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Alphavirus expression vectors
ADVANCES IN VIRUS RESEARCH, VOL. 55
ALPHAVIRUS EXPRESSION VECTORS Sondra Schlesinger Department of Molecular Microbiology Washington University School of Medicine St. Louis, Missouri 63110
I. Introduction II. EngineeringofAlphavirusGenomesto Produce ExpressionVectors A. AlphavirusReplication B. ExpressionVectors C. Translation Enhancement D. NoncytopathicExpression Vectors E. TargetingofAlphavirus F. Inductionof DefectiveAlphavirus Genomesand of Functional Replicons III. SelectedExamples Illustrating the Value ofAlphaviruses as ExpressionVectors References I. INTRODUCTION Alphaviruses are positive strand RNA-enveloped viruses. Three of them, Sindbis virus, Semliki Forest virus, and Venezuelan equine encephalitis virus, are becoming widely recognized as expression vectors. The most versatile of these vectors are replicons in which the structural protein genes are replaced by a heterologous gene. They can be packaged into virus-like particles by the use of defective helpers t h a t provide the structural proteins. Some advances t h a t should extend the use of these vectors include the expression of the structural protein genes from two different defective helpers, which reduces the possibility of recombination between the helper and replicon; the ability to target replicon particles to specific cell types; and the isolation of noncytopathic replicons. Alphaviruses and their replicons are normally cytopathic in cultured vertebrate cells, but replicons t h a t have a mutation in one of the nonstructural protein genes (the nsP2 gene) have a decreased level of viral RNA synthesis and show either reduced or no cytopathogenicity in some cultured cells, in particular baby hamster kidney cells. In addition to these improvements, alphavirus vectors are beginning to demonstrate their value as vaccines and possibly also for expression in neurons. Alphaviruses first received attention as members of a large group of viruses that were classified as arboviruses. These viruses all had one fea565
Copyright © 2000 by Academic Press. All rights of reproduction in any form reserved. 0065-3527/00 $35.00
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ture in common: they replicated in both vertebrates and invertebrates. This characteristic was not one that could be correlated with structure or strategy of replication, and arboviruses have now been divided into several different families of viruses, including flaviviruses, rhabdoviruses, and togaviruses, to which the genus alphaviruses belong. Many alphaviruses are associated with disease as can be imagined from the names Venezuelan equine encephalitis (VEE) virus and the eastern and western encephalitis viruses. The equine part of those names indicates that these viruses were associated with epidemics in horses and that humans are considered a "dead end" host. But, this is not an essay on viruses that cause disease, I am writing about the potential value of alphaviruses, not about how they might cause disease. Some of them, specifically Sindbis virus and Semliki Forest virus, were not considered to be human pathogens and have become important reagents for studies in molecular and cell biology. These two viruses along with VEE virus are now receiving renewed attention in another realm; they are proving to be valuable as vectors for the expression of heterologous genes. The initial incentive for developing alphaviruses as expression vectors was based on two important considerations: their mode of replication (described later) and the cloning of their genomes (1-3). cDNAs of these RNA genomes had been cloned downstream of a bacteriophage promoter. RNA transcribed from cDNA in vitro could then be introduced into cultured cells by transfection leading to the production of new infectious virus particles. These cDNAs could be genetically engineered; viral genes could be deleted and heterologous genes could be added. I have divided this article is divided into two parts. The first and major part describes the mechanics: how alphaviruses function as expression vectors, some features that make them useful, and some of the problems. The second part describes some of the ways in which alphaviruses have been used and some potential ways they can be of value. Sindbis and Semliki Forest viruses are being developed for a wide variety of uses; the main focus of the work with VEE virus has been to develop vaccines. II. ENGINEERING OF ALPHAVIRUS GENOMES TO PRODUCE EXPRESSION VECTORS
A. Alphavirus Replication Alphavirus genomes are positive strand, nonsegmented RNA molecules of approximately 12 x 103 nucleotides (reviewed in ref. 4). They are capped at the 5' terminus, polyadenylated at the 3' terminus and have two long open reading frames (ORF) (Fig. 18.1). The first begins
i
FIG 18.3. Recombinant Semliki Forest virus-mediated expression of green fluorescent protein in neurons of cultured hippocampal slices. (A) Fluorescence illumination of the CA1 region from a living slice at 14 days in culture and 5 days after injection of x 104 infectious particles into the pyramidal cell layer. (B) At higher magnification, green fluorescent protein-positive dendritic spines typical of pyramidal cells are visible, so, Stratum oriens; sp, stratum pyramidale; sr, stratum radiatum. Bars: 80 pJn (A); 25 Hm (B). (Photograph provided by Dr. Markus Ehrengruber, University of Zurich, Zurich, Switzerland.)
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AIphavirus genome
sg promoter I
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nsV2
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sg promoter 5'--~ nsP1
nsP2
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nsP4
~~--A n 3' heterologous gene
Double subgenomic RNA vector ~rs
5'---~ nsP1
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capsid PE2 6K E1 ~-An3'
Defective alphavirus RNA (helper or expression vector)
5 ' - - ' ~ / / / / ~ / ~ capsid PE2 6K E1 ~-An 3' or heterologousgene FIG 18.1. Diagrams of alphavirus genomes. The first diagram illustrates the original alphavirus genome;the others showthe differentexpression vectors. The sg promoter is the subgenomicR N A promoter--those sequences in the complementarystrand that regulate transcription of the subgenomicRNA.
near the 5" terminus and extends for two-thirds the length of the genome where several stop codons ensure termination of translation. This part of the genome codes for the nonstructural proteins (nsPs), required for transcription and replication of the RNA. There is a single site for the initiation of translation near the 5' terminus, and four proteins (nsP1, nsP2, nsP3, and nsP4) are produced by proteolytic cleavage. The protease activity lies in the C-terminal domain of nsP2 and regulates the synthesis of the different viral RNA molecules. Before cleavage, the nsP complex primarily synthesizes the complementary (negative) strand of the genome. This is the template for genomic RNA and also for a subgenomic RNA, which is identical in sequence to the 3' one third of the genome. Cleavage of the nsPs inhibits synthesis of
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negative strands and converts the nsP complex to one that synthesizes high levels of genomic and subgenomic RNA. The second ORF is located in the 3' one third of the genome but is translated only in the context of the subgenomic RNA. It codes for the viral structural proteins: the capsid protein, a hydrophobic 6K protein, and the two viral envelope glycoproteins, E2 and El. The viral structural proteins are translated as a polyprotein, with the amino-terminal capsid protein functioning as an autoprotease to cleave itself from the nascent polypeptide. The capsid protein interacts with genomic RNA, forming a nucleocapsid. The glycoproteins become embedded in the plasma membrane of the cell. Interaction of the nucleocapsid with the glycoproteins initiates assembly and release of infectious extracellular virions. The viral RNAs contain cis-acting recognition sequences required for replication and encapsidation. Sequences at the 3' and at or near the 5' terminus of the genomic RNA are required for replication. Specific recognition signals located within the nsP coding sequence serve as a nucleation site for encapsidation and ensure that genomic RNA is packaged preferentially over subgenomic RNA. The nucleotides spanning the junction between the nsPs and the structural protein genes on the negative strand serve as the promoter for transcription of the subgenomic RNA (Fig. 18.1). Identification of these regions of the genome has played an important part in designing expression vectors and the means to package them.
B. Expression Vectors Three types of alphavirus expression vectors have been designed and are currently in use (5). The "classic" and most popular expression vectors are derived from replicons, defined as self-replicating RNA molecules that lack genetic information for packaging. Replicons become expression vectors when the structural protein genes are replaced by a heterologous gene (Fig. 18.1). These molecules are introduced into cultured cells by transfection--as RNA after transcription in vitro, or as DNA when placed under the control of a eukaryotic promoter. Although replicons lack the structural protein genes, they can be packaged into extracellular particles when cotransfected with defective helper RNAs that contain the structural protein genes (Fig. 18.2) (6,7). Defective helpers lack most of the nsP gene region and will be replicated only in cells that also express the nsPs. Some of these helpers are packaged extremely inefficiently and when they are used essentially only replicons are packaged. Other helpers described for
ALPHAVIRUS EXPRESSION VECTORS
Replicon RNA negative
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Defective RNA
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negative
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helper strand
Subgenomic Genomic RNA RNA
~-~ StprrUt!tural particles
FIG 18.2. Complementation between replicon RNA and a defective helper RNA. The replicon RNA codes for the nonstructural proteins. These proteins replicate and transcribe both the replicon and defective helper RNAs. The subgenomic RNA of the defective helper provides the structural viral proteins. Most of the defective helpers are constructed so that they are not packaged and the particles contain predominantly replicon RNA.
Sindbis virus replicons can be packaged and are also copackaged with the replicon. These copackaged particles are infectious and can spread to uninfected cells (6). Cotransfection of replicons and defective helpers can lead to recombination generating nonsegmented infectious virions by a single crossover event. This problem has been greatly diminished (and perhaps eliminated) by using two helpers: one expressing the capsid protein and the other expressing the membrane proteins (8-11). A second type of expression vector contains two subgenomic RNA promoters, one coding for the structural protein genes and the other for the heterologous gene (12). This type of vector, which is an infectious virus, has been used extensively, in part because it is easy to generate. There are packaging constraints and inserts of less than 2 kb are more stable than larger ones. The level of expression from these vectors is much less than that from replicons, and the insert is less stable. The third expression vector is one in which the heterologous gene is inserted into a defective alphavirus genome. Expression requires that the nsPs be supplied from replicons or virus.
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Earlier reports noted that baby hamster kidney (BHK) cells infected with packaged replicons of Semliki Forest virus or Sindbis virus produced about 50 ~tg of [3-galactosidase per 106 cells. Even though that number seemed impressive, scientists familiar with alphaviruses knew that this was far below the amount of capsid protein produced in virus-infected cells. This difference was due to the presence of an "enhancer" sequence located in the viral subgenomic mRNA within the coding region of the capsid (13-16). These sequences are located about 25 nucleotides downstream of the AUG start codon and form a hairpinlike structure. Translation is stimulated about 10-fold, but only in infected and not in uninfected cells. Factors required for translation become limiting in infected cells, and this structure may impede the movement of ribosomes, allowing limiting factors to bind more effectively. The existence and importance of this structure were clearly demonstrated for replicons derived from Sindbis and Semliki Forest viruses, but not for those from VEE virus (14). D. Noncytopathic Expression Vectors One of the potential problems in using alphaviruses as expression vectors is their cytopathic effect (CPE) on the cells they infect, and there has been much interest in obtaining mutants with reduced pathogenicity. Two different methods were used to obtain such mutants. One, based on earlier work to establish persistently infected cultures of BHK cells, had identified a mutant of Sindbis virus that showed reduced CPE. Dryga et al. (17) showed that a single amino acid change in nsP2 (Pro 726>Ser) conferred this phenotype on the virus. This decrease in CPE was also correlated with an 8- to 10-fold decrease in viral RNA synthesis. That level of inhibition of viral RNA synthesis did not affect virus yields, and when the mutation was inserted into Sindbis virus replicons led to a small increase in the expression of ~galactosidase. The second approach to obtain noncytopathic mutants was to select for cells that had been transfected with Sindbis virus replicons that expressed a gene (the pac gene) that coded for an enzyme that destroyed puromycin. In the presence of puromycin, only cells that could destroy this compound would survive, and only those cells that contained a Sindbis virus replicon that was not cytopathic could survive its replication and transcription, which would be required to produce the enzyme to destroy puromycin. This selection procedure led to
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the isolation of several noncytopathic replicons (18,19). The one that has been analyzed in most detail has a single mutation, which led to the change of a single amino acid (Pro726>Leu) in nsP2; the same amino acid as had been changed in the Sindbis virus m u t a n t with reduced CPE. (Two more m u t a n t s have been sequenced; one is also changed in the same codon and the other in a nearby region of the nsP2 gene.)
E. Targeting of Alphavirus The possible use of alphavirus vectors in h u m a n disease became more promising with the report of Ohno et al. (20) of the targeting of Sindbis vector particles to specific cell types. Mutants of Sindbis virus that contained random insertions in the virion glycoprotein precursor, PE2, had been isolated by Rice and collaborators who showed that the m u t a n t s were defective in their ability to bind to vertebrate cells (21,22). Based on those results, Ohno et al. (20) inserted the immunoglobulin G (IgG)-binding domain of protein A into the PE2 glycoprotein. This allowed them to target these Sindbis virus replicon particles to a particular cell type by adding monoclonal antibodies against a surface marker on those cells. For example, when they used HeLa cells or HeLa cells expressing CD4 ÷ and the SINrep/LacZ replicon, they were only able to detect infection (as measured by the expression of [~-galactosidase) when they used the replicon that had been packaged with the helper containing the protein A insert, anti-CD4 ÷ monoclonal antibody (MAb), and HeLa cells expressing CD4 ÷. They are extending this original work to determine if packaged replicons can be targeted to destroy tumor cells (23).
F. Induction of Defective Alphavirus Genomes and of Functional Replicons Defective alphavirus genomes (Fig. 18.2) will be replicated and their subgenomic mRNA transcribed only when a functional alphavirus replication complex is present in the same cell. It has been possible to obtain stable cell lines that contain defective alphavirus cDNA genomes under the control of a eukaryotic promoter. The defective RNA transcribed in these cells is essentially inert and is probably degraded unless the cells are infected with an alphavirus or a replicon. Olivo et al. (24) were able to show that a B H K cell line containing a defective Sindbis virus genome with the luciferase reporter gene down-
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stream of the subgenomic RNA promoter could be used to detect and quantitate Sindbis virus or replicons. Stable cell lines containing the cDNA genomes of defective helpers have been isolated with the goal of obtaining packaging cell lines. The original packaging cell lines containing Sindbis virus-defective helpers gave rise to recombinants, but in cell lines that had the capsid and viral glycoprotein genes expressed independently, recombinants were not detected (9). The isolation of a cell line containing an inducible cytopathic replicon seemed to be more of a challenge because even one molecule of replicon RNA in the cytoplasm of a cell should be sufficient to initiate the replication cycle and, for the wild-type replicon, would lead to cell death. However, two quite different inducible systems have been described for Sindbis virus replicons. Boorsma et al. (25) took advantage of the Pro726>Ser mutation in the nsP2 gene of the Sindbis virus replicon. They introduced a second mutation, one in the nsP4 gene, that conferred a temperature-sensitive phenotype on the replicon and were then able to establish a BHK cell line that was stably transfected with the Sindbis virus replicon at 37°C. When the temperature was lowered below 35°C, induction of the replicon occurred. The cells were able to recover from induction, a shift-up in temperature reestablished the stable uninduced conditions, and the cells could be reinduced. A completely different method for obtaining an inducible system was described by Ivanova et al. (26), who placed SINrep/LacZ replicon cDNA under the control of promoters of herpesvirus early genes. Herpesvirus early gene promoters require regulatory proteins encoded by immediate-early genes for expression. Sindbis virus replicons were completely silent until the cells were infected by a herpesvirus, in a Vero cell line by herpesvirus type 1, and in a mink lung cell line by human cytomegalovirus (CMV) (26). These cell lines were established as prototypes in which Sindbis virus replicons could be used to devise assays to detect herpesviruses, one of the ways in which alphavirus expression vectors may be of value, which is the next topic.
III. SELECTED EXAMPLES ILLUSTRATINGTHE VALUE OF ALPHAVIRUSES AS EXPRESSION VECTORS
Any discussion of how alphaviruses have been used will be idiosyncratic, a reflection of the author's interests and contributions. Some obvious uses have been in protein production and in tracing the cellu-
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lar localization of specific proteins; these have been described in other reviews (5,27,28). My own prejudices and interests become apparent by my focusing on those uses that, with one exception, involve other viruses. Hepatitis C virus is an important h u m a n pathogen that has not yet been propagated in cultured cells, and many studies have depended on the use of other viral expression systems to identify features of the hepatitis C viral life cycle such as the steps in proteolytic processing (29,30). Sindbis virus replicons have been used in this effort. Filocamo et al. (31,32) constructed a chimeric Sindbis virus, placing the hepatitis C NS3-NS4A gene sequences upstream of the alphavirus structural protein genes making this proteolytic activity required for the correct processing of the Sindbis virus structural proteins and the production of infectious Sindbis virus (31,32). These chimeric viruses were used to isolate and characterize variants of the NS3-NS4A protease. Semliki Forest virus expression vectors have been engineered to package retrovirus vectors (33,34). The system appears to be efficient and permits packaging of a retrovirus genome containing an intron. The retrovirus genome that will be packaged is derived from the alphavirus subgenomic RNA in the cytoplasm and not from transcription in the nucleus where splicing could occur. BHK cells that stably express the noncytopathic replicon of Sindbis virus have provided a useful tool for studies of other viruses. One example is the analysis of the NS1 gene of the flavivirus, yellow fever virus. This gene is essential for yellow fever virus replication, but Lindenbach and Rice (35) were able to recover mutant virus using the noncytopathic Sindbis virus replicon to express the NS1 gene in trans. They were able to establish that the NS1 protein is required for an early step in yellow fever virus replication, the synthesis of the negative strand of the genome. A second example was the development of an assay to detect and quantitate human respiratory syncytial virus (RSV) based on the expression of a reporter gene from an RSV minigenome (36). In this example, BHK cells contained the noncytopathic Sindbis virus replicon expressing the T7 polymerase and the RSV minigenome plus the genes for the RSV-required proteins were introduced as plasmids under the control of the T7 promoter. One of the most promising uses of the alphavirus vectors is in the development of vaccines (37). Initially, the double subgenomic RNA infectious Sindbis and VEE viruses were tested as immunogens (38,39). Although they did evoke a strong immune response, they have several disadvantages, in particular, that as infectious viruses, they
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could spread to other individuals. In addition, these viruses would induce an immune response to the alphavirus structural proteins, limiting the number of times they could be used as an immunogen in the same individual. Packaged replicons t h a t express one or more proteins of another virus have shown great promise as vaccines against several different viruses (37,40). VEE virus glycoproteins target the virus to lymphoid tissue and this m a y aid in the induction of an immune response. The initial reports using a t t e n u a t e d strains of this virus in mice have been encouraging (10). Another w a y of introducing replicons into animals has been to inject n a k e d DNA plasmids in which the replicon genome is u n d e r the control of a eukaryotic promoter. Sindbis and Semliki Forest virus replicon DNAs have been tested in mice. Both were able to induce protective immune responses at much lower concentrations than was seen with a conventional DNA plasmid in which the gene for the immunogen was directly under the control of a eukaryotic promoter (41,42). The two groups realized that one possible reason the replicons were so much more efficient is that they might act to induce cytokines that could stimulate the immune response. It will be important to determine if cotransfection of a replicon t h a t did not express some heterologous protein and a plasmid that did express an immunogen directly would show enhancem e n t of the i m m u n e response. There is one more example that, although not a vaccine, does fit under the category of alphavirus expression vectors as agents that could protect against other viruses, and this is the use of Sindbis virus vectors to inhibit the replication of several viruses, such as flaviviruses, in mosquitoes (43). Alphaviruses m a y turn out to be efficient insecticides! There are obviously many areas in which alphavirus expression vectors can be used that do not involve other viruses. One that now seems promising is in neurobiology. A recent review by Lundstrom provides some examples in which these vectors have been used to express central nervous system proteins (44). Both Sindbis and Semliki Forest virus efficiently infect neurons and can be used to deliver genes to these cells. These vectors have been used to infect rat hippocampal slice cultures (45,46). Live neurons expressing green fluorescent protein have been observed for up to 5 days postinfection (Fig. 18.3, see color insert). Almost all of the cells that express the green fluorescent protein are neurons. F u r t h e r studies using electrophysiological methods will determine the value of these vectors in analyzing neuronal functions.
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ACKNOWLEDGMENTS My research was supported by a grant from the Public Health Service (AIl1377). A special thanks to Markus Ehrengruber for providing the photograph for Fig. 18.3.
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15. Sjoberg, E. M., and Garoff, H. (1996). The translation-enhancing region of the Semliki Forest virus subgenome is only functional in the virus-infected cell. J. Gen. Virol. 77, 1323-1327. 16. Sjoberg, E. M., Suomalainen, M., and Garoff, H. (1994). A significantly improved Semliki Forest virus expression system based on translation enhancer segments from the viral capsid gene. Biotechnology 12, 1127-1131. 17. Dryga, S. A., Dryga, O. A., and Schlesinger, S. (1997). Identification of mutations in a Sindbis virus variant able to establish persistent infection in BHK cells: The importance of a mutation in the nsP2 gene. Virology 228, 74-83. 18. Agapov, E. V., Frolov, I., Lindenbach, B. D., Pragai, B. M., Schlesinger, S., and Rice, C. M. (1998). Noncytopathic Sindbis virus RNA vectors for heterologous gene expression. Proc. Natl. Acad. Sci. U.S.A. 95, 12989-12994. 19. Frolov, I., Agapov, E., Hoffman, T. A., Jr., Pragai, B. M., Lippa, M., Schlesinger, S., and Rice, C. M. (1999). Selection of RNA replicons capable of persistent noncytopathic replication in mammalian cells. J. Virol. 73, 3854-3865. 20. Ohno, K., Sawai, K., Iijima, Y., Levin, B., and Meruelo, D. (1997). Cell-specific targeting of Sindbis virus vectors displaying IgG-binding domains of protein A. Nat. Biotechnol. 15, 763-767. 21. London, S. D., Schmaljohn, A. L., Dalrymple, J. M., and Rice, C. M. (1992). Infectious enveloped RNA virus antigenic chimeras. Proc. Natl. Acad. Sci. U.S,A. 89, 207-211. 22. Dubuisson, J., and Rice, C. M. (1993). Sindbis virus attachment: Isolation and characterization of mutants with impaired binding to vertebrate cells. J. Virol. 67, 3363-3374. 23. Iijima, Y., Ohno, K., Ikeda, H., Sawai, K., Levin, B., and Meruelo, D. (1999). Cell-specific targeting of a thymidine kinase/ganciclovir gene therapy system using a recombinant Sindbis virus vector. Int. J. Cancer 80, 110-118. 24. Olivo, P. D., Frolov, I., and Schlesinger, S. (1994). Acell line that expresses a reporter gene in response to infection by Sindbis virus: A prototype for detection of positive strand RNA viruses. Virology 198, 381-384. 25. Boorsma, M., Nieba, L., Koller, D., Bachmann, M. F., Bailey, J. E., and Renner, W. A. (1999). A temperature-regulated replicon-based DNA expression system. Nat. Biotechol. 18, 429-432. 26. Ivanova, L., Schlesinger, S., and Olivo, P. D. (1999). Regulated expression of a Sindbis virus replicon by herpesvirus promoters. J. Virol. 73, 1998-2005. 27. Garoff, H., and Li, K. J. (1998). Recent advances in gene expression using alphavirus vectors. Curr. Opin. Biotechnol. 9, 464-469. 28. Lundstrom, K. (1997). Alphaviruses as expression vectors. Curr. Opin. Biotechnol. 8, 578-582. 29. Dubuisson, J., Hsu, H. H., Cheung, R. C., Greenberg, H. B., Russell, D. G., and Rice, C. M. (1994). Formation and intracellular localization of hepatitis C virus envelope glycoprotein complexes expressed by recombinant vaccinia and Sindbis viruses. J. Virol. 68, 6147-6160. 30. Lin, C., Lindenbach, B. D., Pragai, B. M., McCourt, D. W., and Rice, C. M. (1994). Processing in the hepatitis C virus E2-NS2 region: Identification of p7 and two distinct E2-specific products with different C termini. J. Virol. 68, 5063-5073. 31. Filocamo, G., Pacini, L., and Migliaccio, G. (1997). Chimeric Sindbis viruses dependent on the NS3 protease of hepatitis C virus. J. Virol. 71, 1417-1427. 32. Filocamo, G., Pacini, L., Nardi, C., Bartholomew, L., Scaturro, M., Delmastro, P., Tramontano, A., De Francesco, R., and Migliaccio, G. (1999). Selection of functional variants of the NS3-NS4A protease of hepatitis C virus by using chimeric Sindbis viruses. J. Virol. 73, 561-575.
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33. Li, K. J., and Garoff, H. (1996). Production of infectious recombinant Moloney murine leukemia virus particles in BHK cells using Semliki Forest virus-derived RNA expression vectors. Proc. Natl. Acad. Sci. U.S.A. 93, 11658-11663. 34. Li, K. J., and Garoff, H. (1998). Packaging ofintron-containinggenes into retrovirus vectors by alphavirus vectors. Proc. Natl. Acad. Sci. U.S.A. 95, 3650-3654. 35. Lindenbach, B. D., and Rice, C. M. (1997). Trans-complementation of yellow fever virus NS1 reveals a role in early RNA replication. J. Virol. 71, 9608-9617. 36. Olivo, P. D., Collins, P. L., Peeples, M. E., and Schlesinger, S. (1998). Detection and quantitation of human respiratory syncytial virus (RSV) using minigenome cDNA and a Sindbis virus replicon: A prototype assay for negative-strand RNA viruses. Virology 251, 198-205. 37. Tubulekas, I., Berglund, P., Fleeton, M., and LiljestrSm, P. (1997). Alphavirus expression vectors and their use as recombinant vaccines: A minireview. Gene 190, 191-195. 38. Caley, I. J., Betts, M. R., Irlbeck, D. M., Davis, N. L., Swanstrom, R., Frelinger, J. A., and Johnston, R. E. (1997). Humoral, mucosal, and cellular immunity in response to a human immunodeficiency virus type 1 immunogen expressed by a Venezuelan equine encephalitis virus vaccine vector. J. Virol. 71, 3031-3038. 39. Davis, N. L., Brown, K. W., and Johnston, R. E. (1996). A viral vaccine vector that expresses foreign genes in lymph nodes and protects against mucosal challenge. J. Virol. 70, 3781-3787. 40. Berglund, P., Fleeton, M. N., Smerdou, C., and LiljestrSm, P. (1999). Immunization with recombinant Semliki Forest virus induces protection against influenza challenge in mice. Vaccine 17, 497-507. 41. Berglund, P., Smerdou, C., Fleeton, M. N., Tubulekas, I., and LiljestrSm, P. (1998). Enhancing immune responses using suicidal DNA vaccines. Nat. Biotechnol. 16, 562-565. 42. Hariharan, M. J., Driver, D. A., Townsend, K., Brumm, D., Polo, J. M., Belli, B. A., Catton, D. J., Hsu, D., Mittelstaedt, D., McCormack, J. E., Karavodin, L., Dubensky, T. W., Jr., Chang, S. M., and Banks, T. A. (1998). DNAimmunization against herpes simplex virus: Enhanced efficacy using a Sindbis virus-based vector. J. Virol. 72, 950-958. 43. Higgs, S., Rayner, J. O., Olson, K. E., Davis, B. S., Beaty, B. J., and Blair, C. D. (1998). Engineered resistance in Aedes aegypti to a West African and a South American strain of yellow fever virus. Am. J. Trop. Med. Hyg. 58, 663-670. 44. Lundstrom, K. (1999). Alphaviruses as tools in neurobiology and gene therapy. J. Recept. Signal Transduct. Res. 19, 673-686. 45. Ehrengruber, M. U., Lundstrom, K., Schweitzer, C., Heuss, C., Schlesinger, S., and G~ihwiler, B. H. (1999). Recombinant Semliki Forest virus and Sindbis virus efficiently infect neurons in hippocampal slice cultures. Proc. Natl. Acad. Sci. U.S.A. 96, 7041-7046. 46. Maletic-Savatic, M., Malinow, R., and Svoboda, K. (1999). Rapid dendritic morphogenesis in CA1 hippocampal dendrites induced by synaptic activity. Science 283, 1923-1927.
ALPHAVIRUS EXPRESSION VECTORS Sondra Schlesinger Department of Molecular Microbiology Washington University School of Medicine St. Louis, Missouri 63110
I. Introduction II. EngineeringofAlphavirusGenomesto Produce ExpressionVectors A. AlphavirusReplication B. ExpressionVectors C. Translation Enhancement D. NoncytopathicExpression Vectors E. TargetingofAlphavirus F. Inductionof DefectiveAlphavirus Genomesand of Functional Replicons III. SelectedExamples Illustrating the Value ofAlphaviruses as ExpressionVectors References I. INTRODUCTION Alphaviruses are positive strand RNA-enveloped viruses. Three of them, Sindbis virus, Semliki Forest virus, and Venezuelan equine encephalitis virus, are becoming widely recognized as expression vectors. The most versatile of these vectors are replicons in which the structural protein genes are replaced by a heterologous gene. They can be packaged into virus-like particles by the use of defective helpers t h a t provide the structural proteins. Some advances t h a t should extend the use of these vectors include the expression of the structural protein genes from two different defective helpers, which reduces the possibility of recombination between the helper and replicon; the ability to target replicon particles to specific cell types; and the isolation of noncytopathic replicons. Alphaviruses and their replicons are normally cytopathic in cultured vertebrate cells, but replicons t h a t have a mutation in one of the nonstructural protein genes (the nsP2 gene) have a decreased level of viral RNA synthesis and show either reduced or no cytopathogenicity in some cultured cells, in particular baby hamster kidney cells. In addition to these improvements, alphavirus vectors are beginning to demonstrate their value as vaccines and possibly also for expression in neurons. Alphaviruses first received attention as members of a large group of viruses that were classified as arboviruses. These viruses all had one fea565
Copyright © 2000 by Academic Press. All rights of reproduction in any form reserved. 0065-3527/00 $35.00
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ture in common: they replicated in both vertebrates and invertebrates. This characteristic was not one that could be correlated with structure or strategy of replication, and arboviruses have now been divided into several different families of viruses, including flaviviruses, rhabdoviruses, and togaviruses, to which the genus alphaviruses belong. Many alphaviruses are associated with disease as can be imagined from the names Venezuelan equine encephalitis (VEE) virus and the eastern and western encephalitis viruses. The equine part of those names indicates that these viruses were associated with epidemics in horses and that humans are considered a "dead end" host. But, this is not an essay on viruses that cause disease, I am writing about the potential value of alphaviruses, not about how they might cause disease. Some of them, specifically Sindbis virus and Semliki Forest virus, were not considered to be human pathogens and have become important reagents for studies in molecular and cell biology. These two viruses along with VEE virus are now receiving renewed attention in another realm; they are proving to be valuable as vectors for the expression of heterologous genes. The initial incentive for developing alphaviruses as expression vectors was based on two important considerations: their mode of replication (described later) and the cloning of their genomes (1-3). cDNAs of these RNA genomes had been cloned downstream of a bacteriophage promoter. RNA transcribed from cDNA in vitro could then be introduced into cultured cells by transfection leading to the production of new infectious virus particles. These cDNAs could be genetically engineered; viral genes could be deleted and heterologous genes could be added. I have divided this article is divided into two parts. The first and major part describes the mechanics: how alphaviruses function as expression vectors, some features that make them useful, and some of the problems. The second part describes some of the ways in which alphaviruses have been used and some potential ways they can be of value. Sindbis and Semliki Forest viruses are being developed for a wide variety of uses; the main focus of the work with VEE virus has been to develop vaccines. II. ENGINEERING OF ALPHAVIRUS GENOMES TO PRODUCE EXPRESSION VECTORS
A. Alphavirus Replication Alphavirus genomes are positive strand, nonsegmented RNA molecules of approximately 12 x 103 nucleotides (reviewed in ref. 4). They are capped at the 5' terminus, polyadenylated at the 3' terminus and have two long open reading frames (ORF) (Fig. 18.1). The first begins
i
FIG 18.3. Recombinant Semliki Forest virus-mediated expression of green fluorescent protein in neurons of cultured hippocampal slices. (A) Fluorescence illumination of the CA1 region from a living slice at 14 days in culture and 5 days after injection of x 104 infectious particles into the pyramidal cell layer. (B) At higher magnification, green fluorescent protein-positive dendritic spines typical of pyramidal cells are visible, so, Stratum oriens; sp, stratum pyramidale; sr, stratum radiatum. Bars: 80 pJn (A); 25 Hm (B). (Photograph provided by Dr. Markus Ehrengruber, University of Zurich, Zurich, Switzerland.)
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AIphavirus genome
sg promoter I
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Defective alphavirus RNA (helper or expression vector)
5 ' - - ' ~ / / / / ~ / ~ capsid PE2 6K E1 ~-An 3' or heterologousgene FIG 18.1. Diagrams of alphavirus genomes. The first diagram illustrates the original alphavirus genome;the others showthe differentexpression vectors. The sg promoter is the subgenomicR N A promoter--those sequences in the complementarystrand that regulate transcription of the subgenomicRNA.
near the 5" terminus and extends for two-thirds the length of the genome where several stop codons ensure termination of translation. This part of the genome codes for the nonstructural proteins (nsPs), required for transcription and replication of the RNA. There is a single site for the initiation of translation near the 5' terminus, and four proteins (nsP1, nsP2, nsP3, and nsP4) are produced by proteolytic cleavage. The protease activity lies in the C-terminal domain of nsP2 and regulates the synthesis of the different viral RNA molecules. Before cleavage, the nsP complex primarily synthesizes the complementary (negative) strand of the genome. This is the template for genomic RNA and also for a subgenomic RNA, which is identical in sequence to the 3' one third of the genome. Cleavage of the nsPs inhibits synthesis of
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negative strands and converts the nsP complex to one that synthesizes high levels of genomic and subgenomic RNA. The second ORF is located in the 3' one third of the genome but is translated only in the context of the subgenomic RNA. It codes for the viral structural proteins: the capsid protein, a hydrophobic 6K protein, and the two viral envelope glycoproteins, E2 and El. The viral structural proteins are translated as a polyprotein, with the amino-terminal capsid protein functioning as an autoprotease to cleave itself from the nascent polypeptide. The capsid protein interacts with genomic RNA, forming a nucleocapsid. The glycoproteins become embedded in the plasma membrane of the cell. Interaction of the nucleocapsid with the glycoproteins initiates assembly and release of infectious extracellular virions. The viral RNAs contain cis-acting recognition sequences required for replication and encapsidation. Sequences at the 3' and at or near the 5' terminus of the genomic RNA are required for replication. Specific recognition signals located within the nsP coding sequence serve as a nucleation site for encapsidation and ensure that genomic RNA is packaged preferentially over subgenomic RNA. The nucleotides spanning the junction between the nsPs and the structural protein genes on the negative strand serve as the promoter for transcription of the subgenomic RNA (Fig. 18.1). Identification of these regions of the genome has played an important part in designing expression vectors and the means to package them.
B. Expression Vectors Three types of alphavirus expression vectors have been designed and are currently in use (5). The "classic" and most popular expression vectors are derived from replicons, defined as self-replicating RNA molecules that lack genetic information for packaging. Replicons become expression vectors when the structural protein genes are replaced by a heterologous gene (Fig. 18.1). These molecules are introduced into cultured cells by transfection--as RNA after transcription in vitro, or as DNA when placed under the control of a eukaryotic promoter. Although replicons lack the structural protein genes, they can be packaged into extracellular particles when cotransfected with defective helper RNAs that contain the structural protein genes (Fig. 18.2) (6,7). Defective helpers lack most of the nsP gene region and will be replicated only in cells that also express the nsPs. Some of these helpers are packaged extremely inefficiently and when they are used essentially only replicons are packaged. Other helpers described for
ALPHAVIRUS EXPRESSION VECTORS
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FIG 18.2. Complementation between replicon RNA and a defective helper RNA. The replicon RNA codes for the nonstructural proteins. These proteins replicate and transcribe both the replicon and defective helper RNAs. The subgenomic RNA of the defective helper provides the structural viral proteins. Most of the defective helpers are constructed so that they are not packaged and the particles contain predominantly replicon RNA.
Sindbis virus replicons can be packaged and are also copackaged with the replicon. These copackaged particles are infectious and can spread to uninfected cells (6). Cotransfection of replicons and defective helpers can lead to recombination generating nonsegmented infectious virions by a single crossover event. This problem has been greatly diminished (and perhaps eliminated) by using two helpers: one expressing the capsid protein and the other expressing the membrane proteins (8-11). A second type of expression vector contains two subgenomic RNA promoters, one coding for the structural protein genes and the other for the heterologous gene (12). This type of vector, which is an infectious virus, has been used extensively, in part because it is easy to generate. There are packaging constraints and inserts of less than 2 kb are more stable than larger ones. The level of expression from these vectors is much less than that from replicons, and the insert is less stable. The third expression vector is one in which the heterologous gene is inserted into a defective alphavirus genome. Expression requires that the nsPs be supplied from replicons or virus.
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Earlier reports noted that baby hamster kidney (BHK) cells infected with packaged replicons of Semliki Forest virus or Sindbis virus produced about 50 ~tg of [3-galactosidase per 106 cells. Even though that number seemed impressive, scientists familiar with alphaviruses knew that this was far below the amount of capsid protein produced in virus-infected cells. This difference was due to the presence of an "enhancer" sequence located in the viral subgenomic mRNA within the coding region of the capsid (13-16). These sequences are located about 25 nucleotides downstream of the AUG start codon and form a hairpinlike structure. Translation is stimulated about 10-fold, but only in infected and not in uninfected cells. Factors required for translation become limiting in infected cells, and this structure may impede the movement of ribosomes, allowing limiting factors to bind more effectively. The existence and importance of this structure were clearly demonstrated for replicons derived from Sindbis and Semliki Forest viruses, but not for those from VEE virus (14). D. Noncytopathic Expression Vectors One of the potential problems in using alphaviruses as expression vectors is their cytopathic effect (CPE) on the cells they infect, and there has been much interest in obtaining mutants with reduced pathogenicity. Two different methods were used to obtain such mutants. One, based on earlier work to establish persistently infected cultures of BHK cells, had identified a mutant of Sindbis virus that showed reduced CPE. Dryga et al. (17) showed that a single amino acid change in nsP2 (Pro 726>Ser) conferred this phenotype on the virus. This decrease in CPE was also correlated with an 8- to 10-fold decrease in viral RNA synthesis. That level of inhibition of viral RNA synthesis did not affect virus yields, and when the mutation was inserted into Sindbis virus replicons led to a small increase in the expression of ~galactosidase. The second approach to obtain noncytopathic mutants was to select for cells that had been transfected with Sindbis virus replicons that expressed a gene (the pac gene) that coded for an enzyme that destroyed puromycin. In the presence of puromycin, only cells that could destroy this compound would survive, and only those cells that contained a Sindbis virus replicon that was not cytopathic could survive its replication and transcription, which would be required to produce the enzyme to destroy puromycin. This selection procedure led to
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the isolation of several noncytopathic replicons (18,19). The one that has been analyzed in most detail has a single mutation, which led to the change of a single amino acid (Pro726>Leu) in nsP2; the same amino acid as had been changed in the Sindbis virus m u t a n t with reduced CPE. (Two more m u t a n t s have been sequenced; one is also changed in the same codon and the other in a nearby region of the nsP2 gene.)
E. Targeting of Alphavirus The possible use of alphavirus vectors in h u m a n disease became more promising with the report of Ohno et al. (20) of the targeting of Sindbis vector particles to specific cell types. Mutants of Sindbis virus that contained random insertions in the virion glycoprotein precursor, PE2, had been isolated by Rice and collaborators who showed that the m u t a n t s were defective in their ability to bind to vertebrate cells (21,22). Based on those results, Ohno et al. (20) inserted the immunoglobulin G (IgG)-binding domain of protein A into the PE2 glycoprotein. This allowed them to target these Sindbis virus replicon particles to a particular cell type by adding monoclonal antibodies against a surface marker on those cells. For example, when they used HeLa cells or HeLa cells expressing CD4 ÷ and the SINrep/LacZ replicon, they were only able to detect infection (as measured by the expression of [~-galactosidase) when they used the replicon that had been packaged with the helper containing the protein A insert, anti-CD4 ÷ monoclonal antibody (MAb), and HeLa cells expressing CD4 ÷. They are extending this original work to determine if packaged replicons can be targeted to destroy tumor cells (23).
F. Induction of Defective Alphavirus Genomes and of Functional Replicons Defective alphavirus genomes (Fig. 18.2) will be replicated and their subgenomic mRNA transcribed only when a functional alphavirus replication complex is present in the same cell. It has been possible to obtain stable cell lines that contain defective alphavirus cDNA genomes under the control of a eukaryotic promoter. The defective RNA transcribed in these cells is essentially inert and is probably degraded unless the cells are infected with an alphavirus or a replicon. Olivo et al. (24) were able to show that a B H K cell line containing a defective Sindbis virus genome with the luciferase reporter gene down-
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stream of the subgenomic RNA promoter could be used to detect and quantitate Sindbis virus or replicons. Stable cell lines containing the cDNA genomes of defective helpers have been isolated with the goal of obtaining packaging cell lines. The original packaging cell lines containing Sindbis virus-defective helpers gave rise to recombinants, but in cell lines that had the capsid and viral glycoprotein genes expressed independently, recombinants were not detected (9). The isolation of a cell line containing an inducible cytopathic replicon seemed to be more of a challenge because even one molecule of replicon RNA in the cytoplasm of a cell should be sufficient to initiate the replication cycle and, for the wild-type replicon, would lead to cell death. However, two quite different inducible systems have been described for Sindbis virus replicons. Boorsma et al. (25) took advantage of the Pro726>Ser mutation in the nsP2 gene of the Sindbis virus replicon. They introduced a second mutation, one in the nsP4 gene, that conferred a temperature-sensitive phenotype on the replicon and were then able to establish a BHK cell line that was stably transfected with the Sindbis virus replicon at 37°C. When the temperature was lowered below 35°C, induction of the replicon occurred. The cells were able to recover from induction, a shift-up in temperature reestablished the stable uninduced conditions, and the cells could be reinduced. A completely different method for obtaining an inducible system was described by Ivanova et al. (26), who placed SINrep/LacZ replicon cDNA under the control of promoters of herpesvirus early genes. Herpesvirus early gene promoters require regulatory proteins encoded by immediate-early genes for expression. Sindbis virus replicons were completely silent until the cells were infected by a herpesvirus, in a Vero cell line by herpesvirus type 1, and in a mink lung cell line by human cytomegalovirus (CMV) (26). These cell lines were established as prototypes in which Sindbis virus replicons could be used to devise assays to detect herpesviruses, one of the ways in which alphavirus expression vectors may be of value, which is the next topic.
III. SELECTED EXAMPLES ILLUSTRATINGTHE VALUE OF ALPHAVIRUSES AS EXPRESSION VECTORS
Any discussion of how alphaviruses have been used will be idiosyncratic, a reflection of the author's interests and contributions. Some obvious uses have been in protein production and in tracing the cellu-
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lar localization of specific proteins; these have been described in other reviews (5,27,28). My own prejudices and interests become apparent by my focusing on those uses that, with one exception, involve other viruses. Hepatitis C virus is an important h u m a n pathogen that has not yet been propagated in cultured cells, and many studies have depended on the use of other viral expression systems to identify features of the hepatitis C viral life cycle such as the steps in proteolytic processing (29,30). Sindbis virus replicons have been used in this effort. Filocamo et al. (31,32) constructed a chimeric Sindbis virus, placing the hepatitis C NS3-NS4A gene sequences upstream of the alphavirus structural protein genes making this proteolytic activity required for the correct processing of the Sindbis virus structural proteins and the production of infectious Sindbis virus (31,32). These chimeric viruses were used to isolate and characterize variants of the NS3-NS4A protease. Semliki Forest virus expression vectors have been engineered to package retrovirus vectors (33,34). The system appears to be efficient and permits packaging of a retrovirus genome containing an intron. The retrovirus genome that will be packaged is derived from the alphavirus subgenomic RNA in the cytoplasm and not from transcription in the nucleus where splicing could occur. BHK cells that stably express the noncytopathic replicon of Sindbis virus have provided a useful tool for studies of other viruses. One example is the analysis of the NS1 gene of the flavivirus, yellow fever virus. This gene is essential for yellow fever virus replication, but Lindenbach and Rice (35) were able to recover mutant virus using the noncytopathic Sindbis virus replicon to express the NS1 gene in trans. They were able to establish that the NS1 protein is required for an early step in yellow fever virus replication, the synthesis of the negative strand of the genome. A second example was the development of an assay to detect and quantitate human respiratory syncytial virus (RSV) based on the expression of a reporter gene from an RSV minigenome (36). In this example, BHK cells contained the noncytopathic Sindbis virus replicon expressing the T7 polymerase and the RSV minigenome plus the genes for the RSV-required proteins were introduced as plasmids under the control of the T7 promoter. One of the most promising uses of the alphavirus vectors is in the development of vaccines (37). Initially, the double subgenomic RNA infectious Sindbis and VEE viruses were tested as immunogens (38,39). Although they did evoke a strong immune response, they have several disadvantages, in particular, that as infectious viruses, they
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could spread to other individuals. In addition, these viruses would induce an immune response to the alphavirus structural proteins, limiting the number of times they could be used as an immunogen in the same individual. Packaged replicons t h a t express one or more proteins of another virus have shown great promise as vaccines against several different viruses (37,40). VEE virus glycoproteins target the virus to lymphoid tissue and this m a y aid in the induction of an immune response. The initial reports using a t t e n u a t e d strains of this virus in mice have been encouraging (10). Another w a y of introducing replicons into animals has been to inject n a k e d DNA plasmids in which the replicon genome is u n d e r the control of a eukaryotic promoter. Sindbis and Semliki Forest virus replicon DNAs have been tested in mice. Both were able to induce protective immune responses at much lower concentrations than was seen with a conventional DNA plasmid in which the gene for the immunogen was directly under the control of a eukaryotic promoter (41,42). The two groups realized that one possible reason the replicons were so much more efficient is that they might act to induce cytokines that could stimulate the immune response. It will be important to determine if cotransfection of a replicon t h a t did not express some heterologous protein and a plasmid that did express an immunogen directly would show enhancem e n t of the i m m u n e response. There is one more example that, although not a vaccine, does fit under the category of alphavirus expression vectors as agents that could protect against other viruses, and this is the use of Sindbis virus vectors to inhibit the replication of several viruses, such as flaviviruses, in mosquitoes (43). Alphaviruses m a y turn out to be efficient insecticides! There are obviously many areas in which alphavirus expression vectors can be used that do not involve other viruses. One that now seems promising is in neurobiology. A recent review by Lundstrom provides some examples in which these vectors have been used to express central nervous system proteins (44). Both Sindbis and Semliki Forest virus efficiently infect neurons and can be used to deliver genes to these cells. These vectors have been used to infect rat hippocampal slice cultures (45,46). Live neurons expressing green fluorescent protein have been observed for up to 5 days postinfection (Fig. 18.3, see color insert). Almost all of the cells that express the green fluorescent protein are neurons. F u r t h e r studies using electrophysiological methods will determine the value of these vectors in analyzing neuronal functions.
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ACKNOWLEDGMENTS My research was supported by a grant from the Public Health Service (AIl1377). A special thanks to Markus Ehrengruber for providing the photograph for Fig. 18.3.
REFERENCES 1. Davis, N. L., Willis, L. V., Smith, J. F., and Johnston, R. E. (1989). In vitro synthesis of infectious Venezuelan equine encephalitis virus RNA from a cDNA clone: analysis of a viable deletion mutant. Virology 171, 189-204. 2. LiljestrSm, P., Lusa, S., Huylebroeck, D., and Garoff, H. (1991). In vitro mutagenesis of a full-length cDNA clone of Semliki Forest virus: The small 6,000-molecularweight membrane protein modulates virus release. J. Virol. 65, 4107-4113. 3. Rice, C. M., Levis, R., Strauss, J. H., and Huang, H. V. (1987). Production of infectious RNA transcripts from Sindbis virus cDNA clones: Mapping of lethal mutations, rescue of a temperature-sensitive marker, and in vitro mutagenesis to generate defined mutants. J. Virol. 61, 3809-3819. 4. Schlesinger, S., and Schlesinger, M. J. (1996). Togaviridae: The viruses and their replication. In Fields Virology, vol. I. (B. N. Fields, D. M. Knipe, and P. M. Howley, eds.) pp. 825-841. Lippincott-Raven, Philadelphia. 5. Frolov, I., Hoffman, T. A., Pragai, B. M., Dryga, S. A., Huang, H. V., Schlesinger, S., and Rice, C. M. (1996). Alphavirus-based expression vectors: Strategies and applications. Proc. Natl. Acad. Sci. U.S.A. 93, 11371-11377. 6. Bredenbeek, P. J., Frolov, I., Rice, C. M., and Schlesinger, S. (1993). Sindbis virus expression vectors: Packaging of RNA replicons by using defective helper RNAs. J. Virol. 67, 6439-6446. 7. LiljestrSm, P., and Garoff, H. (1991). A new generation of animal cell expression vectors based on the Semliki Forest virus replicon. Biotechnology 9, 1356-1361. 8. Frolov, I., Frolova, E., and Schlesinger, S. (1997). Sindbis virus replicons and Sindbis virus: Assembly of chimeras and of particles deficient in virus RNA. J. Virol. 71, 2819-2829. 9. Polo, J. M., Belli, B. A., Driver, D. A., Frolov, I., Sherrill, S., Hariharan, M. J., Townsend, K., Perri, S., Mento, S. J., Jolly, D. J., Chang, S. M., Schlesinger, S., and Dubensky, T. W. Jr. (1999). Stable alphavirus packaging cell lines for Sindbis virus and Semliki Forest virus-derived vectors. Proc. Natl. Acad. Sci. U.S.A. 96, 4598-4603. 10. Pushko, P., Parker, M., Ludwig, G. V., Davis, N. L., Johnston, R. E., and Smith, J. F. (1997). Replicon-helper systems from attenuated Venezuelan equine encephalitis virus: Expression of heterologous genes in vitro and immunization against heterologous pathogens in vivo. Virology 239, 389-401. 11. Smerdou, C., and Liljestr6m, P. (1999). Two-helper RNA system for production of recombinant Semliki Forest virus particles. J. Virol. 73, 1092-1098. 12. Hahn, C. S., Hahn, Y. S., Braciale, T. J., and Rice, C. M. (1992). Infectious Sindbis virus transient expression vectors for studying antigen processing and presentation. Proc. Natl. Acad. Sci. U.S.A. 89, 2679-2683. 13. Frolov, I., and Schlesinger, S. (1994). Translation of Sindbis virus mRNA: Effects of sequences downstream of the initiating codon. J. Virol. 68, 8111-8117. 14. Frolov, I., and Schlesinger, S. (1996). Translation of Sindbis virus mRNA: Analysis of sequences downstream of the initiating AUG codon that enhance translation. J. Virol. 70, 1182-1190.
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