Research On Interaction Between Human Cytomegalovirus Primase And Host Protein DNAJB6 & MicroRNA-122-based Anti-Hepatitis B Virus Research

Human cytomegalovirus (HCMV) is a widely-infected herpersivrus. Health human usually act like latent infections, however in immunocompromised patients (such as organ tansplant patients or HIV-infected individuals) cuold cause serious and even fetal diseases. Due to the social burden of HCMV infections, people are prompted to further understand the life cycle and replication mechanism of HCMV. HCMV viral protein UL70 is thought as primase, playing great roles in HCMV DNA replication. We firstly present the evidence that pUL70 could interact with cell factor DNAJB6, which belongs to the heat shock protein Hsp40/DNAJ family. As a result of splicing, DNAJB6 could be expressed as two isoforms: DNAJB6a and DNAJB6b. We performed yeast two-hybrid screen assay and coimmunoprecipitation in cultured human cells and identified pUL70 could both interact with DNAJB6a and b. Through IFA assay, we found pUL70 could co-localized with DNAJB6a in the nucleus and with DNAJB6b in the cytoplasm. We construted two cell lines which could stably express DNAJB6a and 6b, and we designed several siRNAs to down-regulate the expression of DNAJB6a and 6b. We found upregulate the expression level of DNAJB6a and downregulate the levels of DNAJB6b would facilitate the nuclease localization of pUL70, while upregulate the expression level of DNAJB6b and downregulate the levels of DNAJB6a would help the cytoplasmic accumulation of pUL70. Our studies suggest the relative expression between DNAJB6a and 6b may be related to the regulation of pUL70’s cellular localization, affecting HCMV gene expression, DNA synthesis and virus production.Liver-specific microRNA-122 has been shown to bind to a highly conserved HBV (Hepatitis B virus) pregenomic RNA sequence via base-pairing interactions and inhibit HBV gene expression and replication. However, the transient silencing effects of miR-122 and cytotoxic response it provokes in mammalian cells restricts its applications to the therapeutic potential. Here we introduced a pSUPER vector that directs intracellular synthesis of siRNA-like transcripts to express miR-122 sustainedly and prolong its specific suppression of HBV gene expression. In addition, novel attenuated Salmonella strain △msbB&△ssrA/B was constructed and used for safe, effective and tissue-specific delivery in SCID (Severe Combined Immunodeficiency) mice. In this study, Salmonella strain performed efficient delivery of constructed pSUPER plasmids into livers, leading to substantial expression of miR-122 with little adverse effects in mice. Moreover, suppressions of viral gene expression and replication occurred in the HBV-infected mice that were treated with Salmonella carrying miR-122 expressing vector. These results indicate that Salmonella-mediated gene target therapy with miR-122 expressing vector could inhibit HBV gene expression and replication in vivo.