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.

Bc1-2 protects mice against fatal alphavirus encephalitis.

Virus-induced apoptosis has been well characterized in vitro, but the role of apoptosis in viral pathogenesis is not well understood. The suicide of a cell in response to viral infection is postulated to be an important host defense for the organism, leading to a reduction in its total viral burden. However, virus-induced death of nonregenerating cells in the central nervous system may be detrimental to the host. Therefore, to investigate the role of apoptosis in the pathogenesis of fatal encephalitis, we constructed a recombinant alphavirus chimera that expresses the antiapoptotic gene, bcl-2, in virally infected neural cells. Infection of neonatal mice with the alphavirus chimera expressing human bcl-2 [Sindbis virus (SIN)/bcl-2] resulted in a significantly lower mortality rate (7.5%) as compared with infection with control chimeric viruses containing a chloramphenicol acetyltransferase (CAT) reporter gene (SIN/CAT) (78.1%) or bcl-2 containing a premature stop codon (SIN/bcl-2stop) (72.1%) (P < 0.001). Viral titers were reduced 5-fold 1 day after infection and 10-fold 6 days after infection in the brains of SIN/bcl-2-infected mice as compared to SIN/CAT or SIN/bcl-2stop-infected mice. In situ end labeling to detect apoptotic nuclei demonstrated a reduction in the number of foci of apoptotic cells in the brains of mice infected with SIN/bcl-2 as compared with SIN/bcl-2stop. The reduction in apoptosis was associated with a reduction in the number of foci of cells expressing alphavirus RNA. Thus, the antiapoptotic gene, bcl-2, suppresses viral replication and protects against a lethal viral disease, suggesting an interaction between cellular genetic control of viral replication and cell death.

Inhibition of luciferase expression in transgenic Aedes aegypti mosquitoes by Sindbis virus expression of antisense luciferase RNA

A rapid and reproducible method of inhibiting the expression of specific genes in mosquitoes should further our understanding of gene function and may lead to the identification of mosquito genes that determine vector competence or are involved in pathogen transmission. We hypothesized that the virus expression system based on the mosquito-borne Alphavirus, Sindbis (Togaviridae), may efficiently transcribe effector RNAs that inhibit expression of a targeted mosquito gene. To test this hypothesis, germ-line-transformed Aedes aegypti that express luciferase (LUC) from the mosquito Apyrase promoter were intrathoracically inoculated with a double subgenomic Sindbis (dsSIN) virus TE/3′2J/anti-luc (Anti-luc) that transcribes RNA complementary to the 5′ end of the LUC mRNA. LUC activity was monitored in mosquitoes infected with either Anti-luc or control dsSIN viruses expressing unrelated antisense RNAs. Mosquitoes infected with Anti-luc virus exhibited 90% reduction in LUC compared with uninfected and control dsSIN-infected mosquitoes at 5 and 9 days postinoculation. We demonstrate that a gene expressed from the mosquito genome can be inhibited by using an antisense strategy. The dsSIN antisense RNA expression system is an important tool for studying gene function in vivo.

Yeast mutations in multiple complementation groups inhibit brome mosaic virus RNA replication and transcription and perturb regulated expression of the viral polymerase-like gene

Brome mosaic virus (BMV), a member of the alphavirus-like superfamily of positive-strand RNA viruses, encodes two proteins, 1a and 2a, that interact with each other, with unidentified host proteins, and with host membranes to form the viral RNA replication complex. Yeast expressing 1a and 2a support replication and subgenomic mRNA synthesis by BMV RNA3 derivatives. Using a multistep selection and screening process, we have isolated yeast mutants in multiple complementation groups that inhibit BMV-directed gene expression. Three complementation groups, represented by mutants mab1–1, mab2–1, and mab3–1 (for maintenance of BMV functions), were selected for initial study. Each of these mutants has a single, recessive, chromosomal mutation that inhibits accumulation of positive- and negative-strand RNA3 and subgenomic mRNA. BMV-directed gene expression was inhibited when the RNA replication template was introduced by in vivo transcription from DNA or by transfection of yeast with in vitro transcripts, confirming that cytoplasmic RNA replication steps were defective. mab1–1, mab2–1, and mab3–1 slowed yeast growth to varying degrees and were temperature-sensitive, showing that the affected genes contribute to normal cell growth. In wild-type yeast, expression of the helicase-like 1a protein increased the accumulation of 2a mRNA and the polymerase-like 2a protein, revealing a new level of viral regulation. In association with their other effects, mab1–1 and mab2–1 blocked the ability of 1a to stimulate 2a mRNA and protein accumulation, whereas mab3–1 had elevated 2a protein accumulation. Together, these results show that BMV RNA replication in yeast depends on multiple host genes, some of which directly or indirectly affect the regulated expression and accumulation of 2a.