Sequence analysis of a substantial part of the polymerase gene of the murine coronavirus MHV-A59 revealed the 3' end of an open reading frame (ORF1a) overlapping with a large ORF (ORF1b; 2733 amino acids) which covers the 3' half of the polymerase gene. The expression of ORF1b occurs by a ribosomal frameshifting mechanism since the ORF1a/ORF1b overlapping nucleotide sequence is capable of inducing ribosomal frameshifting in vitro as well as in vivo. A stem-loop structure and a pseudoknot are predicted in the nucleotide sequence involved in ribosomal frameshifting. Comparison of the predicted amino acid sequence of MHV ORF1b with the amino acid sequence deduced from the corresponding gene of the avian coronavirus IBV demonstrated that in contrast to the other viral genes this ORF is extremely conserved. Detailed analysis of the predicted amino acid sequence revealed sequence elements which are conserved in many DNA and RNA polymerases.

Complementary DNA (cDNA) libraries were constructed representing the genome RNA of the coronavirus mouse hepatitis virus, strain A59 (MHV-A59). From these libraries clones were selected to form a linear map across the entire gene A, the putative viral polymerase gene. This gene is approximately 23 kb in length, considerably larger than earlier estimates. Sequence analysis of the 5' terminal region of the genome indicates the presence of the 66-nucleotide leader that is found on all mRNAs. Secondary structure analysis of the 5' terminal region suggests that transcription of leader terminates in the region of nucleotide 66. The sequence of the first 2000 nucleotides is very similar to that reported for the closely related JHM strain of MHV and potentially encodes p28, a basic protein thought to be a component of the viral polymerase (L. Soe, C. K. Shieh, S. Baker, M. F. Chang, and M. M. C. Lai, 1987, J. Virol., 61, 3968-3976). Gene A contains two of the consensus sequences found in intergenic regions. One is adjacent to the 5' leader sequence and the other is upstream from the initiation codon for translation of gene B.

؉ T-cell responses, which result in reduced HSV-2-derived morbidity, as well as reduced mortality. However, coinjection with DNAs expressing MCP-1, IP-10, and MIP-1␣ increased mortality in the challenged mice. Chemokine DNA coinjection also modulated its own production as well as the production of cytokines. These studies demonstrate that chemokines can dominate and drive immune responses with defined phenotypes, playing an important role in the generation of protective antigen-specific immunity.

It has previously been reported that herpes simplex virus (HSV)-2 gD DNA vaccine preferentially induces T-helper (Th) 1-type cellular immune responses, whereas the literature supports the view that subunit vaccines tend to induce potent antibody responses, supporting a Th2 bias. Here, using an HSV gD vaccine model, we investigated whether priming and boosting with a DNA or protein vaccine could induce both potent antibody and Th1-type cellular immune responses. When animals were primed with DNA and boosted with protein, both antibody and Th-cell proliferative responses were significantly enhanced. Furthermore, production of Th1-type cytokines (interleukin-2, interferon-gamma) was enhanced by DNA priming-protein boosting. In contrast, protein priming-DNA boosting produced antibody levels similar to those following protein-protein vaccination but failed to further enhance Th-cell proliferative responses or cytokine production. DNA priming-protein boosting resulted in an increased IgG2a isotype (a Th1 indicator) profile, similar to that induced by DNA-DNA vaccination, whereas protein priming-DNA boosting caused an increased IgG1 isotype (a Th2 indicator) profile similar to that seen after protein-protein vaccination. This result indicates that preferential induction of IgG1 or IgG2a isotype is determined by the type of priming vaccine used. Thus, this study suggests that HSV DNA priming-protein boosting could elicit both potent Th1-type cellular immune responses and antibody responses, both of which likely are important for protection against HSV infection.

RNA interference (RNAi) is a process that can target intracellular RNAs for degradation in a highly sequence specific manner, making it a powerful tool that is being pursued in both research and therapeutic applications. Hepatitis B virus (HBV) is a serious public health problem in need of better treatment options, and aspects of its life cycle make it an excellent target for RNAi-based therapeutics. We have designed a vector that expresses interfering RNAs that target HBV transcripts, including both viral RNA replicative intermediates and mRNAs encoding viral proteins. Our vector design incorporates many features of endogenous microRNA (miRNA) gene organization that are proving useful for the development of reagents for RNAi. In particular, our vector contains an RNA pol II driven gene cassette that leads to tissue specific expression and efficient processing of multiple interfering RNAs from a single transcript, without the co-expression of any protein product. This vector shows potent silencing of HBV targets in cell culture models of HBV infection. The vector design will be applicable to silencing of additional cellular or disease-related genes.