Sequence requirements for Sindbis virus subgenomic mRNA promoter function in cultured cells.

The Sindbis virus minimal subgenomic mRNA promoter (spanning positions -19 to +5 relative to the subgenomic mRNA start site) is approximately three- to sixfold less active than the fully active -98 to +14 promoter region. We identified two elements flanking the -19 to +5 region which increase its transcription to levels comparable to the -98 to +14 region. These elements span positions -40 to -20 and +6 to +14 and act synergistically to enhance transcription. Nine different virus libraries were constructed containing blocks of five randomized nucleotides at various positions in the -40 to +14 region. On passaging these libraries in mosquito cells, a small subset of the viruses came to dominate the population. Sequence analysis at the population level and for individual clones revealed that in general, wild-type bases were preferred for positions -15 to +5 of the minimal promoter. Base mutagenesis experiments indicated that the selection of wild-type bases in this region was primarily due to requirements for subgenomic mRNA transcription. Outside of the minimal promoter, the -35 to -29 region contained four positions which also preferred wildtype bases. However, the remaining positions generally preferred non-wild-type bases. On passaging of the virus libraries on hamster cells, the -15 to +5 region again preferred the wild-type base but most of the remaining positions exhibited almost no base preference. The promoter thus consists of an essential central region from -15 to +5 and discrete flanking sites that render it fully active, depending on the host environment.

A novel viral RNA species in Sindbis virus-infected cells.

Sindbis virus (SIN), the type alphavirus, has been studied extensively to identify the viral cis-acting sequences and proteins involved in RNA transcription and replication. However, very little is known about how these processes are coordinated. For example, synthesis of the genomic RNA and the subgenomic mRNA depends on the minus strand. Do these activities occur independently on different templates, or can replication and transcription take place simultaneously on the same template? We describe the appearance of a SIN-specific, plus-sense RNA that is intermediate in size between the genomic and subgenomic RNA species. This RNA, designated RNA II, is observed in a number of different cell lines, both early and late in infection. The number of RNA II species, their sizes, and their abundances are influenced by the subgenomic promoter. We have mapped the 3' end of RNA II to a site within the subgenomic promoter, four nucleotides before the initiation site of the subgenomic mRNA. Our results indicate that the appearance of RNA II is correlated with subgenomic mRNA transcription, such that strong or active promoters tend to increase the abundance of RNA II, relative to weak or less active promoters. RNA II is most abundantly detected with the full promoter and is at much lower abundance with the minimal promoter. The possible origins of RNA II are discussed.

Alphavirus-based expression vectors: strategies and applications.

Alphaviruses are positive-strand RNA viruses that can mediate efficient cytoplasmic gene expression in insect and vertebrate cells. Through recombinant DNA technology, the alphavirus RNA replication machinery has been engineered for high-level expression of heterologous RNAs and proteins. Amplification of replication-competent alpha-virus RNAs (replicons) can be initiated by RNA or DNA transfection and a variety of packaging systems have been developed for producing high titers of infectious viral particles. Although normally cytocidal for vertebrate cells, variants with adaptive mutations allowing noncytopathic replication have been isolated from persistently infected cultures or selected using a dominant selectable marker. Such mutations have been mapped and used to create new alphavirus vectors for noncytopathic gene expression in mammalian cells. These vectors allow long-term expression at moderate levels and complement previous vectors designed for short-term high-level expression. Besides their use for a growing number of basic research applications, recombinant alphavirus RNA replicons may also facilitate genetic vaccination and transient gene therapy.

Sindbis virus vectors for expression in animal cells.

Sindbis virus and other alphavirus gene expression vectors have recently been used to express and study the functions of proteins and RNA, to evaluate classical vaccine and novel antiviral approaches, and for nucleic acid immunization. The vectors will likely attract continuing, innovative applications that exploit their useful features: rapid and efficient gene expression, wide host range, and RNA genomes.

Evolution of the Sindbis virus subgenomic mRNA promoter in cultured cells.

Transcription of the subgenomic mRNA of alphaviruses initiates at an internal site, called the promoter, which is highly conserved. To determine the functional significance of this conservation, we used an approach that randomizes positions -13 to -9 of the promoter to generate a library containing all possible sequences within this region, including the wild-type sequence. Viruses in the mixed population with more-efficient promoters were selected for during passaging in mammalian (BHK-21) cells. Results from early passage populations indicate that a large number of different promoters are functionally active. Analysis of eight individual viruses found that although each contained a promoter with different degrees of sequence identity to the wild-type sequence, all eight viruses produced progeny. This suggests that the mechanism for transcription allows for a diversity of sequences to serve as promoters. Further passaging of the viral library led to a population consensus sequence that increasingly resembled the wild-type sequence, despite the fact that these promoters are not constrained by the need to encode the carboxyl terminus of the nsP4 protein. Thus, conservation of the region of the promoter from -13 to -9 is in large part due to selection for promoter function, and the wild-type sequence and sequences closely similar to it seem to be optimal for promoter function in BHK-21 cells.

Host-dependent evolution of the Sindbis virus promoter for subgenomic mRNA synthesis.

Alphaviruses are alternately transmitted between arthropod and vertebrate hosts. In each host, the virus transcribes a subgenomic mRNA that encodes the viral structural proteins which encapsidate the genome to form progeny virions. Transcription initiates at an internal site called the promoter. To determine if promoter utilization varies in mammalian versus mosquito cells, we used these cells as hosts to select for active promoters among a library of different mutant promoters. Compared with that in BHK-21 cells, selection was more rapid in mosquito (C7-10) cells, with much less diversity of promoters remaining after fewer passages. Thus, promoter selection is host dependent. With further passaging, both BHK-21 and C7-10 cells selected for similar sequences that closely resemble the wild-type promoter sequence. The difference in the rates of selection is not because BHK-21-derived promoters cannot function in mosquito cells. Instead, part of the host dependence is probably due to posttranscriptional differences between BHK-21 and C7-10 cells that may require more active promoters in mosquito cells. Part of the host dependence may also be attributed to the decreased rate of transcription versus that of replication in mosquito cells. This change in regulation of subgenomic to genomic RNA synthesis appears to correlate with the extent of cleavage or pausing of the genomic RNA synthesis at or close to the promoter.

Utilization of heterologous alphavirus junction sequences as promoters by Sindbis virus.

We used Sindbis virus, an alphavirus, as a model to study the evolution of the recognition of viral cis-acting sequences. During the life cycle of alphaviruses, a full-length minus-strand RNA is made and serves as a template for both genomic RNA replication and subgenomic mRNA transcription. Transcription initiates at an internal promoter site, the junction sequence, to produce a subgenomic mRNA. The junction sequences of alphaviruses are highly conserved, but they do contain a number of base differences. These could have been essentially neutral mutations during evolution, such that any of the contemporary sequences can be recognized efficiently by any of the alphaviruses. Alternately, the changes could have resulted in significant functional divergence, such that the contemporary viruses can no longer recognize heterologous junction sequences as promoters. To distinguish between these possibilities, we constructed Sindbis virus derivatives with two subgenomic mRNA promoters. One is the wild-type Sindbis virus promoter used for expression of the structural proteins. The other is either the minimal Sindbis virus promoter or the corresponding junction sequences from other alphaviruses, which are placed upstream of the bacterial chloramphenicol acetyltransferase (CAT) gene. RNA analyses were used to determine the relative promoter strengths of the various junction sequences. The results showed that all but two were recognized as promoters by Sindbis virus. CAT enzyme assays were used to measure the accumulation of CAT protein made from mRNAs transcribed by using the heterologous junction sequences as promoters. Most of the viruses expressed amounts of CAT enzyme within 10-fold of each other. The two viruses with junction sequences that were not recognized as promoters did not give significant CAT expression. We conclude that, with respect to Sindbis virus, the junction sequences are functionally conserved; i.e., most of the contemporary nucleotide differences in the junction sequences are neutral or near-neutral mutations. The functional conservation suggests that neither the cis-acting sequence nor the cognate binding site of the transcription factor can change independently. This type of coupled evolution between cis-acting sequences and their cognate viral protein binding sites may be a general phenomenon. For example, it explains the ubiquitous presence of conserved cis-acting sequences in each of the families of RNA viruses. There are implications of this hypothesis for the design of antiviral drugs.

Analysis of Sindbis virus promoter recognition in vivo, using novel vectors with two subgenomic mRNA promoters.

Four types of Sindbis virus vectors, each carrying two promoters for subgenomic mRNA synthesis, were designed to measure relative promoter strengths and to survey potential contextual effects on promoter strengths. One of the promoters in each vector was used as the reference promoter, while the other was the one being tested. We used these vectors to measure the relative strengths of four promoters: the minimal promoter, an extended sequence believed to have full promoter activity, and two mutant promoters, one with an inactivating 3-nucleotide insertion called CR4.1 and the other with a 4-nucleotide deletion called delta 4. The strengths of the promoters were measured by quantitating the RNA transcribed from each promoter in vivo and also by assaying for chloramphenicol acetyltransferase activity encoded by one of the two transcripts. We found that the relative strengths of the promoters were similar in different contexts. The complete promoter was 6-fold more active, the delta 4 promoter was (surprisingly) about twice as active, and the CR4.1 promoter was 100-fold less active than the minimal promoter. At least two contextual effects were identified that can alter the activity of one or both promoters in the vectors. One effect is that given identical promoters, the 3'-proximal promoter on the minus-strand template can be more active than the 5'-proximal promoter. This may be due to preferential association of one or more components of the transcription complex for the 3' end of the minus-strand template. A second effect is promoter competition, particularly when the promoters are closely spaced.

Promoter for Sindbis virus RNA-dependent subgenomic RNA transcription.

Sindbis virus is a positive-strand RNA enveloped virus, a member of the Alphavirus genus of the Togaviridae family. Two species of mRNA are synthesized in cells infected with Sindbis virus; one, the 49S RNA, is the genomic RNA; the other, the 26S RNA, is a subgenomic RNA that is identical in sequence to the 3' one-third of the genomic RNA. Ou et al. (J.-H. Ou, C. M. Rice, L. Dalgarno, E. G. Strauss, and J. H. Strauss, Proc. Natl. Acad. Sci. USA 79:5235-5239, 1982) identified a highly conserved region 19 nucleotides upstream and 2 nucleotides downstream from the start of the 26S RNA and proposed that in the negative-strand template, these nucleotides compose the promoter for directing the synthesis of the subgenomic RNA. Defective interfering (DI) RNAs of Sindbis virus were used to test this proposal. A 227-nucleotide sequence encompassing 98 nucleotides upstream and 117 nucleotides downstream from the start site of the Sindbis virus subgenomic RNA was inserted into a DI genome. The DI RNA containing the insert was replicated and packaged in the presence of helper virus, and cells infected with these DI particles produced a subgenomic RNA of the size and sequence expected if the promoter was functional. The initiating nucleotide was identical to that used for Sindbis virus subgenomic mRNA synthesis. Deletion analysis showed that the minimal region required to detect transcription of a subgenomic RNA from the negative-strand template of a DI RNA was 18 or 19 nucleotides upstream and 5 nucleotides downstream from the start of the subgenomic RNA.

A cis-acting mutation in the Sindbis virus junction region which affects subgenomic RNA synthesis.

The synthesis of Sindbis virus minus-strand and genomic and subgenomic RNAs is believed to require specific cis-acting sequences or structures in the template RNAs and a combination of virus-specific proteins and host components which act in trans. A conserved sequence of about 21 nucleotides in the junction region and encompassing the start site for the subgenomic RNA has been proposed to function as the promoter on the minus-strand template for synthesis of the subgenomic RNA (J.-H. Ou, C. M. Rice, L. Dalgarno, E. G. Strauss, and J. H. Strauss, Proc. Natl. Acad. Sci. USA 79:5235-5239, 1982). We introduced a three-base insertion in this sequence, which also inserts a single amino acid near the COOH terminus of nsP4, in a cDNA clone of Sindbis virus from which infectious RNA transcripts can be generated. The phenotype of this mutant, called Toto1100CR4.1, was studied after RNA transfection of chicken embryo fibroblasts or BHK cells. The mutation leads to a drastic reduction in the level of the subgenomic RNA but does not alter the start site of the RNA. Probably as a consequence of depressed structural-protein synthesis, very few progeny virions are released and the mutant makes tiny or indistinct plaques even after prolonged incubation. The cis-acting effect of this mutation was demonstrated by incorporating either a wild-type or mutant junction region into a defective-interfering RNA and examining the relative synthesis of defective-interfering RNA-derived subgenomic RNA in vivo in the presence of wild-type helper virus. These results show that the junction region is recognized by yet unidentified viral trans-acting components for subgenomic RNA synthesis. When the Toto1100CR4.1 mutant was passaged in culture, plaque morphology variants readily arose. A total of 24 independent revertants were isolated, and 16 were characterized in detail. All revertants analyzed showed an increase in the level of subgenomic RNA synthesis. Sequence analysis of the junction region showed that all were pseudorevertants, with only two containing potentially compensating changes in the junction region. An assay was developed to identify revertants with second-site changes in trans-acting viral components involved in subgenomic RNA synthesis. At least two such revertants were identified. Mapping of these and other second-site compensating mutations may provide genetic clues as to which virus-specific protein(s) is responsible for interaction with the conserved junction region to promote subgenomic RNA synthesis.

Tn5supF, a 264-base-pair transposon derived from Tn5 for insertion mutagenesis and sequencing DNAs cloned in phage lambda.

We constructed a derivative of transposon Tn5 called Tn5supF for insertion mutagenesis and sequencing DNAs cloned in phage lambda. This element carries a supF amber-suppressor tRNA gene. Its insertion into lambda can be selected by plaque formation by using nonsuppressing (sup0) Escherichia coli for amber mutant lambda phage and sup0 dnaB-amber E. coli for nonamber lambda phage. Tn5supF is just 264 base pairs long. It transposes efficiently and inserts quasi-randomly into DNA targets. The unique sequences near its termini can be used as primer binding sites for dideoxynucleotide DNA sequencing, thus permitting the direct sequencing of DNAs cloned in phage lambda without subcloning.

Effect of base pair mismatches on recombination via the RecBCD pathway.

The effect of base pair mismatches on recombination via the RecBCD pathway was studied in mutS and wild-type Escherichia coli, using substrates that contain single or multiple mismatches. Recombination between homologous DNA inserts in lambda phage and pBR322-derived plasmids forms phage-plasmid cointegrates that result from an odd numbers of crossovers. In the mutS host, when the sequence homology of a pair of 405 bp substrates decreased from 100% to 89%, the recombinant frequency decreased by about 9-fold, while in the wild-type host the decrease was about 240-fold. These results suggest that multiple mismatches can reduce recombinant frequencies by impeding the mechanism of recombination itself, and by provoking mismatch repair. Single mismatches in 31 bp substrates caused reductions in recombinant frequencies of 2- or 12-fold, depending on the location of the mismatch. However, unlike the reduction by multiple mismatches, the reduction of the recombinant frequencies by single mismatches was the same in both mutS and wild-type hosts. Thus a single mismatch is sufficient to impede recombination, and mismatch repair seems unable to act on single mismatches in very short homologies during recombination.

Association of the Sindbis virus RNA methyltransferase activity with the nonstructural protein nsP1.

SVLM21 is a mutant of Sindbis virus, which in contrast to SVSTD, is able to replicate in Aedes albopictus mosquito cells deprived of methionine. We have obtained evidence that the basis of this low methionine-resistance (LMR) phenotype is the generation of an altered RNA methyltransferase with an increased affinity for S-adenosylmethionine (ado met). We now report that following the substitution of the nucleotide sequence, 126-504, from SVLM21 cDNA for the corresponding sequence of the Toto 1101 plasmid (infectious Sindbis viral RNA can be transcribed from this plasmid) we were able to generate recombinant Sindbis virus (SVMS-65a) with the LMR phenotype. (SVTOTO virus derived from Toto 1101, like SVSTD, lacks the LMR phenotype.) As was the case with SVLM21, SVMS-65a not only possessed the LMR phenotype but also showed an increased sensitivity to Neplanocin A, a potent inhibitor of S-adenosylhomocysteine (ado hcy) hydrolase. Sequencing of the nucleotide 126-504 region from SVLM21 revealed two mutations; these mutations occurred in adjacent codons and lead to two predicted amino acid changes in the SV nsPl protein; at residue 87, from Arg to Leu, and at residue 88 from Ser to Cys. Since the nucleotide sequence 126-504 lies entirely within the gene for nsP1, we conclude that the RNA methyltransferase activity generated by SV is associated with nsP1. We suggest that residues 87 and 88 in nsP1, where the amino acid changes in SVLM21 nsP1 have occurred, are at or near the binding site for ado met; we also suggest that these changes in nsP1 are responsible for the increased affinity of the SVLM21 RNA methyltransferase for ado met and thereby for the LMR phenotype. Alternatively, it is possible that the binding site for ado met is elsewhere on nsP1 or even on another protein, and that the changes at residues 87 and 88 lead to an alteration of the binding site.

Sindbis virus: an efficient, broad host range vector for gene expression in animal cells.

Sindbis virus, an enveloped virus with a single-stranded RNA genome, was engineered to express a bacterial protein, chloramphenicol acetyltransferase (CAT), in cultured insect, avian, and mammalian cells. The vectors were self-replicating and gene expression was efficient and rapid; up to 10(8) CAT polypeptides were produced per infected cell in 16 to 20 hours. CAT expression could be made temperature-sensitive by means of a derivative that incorporated a temperature-sensitive mutation in viral RNA synthesis. Vector genomic RNAs were packaged into infectious particles when Sindbis helper virus was used to supply virion structural proteins. The vector RNAs were stable to at least seven cycles of infection. The expression of CAT increased about 10(3)-fold, despite a 10(15)-fold dilution during the passaging. Sindbis virus vectors should prove useful for expressing large quantities of gene products in a variety of animal cells.

Molecular analysis of Sindbis virus pathogenesis in neonatal mice by using virus recombinants constructed in vitro.

Genetic loci affecting Sindbis virus pathogenesis in neonatal mice have been examined by using a full-length cDNA clone of the virus (Toto1101). The full-length cDNA is linked to a bacteriophage SP6 promoter to facilitate the synthesis of infectious RNA transcripts in vitro. Virus derived from Toto1101 showed reduced virulence (attenuation) in neonatal mice. Replacement of the E1 glycoprotein and 6K genes of Toto1101 with cloned E1 and 6K genes derived from a virulent Sindbis virus strain, AR339 (SB), resulted in a new construct, TR2000, that gave rise to virulent virus. Sequence determinations for the entire substituted regions of TR2000, Toto1101, and related virulent and attenuated strains identified three coding differences in E1 between Toto1101 and TR2000. These differences, individually or in combination, may be responsible for the attenuated phenotype. Previous studies in this laboratory identified another attenuating mutation at amino acid position 114 of the E2 glycoprotein (N.L. Davis, F.J. Fuller, W.G. Dougherty, R.A. Olmsted, and R.E. Johnston, Proc. Natl. Acad. Sci. USA 83:6771-6775, 1986). Substitution of Arg-114 in the mutant SB-RL for Ser-114 of SB appears to confer three distinguishing phenotypes: attenuation in neonatal mice, increased sensitivity to specific E2 monoclonal antibodies, and accelerated penetration of BHK cells. Replacement of TR2000 sequences containing the codon for amino acid 114 of E2 with corresponding fragments from cDNA clones of SB or SB-RL produced two strains of Sindbis virus (TR2100 and TR2200) which were isogenic except for the E2 114 codon (Ser and Arg, respectively). The three diagnostic phenotypes cosegregated according to the origin of the codon for amino acid 114 of E2, confirming the dramatic effect of this single amino acid substitution on these three phenotypes.

Bidirectional chain-termination nucleotide sequencing: transposon Tn5seq1 as a mobile source of primer sites.

The sequencing of large DNA fragments by the chain-termination method [Sanger et al., Proc. Natl. Acad. Sci. USA 74 (1977) 5463-5467] has generally required extensive manipulations to bring all parts of the fragment near a specific primer-binding site, or the repeated synthesis of new oligodeoxynucleotide primers. Here we develop a more efficient approach, the use of a transposable element to insert primer binding sites at random in the DNA of interest. We constructed a Tn5 derivative called Tn5seq1 with unique DNA segments near each end so that oligodeoxynucleotides matching them could serve as primers for sequencing in each direction from any Tn5seq1 insertion site. Our experiments demonstrate the use of Tn5seq1 for sequencing in pBR322 plasmids and also in uncloned DNAs of the Escherichia coli chromosome. The unique segments near the left and right ends of Tn5seq1 are promoters from phages T7 and SP6, respectively, to permit the efficient transcription of adjacent DNAs in vivo or in vitro.