whereas after expression at 15 degrees C, both nsP1 and methyltransferase activity were in the supernatant fraction. Paradoxically, the activity from E. coli was completely inhibited by Triton X-100 and DOC. Sucrose gradient analysis showed that even the "soluble" nsP1-methyltransferase was in aggregates. The methyltransferase activities in the P15 fractions of SFV-infected BHK cells and AcNPV-nsP1-infected Sf9 cells and in E. coli catalyzed linear incorporation of the [3H]methyl group from S-adenosylmethionine to GTP for a 60-min period. The enzymes from the three sources had similar substrate specificities and Km values for S-adenosylmethionine. In addition to GTP, they all methylated dGTP and GpppG, but not m7GTP or GpppA, or in vitro-transcribed RNAs with GpppA and GpppG caps. The unique properties of SFV-specific nsP1 methyltransferase are discussed.     

Synthesis of Semliki Forest virus RNA polymerase components nsP1 through nsP4 in Saccharomyces cerevisiae by expression of cDNA encoding the nonstructural polyprotein.

A Semliki Forest virus nonstructural polyprotein, P1234, expressed in the yeast Saccharomyces cerevisiae in the absence of a replicative RNA template appeared to be properly cleaved into nsP1 to nsP4. All nsPs were membrane associated, and nsP2 was also transported to the nucleus. The membrane fraction containing nsPs showed guanine-7-methyltransferase and guanylyltransferase-like activities, typical for Semliki Forest virus nsP1.     

Mechanisms of mutations inhibiting fusion and infection by Semliki Forest virus

Semliki Forest virus (SFV) infects cells by an acid-dependent membrane fusion reaction catalyzed by the virus spike protein, a complex containing E1 and E2 transmembrane subunits. E1 carries the putative virus fusion peptide, and mutations in this domain of the spike protein were previously shown to shift the pH threshold of cell-cell fusion (G91A), or block cell-cell fusion (G91D). We have used an SFV infectious clone to characterize virus particles containing these mutations. In keeping with the previous spike protein results, G91A virus showed limited secondary infection and an acid-shifted fusion threshold, while G91D virus was noninfectious and inactive in both cell- cell and virus-liposome fusion assays. During the low pH- induced SFV fusion reaction, the E1 subunit exposes new epitopes for monoclonal antibody (mAb) binding and forms an SDS-resistant homotrimer, the virus associates hydrophobically with the target membrane, and fusion of the virus and target membranes occurs. After low pH treatment, G91A spike proteins were shown to bind conformation-specific mAbs, associate with target liposome membranes, and form the E1 homotrimer. However, both G91A membrane association and homotrimer formation had an acid-shifted pH threshold and reduced efficiency compared to wt virus. In contrast, studies of the fusion-defective G91D mutant showed that the virus efficiently reacted with low pH as assayed by mAb binding and liposome association, but was essentially inactive in homotrimer formation. These results suggest that the G91D mutant is noninfectious due to a block in a late step in membrane fusion, separate from the initial reaction to low pH and interaction with the target membrane, and involving the lack of efficient formation of the E1 homotrimer.     

Functional defects of RNA-negative temperature-sensitive mutants of Sindbis and Semliki Forest viruses.

Defects in RNA and protein synthesis of seven Sindbis virus and seven Semliki Forest virus RNA-negative, temperature-sensitive mutants were studied after shift to the restrictive temperature (39 degrees C) in the middle of the growth cycle. Only one of the mutants, Ts-6 of Sindbis virus, a representative of complementation group F, was clearly unable to continue RNA synthesis at 39 degrees C, apparently due to temperature-sensitive polymerase. The defect was reversible and affected the synthesis of both 42S and 26S RNA equally, suggesting that the same polymerase component(s) is required for the synthesis of both RNA species. One of the three Sindbis virus mutants of complementation group A, Ts-4, and one RNA +/- mutant of Semliki Forest virus, ts-10, showed a polymerase defect even at the permissive temperature. Seven of the 14 RNA-negative mutants showed a preferential reduction in 26S RNA synthesis. The 26S RNA-defective mutants of Sindbis virus were from two different complementation groups, A and G, indicating that functions of two viral nonstructural proteins ("A" and "G") are required in the regulation of the synthesis of 26S RNA. Since the synthesis of 42S RNA continued, these functions of proteins A and G are not needed for the polymerization of RNA late in infection. The RNA-negative phenotype of 26S RNA-deficient mutants implies that proteins regulating the synthesis of this subgenomic RNA must have another function vital for RNA synthesis early in infection or in the assembly of functional polymerase. Several of the mutants having a specific defect in the synthesis of 26S RNA showed an accumulation of a large nonstructural precursor protein with a molecular weight of about 200,000. One even larger protein was demonstrated in both Semliki Forest virus- and Sindbis virus-infected cells which probably represents the entire nonstructural polyprotein.     

ATPase and GTPase activities associated with Semliki Forest virus nonstructural protein nsP2.

The replication of Semliki Forest virus requires four nonstructural proteins (nsP1 to nsP4), all derived from the same polyprotein. One of these, nsP2, is a multifunctional protein needed in RNA replication and in the processing of the nonstructural polyprotein. On the basis of amino acid sequence homologies, nsP2 was predicted to possess nucleoside triphosphatase and RNA helicase activities. Here, we report the engineered expression in Escherichia coli of nsP2 and of an amino-terminal fragment of it by use of the highly efficient T7 expression system. Both polypeptides were produced as fusion proteins with a histidine tag at the amino terminus and purified by immobilized-metal affinity chromatography. The two recombinant proteins exhibited ATPase and GTPase activities, which were further stimulated by the presence of single-stranded RNA. The activities were not found in similarly prepared fractions from uninduced control cells or cells expressing an unrelated polypeptide. Radiolabeled ribonucleoside triphosphates could be cross-linked to both the full-length and the carboxy-terminally truncated nsP2 protein, and both polypeptides had RNA-binding capacity. We also expressed and purified an nsP2 variant which had a single amino acid substitution in the nucleotide-binding motif (Lys-192-->Asn). No nucleoside triphosphatase activity was associated with this mutant protein.     

Characterization of the cysteine protease domain of Semliki Forest virus replicase protein nsP2 by in vitro mutagenesis

The function of Semliki Forest Virus nsP2 protease was investigated by site-directed mutagenesis. Mutations were introduced in its protease domain, Pro39, and the mutated proteins were expressed in Escherichia coli, purified and their activity in vitro was compared to that of the wild type Pro39. Mutations M781T, A662T and G577R, found in temperature-sensitive virus strains, rendered the enzyme temperature-sensitive in vitro as well. Five conserved residues were required for the proteolytic activity of Pro39. Changes affecting Cys⁴⁷⁸, His⁵⁴⁸, and Trp⁵⁴⁹ resulted in complete inactivation of the enzyme, whereas the replacements N600D and N605D significantly impaired its activity. The importance of Trp⁵⁴⁹ for the proteolytic cleavage specificity is discussed and a new structural motif involved in substrate recognition by cysteine proteases is proposed.     

Nuclear localization of Semliki Forest virus-specific nonstructural protein nsP2.

About 50% of Semliki Forest virus-specific nonstructural protein nsP2 is associated with the nuclear fraction in virus-infected BHK cells. Transport into the nucleus must be specific, since only trace amounts of nsP3 and nsP4 and about 13% of nsP1, all derived from the same polyprotein, were found in the nucleus. Subfractionation of [35S]methionine-labeled Semliki Forest virus-infected cells showed that 80 to 90% of the nuclear nsP2 was associated with the nuclear matrix. Indirect immunofluorescence, with anti-nsP2 antiserum, showed the most intensive staining of structures which by Nomarski optics appeared to be nucleoli. In the presence of 1 to 5 micrograms of dactinomycin per ml the nuclei were stained evenly and no nucleoli could be found. Transport of nsP2 into the nucleus occurred early in infection and was fairly rapid. A cDNA encoding the complete nsP2 was isolated by the polymerase chain reaction technique and ligated into a simian virus 40 expression vector derivative. When BHK cells were transfected with this pSV-NS2 vector by the lipofection procedure, nsP2 was expressed in about 1 to 5% of the cells, as shown by indirect immunofluorescence. In positively transfected cells the immunofluorescence stain was most intensive in the nucleoli. Thus, Semliki Forest virus-specific nsP2 must have information which directs it into the nuclear matrix and, more specifically, into the nucleoli.     

Molecular determinants of substrate specificity for Semliki Forest virus nonstructural protease

The C-terminal cysteine protease domain of Semliki Forest virus nonstructural protein 2 (nsP2) regulates the virus life cycle by sequentially cleaving at three specific sites within the virus-encoded replicase polyprotein P1234. The site between nsP3 and nsP4 (the 3/4 site) is cleaved most efficiently. Analysis of Semliki Forest virus-specific cleavage sites with shuffled N-terminal and C-terminal half-sites showed that the main determinants of cleavage efficiency are located in the region preceding the cleavage site. Random mutagenesis analysis revealed that amino acid residues in positions P4, P3, P2, and P1 of the 3/4 cleavage site cannot tolerate much variation, whereas in the P5 position most residues were permitted. When mutations affecting cleavage efficiency were introduced into the 2/3 and 3/4 cleavage sites, the resulting viruses remained viable but had similar defects in P1234 processing as observed in the in vitro assay. Complete blockage of the 3/4 cleavage was found to be lethal. The amino acid in position P1' had a significant effect on cleavage efficiency, and in this regard the protease markedly preferred a glycine residue over the tyrosine natively present in the 3/4 site. Therefore, the cleavage sites represent a compromise between protease recognition and other requirements of the virus life cycle. The protease recognizes at least residues P4 to P1', and the P4 arginine residue plays an important role in the fast cleavage of the 3/4 site.     

Protein-bound oligosaccharides of Semliki Forest virus

Semliki Forest virus was grown in BHK cells and labeled in vivo with radio-active monosaccharides. promnase digenst of the virus chromatographer on Bio-Gel P 6 revealed glycopeptides of A-type and B-type. (For the nomenclature see Johnson J. and Clamp J.R. (1971) Biochem. J. 123, 739–745) The former was labeled with [³H]fucose, [³H]galactose, [³H]mannose and [¹⁴C]glucosamine, the latter only with [³H]mannose and [¹⁴C]glucosamine. The three envelope glycoproteins E₁, E₂ and E₃ were isolated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and subjected to pronase digestion. The glycoproteins E₁ and E₃ revealed glycopeptides of A-type. E₂ revealed glycopeptides of B-type. E₂ yielded additionally a glycopeptide (M_r3100) which was heavily labeled from [³H]galactose, but only marginally from [¹⁴C]glucosamine, [³H]fucose and [³H]mannose. Wether this glycopeptide belongs to the A-type or not remains uncertain. The apparent molecular weights of the A-type units measured by gel filtration were 3400 in E₁ and 4000 in E₃; the B-type unit of E₂ had an apparent molecular weight of 2000. Combined with the findings of our earlier chemical analysis these data suggast that E₁ and E₃ contain on the average one A-type unit; E₂ probably contains one 3100 dalton unit plus one or two B-type units.     

Role of the amphipathic peptide of Semliki forest virus replicase protein nsP1 in membrane association and virus replication

Semliki Forest virus RNA replication takes place in association with specific cytoplasmic vacuoles, derived from the endosomal apparatus. Of the four virus-encoded replicase proteins, nsP1 serves as the membrane anchor of the replication complex. An amphipathic peptide segment, G₂₄₅STLYTESRKLLRSWHLPSV₂₆₄, has been implicated in the membrane binding of nsP1. nsP1 variants with changes within the peptide were studied after protein expression and in the context of virus infection. Proteins with mutations R253E and W259A accumulated in the cytoplasm and were very poorly palmitoylated. The same mutations also drastically affected the localization of the precursor polyprotein P123, and they were lethal when introduced into the virus genome. Mutations R253A and L255A+L256A partially changed the localization of nsP1, and the respective viruses acquired compensatory changes. L255A+L256A only yielded virus encoding L255A+L256V, indicating the importance of a hydrophobic residue in the central 256 position. When fused to green fluorescent protein, the peptide was required in at least two tandem copies to effect a change in localization, but even then the fusion protein was associated with membranes in a nonspecific manner. Thus, the amphipathic peptide is a crucial element for the membrane association of nsP1 and the replication complex. It provides essential affinity for membranes, and other regions of nsP1 also appear to contribute to the localization of the protein.     

Cholesterol is required for infection by Semliki Forest virus

Semliki Forest virus (SFV) and many other enveloped animal viruses enter cells by a membrane fusion reaction triggered by the low pH within the endocytic pathway. In vitro, SFV fusion requires cholesterol in the target membrane, but the role of cholesterol in vivo is unknown. In this paper, the infection pathway of SFV was studied in mammalian and inset cells substantially depleted of sterol. Cholesterol-depleted cells were unaltered in their ability to bind, internalize, and acidify virus, but were blocked in SFV fusion and subsequent virus replication. Depleted cells could be infected by the cholesterol-independent vesicular stomatitis virus, which also enters cells via endocytosis and low pH-mediated fusion. The block in SFV infection was specifically reversed by cholesterol but not by cholestenone, which lacks the critical 3 beta-hydroxyl group. Cholesterol thus is central in the infection pathway of SFV, and may act in vivo to modulate infection by SFV and other pathogens.     

Molecular dynamics simulations of the E1/E2 transmembrane domain of the Semliki Forest virus

Transmembrane (TM) helix-helix interactions are important for virus budding and fusion. We have developed a simulation strategy that reveals the main features of the helical packing between the TM domains of the two glycoproteins E1 and E2 of the alpha-virus Semliki Forest virus and that can be extrapolated to sketch TM helical packing in other alpha-viruses. Molecular dynamics simulations were performed in wild-type and mutant peptides, both isolated and forming E1/E2 complexes. The simulations revealed that the isolated wild-type E1 peptide formed a more flexible helix than the rest of peptides and that the wild-type E1/E2 complex consists of two helices that intimately pack their N-terminals. The residues located at the interhelical interface displayed the typical motif of the left-handed coiled-coils. These were small and medium residues as Gly, Ala, Ser, and Leu, which also had the possibility to form interhelical Calpha-H...O hydrogen bonds. Results from the mutant complexes suggested that correct packing is a compromise between these residues at both E1 and E2 interhelical interfaces. This compromise allowed prediction of E1-E2 contact residues in the TM spanning domain of other alphaviruses even though the sequence identity of E2 peptides is low in this domain.     

Short-lived minus-strand polymerase for Semliki Forest virus

Semliki Forest virus (SFV)-infected BHK-21, Vero, and HeLa cells incorporated [3H]uridine into 42S and 26S plus-strand RNA and into viral minus-strand RNA (complementary to the 42S virion RNA) early in the infectious cycle. Between 3 and 4 h postinfection, the synthesis of minus-strand RNA ceased in these cultures, although the synthesis of plus-strand RNA continued at a maximal rate. At the time of cessation of minus-strand RNA synthesis, two changes in the pattern of viral protein synthesis were detected: a decrease in the translation of nonstructural proteins and an increase in the translation of the viral structural proteins. Addition of cycloheximide and puromycin to cultures of SFV-infected BHK cells actively synthesizing both viral plus- and minus-strand RNA resulted within 15 to 30 min in the selective shutoff of minus-strand RNA synthesis. Removal of the cycloheximide-containing medium led to the resumption of minus-strand synthesis and to an increased rate of viral RNA synthesis. We conclude that the minus-strand polymerase regulates the rate of SFV plus-strand RNA synthesis by determining the number of minus-strand templates and that the synthesis of the minus-strand templates is regulated at the level of translation by a mechanism which utilizes one or more short-lived polymerase proteins.