Gene Rearrangement Attenuates Expression and Lethality of a Nonsegmented Negative Strand RNA Virus
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Proc. Natl. Acad. Sci. USA Vol. 95, pp. 3501–3506, March 1998 Biochemistry Gene rearrangement attenuates expression and lethality of a nonsegmented negative strand RNA virus GAIL WILLIAMS WERTZ*, VICTORIA P. PEREPELITSA, AND L. ANDREW BALL Department of Microbiology, University of Alabama School of Medicine, Birmingham, AL 35294 Communicated by George Bruening, University of California at Davis, Davis, CA, January 2, 1998 (received for review November 24, 1997) ABSTRACT The nonsegmented negative strand RNA vi- positions are transcribed less abundantly as their distance from ruses comprise hundreds of human, animal, insect, and plant the promoter increases (3–6). Genes that encode proteins pathogens. Gene expression of these viruses is controlled by required in stoichiometric amounts such as the nucleocapsid the highly conserved order of genes relative to the single protein N, which is necessary for genome encapsidation and transcriptional promoter. We utilized this regulatory mech- replication, are located at or near the 39 terminus, whereas anism to alter gene expression levels of vesicular stomatitis genes that encode proteins needed in catalytic amounts such virus by rearranging the gene order. This report documents as the RNA dependent RNA polymerase L are more promoter that gene expression levels and the viral phenotype can be distal. The importance of gene regulation is shown by the manipulated in a predictable manner. Translocation of the observation that the relative molar ratios of the nucleocapsid promoter–proximal nucleocapsid protein gene N, whose prod- (N) protein, phosphoprotein (P), and polymerase (L) proteins uct is required stoichiometrically for genome replication, to are critical for optimal VSV viral RNA replication (7–9) and successive positions down the genome reduced N mRNA and that RNA replication in vitro and in cultured cells is propor- protein expression in a stepwise manner. The reduction in N tional to the amount of N protein synthesized (10, 11). gene expression resulted in a stepwise decrease in genomic Gene regulation by progressive transcriptional attenuation RNA replication. Translocation of the N gene also attenuated provides a possible explanation for the strong conservation of the viruses to increasing extents for replication in cultured gene order among the Mononegavirales, and it suggested a cells and for lethality in mice, without compromising their novel way to manipulate the viral phenotype—by rearranging ability to elicit protective immunity. Because monopartite the genes. Because both phylogenetic and experimental studies negative strand RNA viruses have not been reported to of these viruses indicate that homologous RNA recombination undergo homologous recombination, gene rearrangement occurs rarely if at all (2, 12, 13), deliberate rearrangement of should be irreversible and may provide a rational strategy for the viral genes should be irreversible by nature and have the developing stably attenuated live vaccines against this type of potential to perturb the properties of the virus profoundly. virus. Infectious viruses have recently been recovered from cDNA clones of the rhabdoviruses rabies and VSV (14–16) as well as The ability to alter the genotype of a virus in a manner that several other of the Mononegavirales (17), and this recovery results in a predictable change in gene expression would be makes possible the introduction of deliberate genetic changes. invaluable for studies of gene function and control. However, We examined whether the expression of a gene product critical the genetic mobility of many viruses and RNA viruses in for replication could be decreased and viral replication levels particular, engendered by homologous recombination, cou- reduced in a systematic and predictable manner. We tested this pled with the innate polymerase error rate usually has a high hypothesis by moving the gene for the nucleocapsid protein of potential to reverse both natural and engineered genetic VSV from its wild-type promoter proximal position to succes- changes. We show that it is possible to utilize the general sive positions down the viral genome. This paper describes how mechanism for control of gene expression of a negative strand gene translocation reduced N gene expression in a stepwise RNA virus to alter gene expression levels in a predictable way. manner thereby lowering the viral growth potential in cell In the present report, we describe how manipulation of the culture and attenuating its lethality for mice. Importantly, genome of a nonsegmented, negative strand RNA virus to these changes occurred without compromising the ability of translocate an essential gene for replication can be utilized to the virus to elicit a protective host response, suggesting that alter systematically the phenotype of the virus in a manner that this approach may provide a rational method to achieve a should be irreversible. measured and stable degree of attenuation of this type of virus. The nonsegmented negative strand RNA viruses (order Mononegavirales) comprise many significant pathogens of hu- MATERIALS AND METHODS mans, animals, plants, and insects. The four virus families in the order are: the Rhabdoviridae, such as rabies virus, vesicular Viruses and Cells. The San Juan isolate of the Indiana stomatitis virus (VSV), and potato yellow dwarf virus; the serotype of VSV provided the original template for all cDNA Paramyxoviridae, such as measles, human, and bovine respira- clones used in this work except the G protein gene, which was tory syncytial viruses; the Filoviridae, Ebola, and Marburg derived from the Orsay isolate of VSV Indiana (15). Baby viruses, and the Bornaviridae. These viruses have an elegantly hamster kidney (BHK-21) cells were used to recover viruses simple means of controlling gene expression by the highly from cDNAs, for single step growth experiments, and radio- conserved order of the genes relative to the single transcrip- isotopic labeling of RNAs and proteins. BSC-40 cells were used tional promoter (1, 2). Genes proximal to the 39 promoter site for plaque assays. Plasmid Construction and Recovery of Infectious Viruses. are transcribed at high levels whereas those at more distal Each of the five genes of VSV contains the same sequence of The publication costs of this article were defrayed in part by page charge Abbreviations: VSV, vesicular stomatitis virus; pfu, plaque-forming payment. This article must therefore be hereby marked ‘‘advertisement’’ in unit; IC, intracerebral; IN, intranasal; N, nucleocapsid protein; P, accordance with 18 U.S.C. §1734 solely to indicate this fact. phosphoprotein; L, polymerase. © 1998 by The National Academy of Sciences 0027-8424y98y953501-6$2.00y0 *To whom reprint requests should be addressed. e-mail: gailowertz@ PNAS is available online at http:yywww.pnas.org. microbio.uab.edu. 3501 Downloaded by guest on October 5, 2021 3502 Biochemistry: Wertz et al. Proc. Natl. Acad. Sci. USA 95 (1998) the first five nucleotides: 39-UUGUC-59. We used this com- mon sequence to construct molecular clones of individual genes from which DNA fragments precisely encompassing each gene could be released by restriction enzyme digestion and the genes could be reassembled without introducing other changes to the genome as described (L.A.B., C. Pringle, V.P.P., and G.W.W., unpublished work). The DNA segments that encompassed the individual genes were reassembled in any desired order to create a family of DNA plasmids whose nucleotide sequences corresponded precisely to that of wild- type VSV, except for the fact that their genes were rearranged. One deliberate mutation was introduced: the untranscribed, intergenic dinucleotide after the P gene is 39-CA-59 in the wild-type sequence, and this was made 39-GA-59 to conform with all the other gene junctions. These cDNA plasmids contained T7 promoters that expressed precise copies of the positive sense antigenomic VSV RNA (by use of a hepatitis d virus ribozyme positioned to generate an exact 39 terminus), and they were used for recovery of infectious viruses by transfection into VTF7–3-infected cells as described (15, 18, FIG. 1. Gene order and plaque phenotype for wild-type (N1) VSV and rearranged viruses N2, N3, and N4. 19). The recovered viruses were amplified by low-multiplicity passage on BHK-21 cells in the presence of cytosine arabino- m third, or fourth in the gene order (Fig. 1). All other aspects of side to inhibit replication of VTF7–3, filtered through 0.2- the viral nucleotide sequence were unaltered with the excep- filters to remove VTF7–3 and the process repeated until tion of the deliberate change of one nucleotide. The untran- VTF7–3 was not detectable by plaque assay. The gene orders scribed, intergenic dinucleotide after the P gene that is 39- of the recovered viruses were verified by amplifying the CA-59 in the wild-type virus was made 39-GA-59 to conform rearranged portions of the viral genomes using reverse tran- with all of the other intergenic junctions. Previous work scription and PCR followed by restriction enzyme analysis indicates this change has little effect on transcription (21). The (Fig. 2). wild-type gene order also was reconstructed from the same Single Cycle Virus Replication. BHK-21 cells were infected components as a control for the validity of the strategy, and at a multiplicity of three. After 1 hr of adsorption, the inoculum this virus was utilized as the wild-type virus control in all was removed, cultures were washed twice, and fresh medium subsequent experiments.