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Journal of Virology, March 2006, p. 3108-3111, Vol. 80, No. 6
0022-538X/06/$08.00+0     doi:10.1128/JVI.80.6.3108-3111.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Identification of Mutations Causing Temperature-Sensitive Defects in Semliki Forest Virus RNA Synthesis

Valeria Lulla,1,2 Andres Merits,2 Peter Sarin,1 Leevi Kääriäinen,1 Sirkka Keränen,1 and Tero Ahola1*

Program in Cellular Biotechnology, Institute of Biotechnology, University of Helsinki, Helsinki, Finland,1 Estonian Biocentre and Institute of Molecular and Cellular Biology, Tartu, Estonia2

Received 7 September 2005/ Accepted 21 December 2005


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ABSTRACT
 
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.


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TEXT
 
The alphaviruses are enveloped positive-strand RNA viruses, whose 42S genome of approximately 11.5 kb encodes four nonstructural (ns) proteins, nsP1 to nsP4, starting from the 5' end. In Semliki Forest virus (SFV), the ns proteins are produced as a large polyprotein, P1234, of 2,432 residues, which is processed to the final products in a regulated sequential order (23). The nsPs have multiple enzymatic and nonenzymatic functions required in viral RNA replication (9; see below). The structural proteins, encoded by the 3' third of the genome, are translated from a subgenomic 26S mRNA generated by internal initiation on the complementary minus-strand template. Temperature-sensitive (ts) mutants of Sindbis virus (SIN) and SFV (2, 10, 19) have been used in studies of RNA synthesis, processing and intracellular transport of viral proteins, and maturation of virus particles. They have yielded important insights into the different stages of viral RNA replication, such as the regulation of minus-strand and subgenomic RNA syntheses (reviewed in reference 9).

To tie the virus replication phenotypes observed in ts mutants with the properties of the individual ns proteins, we have initiated a systematic analysis of those SFV ts mutants, which displayed a significant phenotype in RNA synthesis and therefore are expected to have lesions in the ns proteins. We have now sequenced the entire ns region of the second passage of SFV ts1, ts6, ts9, ts10, ts11, ts13, and ts14 that had been stored at –70°C for over three decades (10). Viral RNAs were isolated by using RNeasy Minikit (QIAGEN) and reverse transcription-PCR amplified by a set of 13 forward primers and 13 reverse primers altogether amplifying nucleotides 1 to 7900 of the SFV genome. The sequences of all of the primers used are available from the authors upon request. The PCR products were subjected to automated DNA sequencing without cloning. When differences from the wild-type (wt) SFV sequence were detected, the amplification and sequencing procedures were repeated, and only the confirmed changes were considered to be mutations present in the virus stock.

The mutations and consequent amino acid changes found in the mutant stocks are detailed in Table 1. The presence of only a single substitution in most mutants may reflect the way in which the stocks were originally prepared, involving the minimum possible number of passages (10). To confirm that the mutations causing amino acid changes were responsible for the ts phenotype, the individual changes were transferred to the infectious cDNA clone pSP6-SFV4 (14), starting from the appropriate PCR fragment and utilizing available restriction sites. All of the clones were verified by sequencing the entire fragment transferred. According to the nomenclature of Suopanki et al. (20), the recombinant putatively ts viruses are designated SFots6, SFots9, SFots11, and SFots14. When the virus contains the mutation(s) present in the ns protein region, originating from mutants initially classified as RNA positive or "±" (implying the presence of additional mutations in the structural proteins), the recombinant virus is named SFons1, SFons10, or SFons13. For ts13, which had two amino acid changes in the nsPs (Table 1), recombinant SFons13ab contained both changes, whereas SFons13a and SFons13b carried them separately. Capped transcripts were prepared with SP6 polymerase after linearization of the plasmids with SpeI (14) and used for transfection of BHK21 cells by the aid of Lipofectin (Invitrogen). The virus stock was collected after growth for 60 h at the permissive temperature of 28°C, diluted 1 to 100, and used to infect fresh BHK cells. After a further 60 h, the second passage stock was harvested. These stocks were used in all subsequent experiments. The presence of the desired mutation in these second passage stocks was verified by sequencing after reverse transcription-PCR of isolated RNA. New passages of the original ts virus stocks, grown in BHK cells for 60 h at 28°C starting at multiplicity of infection (MOI) of 0.01, were used as controls in all experiments.


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  		TABLE 1. Nucleotide and encoded amino acid changes between the parental wt strain and ts mutants

(i) All of the virus stocks were titrated in BHK cells at 28°C and at the restrictive temperature of 39°C by plaque assay (Table 2) as described previously (10). (ii) To measure leak yield at the nonpermissive temperature, two parallel dishes were infected with each virus stock at an MOI of 10: one incubated at 28°C for 16 h and the other incubated at 39°C for 8 h. The accumulated virus stocks were titrated by plaque assay at 28°C (Table 2). (iii) To study RNA synthesis at the restrictive temperature, BHK cells in 35-mm dishes were infected at an MOI of 50 and labeled with 25 µCi of [3H]uridine/dish in the presence of 2 µg of actinomycin D/ml between 2 and 4 h postinfection. The cells were lysed in 1% sodium dodecyl sulfate, and acid-insoluble radioactivity was determined by liquid scintillation. Based on these results, the viruses were classified as RNA positive (>40% of wt RNA synthesis), negative (<5% of wt), or "±" (5 to 40% of wt) (Table 2). (iv) To measure subgenomic RNA synthesis, cells infected as described above were kept at 28°C for 6 h and then transferred to 39°C for 1 h and labeled for a further 1 h at 39°C with 50 µCi of [3H]uridine/ml. Actinomycin D was added at the time of shift-up. RNA was extracted with TRIzol (Invitrogen), and the samples were denatured with glyoxal-dimethyl sulfoxide and analyzed in 0.8% agarose gels. For the wt, the molar ratio of 42S to 26S RNA was close to 0.5 in several repeated experiments. Mutants with a ratio of >1.0 were classified as defective, and ts11 with a ratio close to 0.75 was classified as partially defective ("±") (Table 2 and Fig. 1A). (v) Polyprotein processing was studied after shift-up to the restrictive temperature at 5.5 h by pulse-labeling the cells with [35S]methionine starting at 6 h for 15 min. Mutants that showed a >2-fold increase in the accumulation of both of the largest precursors, P1234 and P123, compared to the wt at the 1-h chase point were classified as defective (Table 2 and Fig. 1B). It remains possible that more careful quantitative studies will reveal additional defects for the mutants.


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  		TABLE 2. Properties of the original and recombinant virus strains


Figure 1
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  		FIG. 1. RNA and protein analysis of the recombinant virus strains after shift-up and equilibration to the restrictive temperature. (A) Viral RNAs labeled for 1 h with [3H]uridine were extracted, analyzed in agarose gels, and visualized by fluorography. Virus strains are indicated at the top, and the positions of 42S and 26S RNAs are marked. (B) Proteins were labeled for 15 min with [35S]methionine. The sample from one parallel plate was extracted immediately after the pulse, while the other plate was chased with excess cold methionine for 1 h before extraction. The samples were immunoprecipitated with combinations of antibodies against the ns proteins and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fluorography to detect all of the polyprotein precursors and mature proteins (16). The virus strain, sample (pulse or chase), and antibody combinations used for immunoprecipitation (IP, numbers denote antibodies against the ns proteins) are indicated at the top. The positions of proteins are marked on the left. NsP1 and nsP3 migrate very similarly but can be visualized on adjacent lanes due to the antibody combinations used.

The wt viruses behaved as expected and did not present any significant temperature-dependent reduction in virus titer or yield. The RNA-positive ts1 mutant has a defect in particle formation due to a mutation in the envelope protein E3 (21) and makes a reduced amount of 26S RNA. The latter defect is clearly due to the mutation S308N now uncovered in the nsP2 protein (Table 2): SFons1 containing this alteration alone grew like wt at 39°C but produced less 26S RNA (Fig. 1A). The mutation in ts4 was mapped previously (20), and both ts4 and SFots4 were included in the current investigation as additional controls. The original ts6 behaved as an RNA negative mutant with defects in polyprotein processing and 26S RNA synthesis as described previously (11). The recombinant virus SFots6 showed lesser defects in virus titer and yield and had considerable RNA synthesis at 39°C (Table 2). However, it was still defective in polyprotein processing and 26S RNA synthesis, showing that these phenotypes were due to the mutation A662T in nsP2 (Fig. 1). At present, we do not know the reason for the difference between ts6 and SFots6, but it should be pointed out that the strain backgrounds of the recombinant infectious clone and the original cloned wt, the parent strain of the mutants, have several sequence differences.

The mutations in SFots9, SFots11, and SFots14 fully recapitulated the phenotype of the original viruses ts9, ts11, and ts14, respectively (Table 2). Mutant ts10 is RNA± and, accordingly, the ns mutation of SFons10 did not reduce virus yield but gave a corresponding "±" phenotype in RNA synthesis (Table 2). The structural protein region of the mutants ts10 and ts13, originally classified as RNA±, was also sequenced. In both cases it contained several mutations (see GenBank accession numbers listed in Table 1), some of which are presumably responsible for the ts phenotype of virus production. ts13 turned out to contain two amino acid changes in the ns protein region (Table 1). Their combination in SFons13ab was required to reproduce the phenotype of ts13, which now scored as RNA negative. Either mutation alone, in SFons13a and SFons13b, led to reduced RNA synthesis at the restrictive temperature, and the mutation in nsP2 caused a stronger defect (Table 2). Interestingly, there is also a SIN ts mutant in which mutations both in nsP2 and nsP4 are required for the ts phenotype, and this has been interpreted to support a functional interaction between these proteins (5).

The distribution of the ts mutations between the different functional domains of the nonstructural proteins (Fig. 2) leads to some straightforward conclusions. All of the mutations located within the C-terminal protease domain of nsP2 cause a defect in polyprotein processing. All mutations resulting in defective 26S RNA synthesis are found within nsP2 and, at least in this case, both of the domains contain mutations causing this alteration. Previously, SIN mutations within the protease domain often showed an associated defect in subgenomic RNA synthesis, but this is not the case for SIN helicase domain mutants, with the possible exception of SIN ts21 (3, 6, 17, 20). Now, especially ts1, whose 26S RNA defect has been well documented (10, 11), and also ts9 and ts13 (Fig. 1A) show that the helicase domain also influences 26S RNA synthesis. This may not be surprising, given that nsP2 has to act as a single unit, and thus a mutation in one domain could affect also functions of the other, for instance by altering protein conformation. Since all three mutations now mapped to the helicase domain cause a 26S RNA phenotype, a more direct role of the helicase in 26S RNA synthesis also needs to be considered. ts1 still appears to be unique in overproducing the ns proteins (11, 18) and thus may constitute a unique functional class. It will be interesting to study the multiple mutations now found in nsP2 in terms of its known biochemical activities of NTPase, RNA triphosphatase, RNA helicase, and protease (4, 15, 22, 24) and thereby attempt to connect the phenotypic changes of the virus to functional alterations at the molecular level.


Figure 2
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  		FIG. 2. Functional domains of SFV nonstructural proteins and the location of mutations causing ts phenotypes. The domains responsible for the enzymatic activities are given approximately, as defined by functional studies or sequence conservation (9). Domain X is a highly conserved domain of unknown function. Amino acids (aa) are numbered separately for each of the ns proteins.

No new insights into the poorly understood functions of nsP3 were gained in the present study, and only one mutation by itself causing a relatively mild phenotype was found in the core polymerase subunit nsP4. The mutations found in nsP1 also merit further study. The ts10 lesion maps close to the processing site nsP1/2, and although no easily perceived defect in polyprotein processing was apparent in either this or the previous investigations made on ts10, this issue requires careful quantitative study under a variety of conditions. The 1/2 site cleavage is central in regulating the plus versus minus strand synthesis in alphaviruses (12, 13), and a mutation in SIN affecting pathogenesis has been mapped to this cleavage site (7, 8). The mutation in ts14 is located in the methyltransferase/capping enzyme domain of nsP1 (1) and should offer further insight into the role of RNA capping in the virus life cycle, if this process proves to be defective in ts14. In conclusion, the mutations now uncovered will help to clarify the multiple roles of the ns proteins in the different stages of alphavirus RNA synthesis and connect the in vitro observed enzymatic activities with events in infected cells.


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ACKNOWLEDGMENTS
 
We thank Giuseppe Balistreri for help in RNA analysis and Airi Sinkko for technical assistance.

This study was supported by the European Union 5th Framework Programme project SFvectors, by Academy of Finland grant 201687, by Estonian Science Foundation grant 5055, and by grant 067575 from The Wellcome Trust.


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FOOTNOTES
 
* Corresponding author. Mailing address: Institute of Biotechnology, P.O. Box 56 (Viikinkaari 9), University of Helsinki, 00014 Helsinki, Finland. Phone: 358-9-19159403. Fax: 358-9-19159560. E-mail: tero.ahola{at}helsinki.fi. Back


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Journal of Virology, March 2006, p. 3108-3111, Vol. 80, No. 6
0022-538X/06/$08.00+0     doi:10.1128/JVI.80.6.3108-3111.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Lulla, V., Sawicki, D. L., Sawicki, S. G., Lulla, A., Merits, A., Ahola, T. (2008). Molecular Defects Caused by Temperature-Sensitive Mutations in Semliki Forest Virus nsP1. J. Virol. 82: 9236-9244 [Abstract] [Full Text]  
  • Kiiver, K., Tagen, I., Zusinaite, E., Tamberg, N., Fazakerley, J. K., Merits, A. (2008). Properties of non-structural protein 1 of Semliki Forest virus and its interference with virus replication. J. Gen. Virol. 89: 1457-1466 [Abstract] [Full Text]  
  • Balistreri, G., Caldentey, J., Kaariainen, L., Ahola, T. (2007). Enzymatic Defects of the nsP2 Proteins of Semliki Forest Virus Temperature-Sensitive Mutants. J. Virol. 81: 2849-2860 [Abstract] [Full Text]  

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