Hongjian Li

Effective inhibition of HCMV UL49 gene expression and viral replication by oligonucleotide external guide sequences and RNase P

BackgroundHuman cytomegalovirus (HCMV) is a ubiquitous herpesvirus that typically causes asymptomatic infections in healthy individuals but may lead to serious complications in newborns and immunodeficient individuals. The emergence of drug-resistant strains of HCMV has posed a need for the development of new drugs and treatment strategies. Antisense molecules are promising gene-targeting agents for specific regulation of gene expression. External guide sequences (EGSs) are oligonucleotides that consist of a sequence complementary to a target mRNA and recruit intracellular RNase P for specific degradation of the target RNA. The UL49-deletion BAC of HCMV was significantly defective in growth in human foreskin fibroblasts. Therefore, UL49 gene may serve as a potential target for novel drug development to combat HCMV infection. In this study, DNA-based EGS molecules were synthesized to target the UL49 mRNA of human cytomegalovirus (HCMV).ResultsBy cleavage activity assessing in vitro, the EGS aimed to the cleavage site 324 nt downstream from the translational initiation codon of UL49 mRNA (i.e. EGS324) was confirmed be efficient to direct human RNase P to cleave the target mRNA sequence. When EGS324 was exogenously administered into HCMV-infected human foreskin fibroblasts (HFFs), a significant reduction of ~76% in the mRNA and ~80% in the protein expression of UL49 gene, comparing with the cells transfected with control EGSs. Furthermore, a reduction of about 330-fold in HCMV growth were observed in HCMV-infected HFFs treated with the EGS.ConclusionsThese results indicated that UL49 gene was essential for replication of HCMV. Moreover, our study provides evidence that exogenous administration of a DNA-based EGS can be used as a potential therapeutic approach for inhibiting gene expression and replication of a human virus.

Inhibition of human cytomegalovirus DNA replication by small interfering RNAs targeted to UL49

Human cytomegalovirus (HCMV) is a ubiquitous virus. Although the infection in healthy children and adults is usually asymptomatic, in immunocompromised individuals and newborns it is a significant cause of morbidity and mortality. UL49, an essential gene of HCMV, is highly conserved among various HCMV strains. The expression of UL49 is correlated with the production of virions. When UL49 is inhibited in the HCMV, the production of virions is reduced severely. In this study, RNA interference was applied to further investigate the roles of UL49 in viral replication. Two effective small interfering RNAs against UL49 were selected. Silencing of UL49 in HCMV-infected human foreskin fibroblast cells reduced the transcription levels of early and late genes, but not immediate-early ones. In addition, the viral DNA content was significantly reduced. This is the first time to uncover the role of UL49 in viral DNA synthesis, which indicates that UL49 might play an important role in this period. So the down-regulation of UL49 mRNA using RNAi might be a potential clinical therapy against the virus.

Identification of Prosaposin as a Novel Interaction Partner for Rhox5

Prosaposin (Psap) has multiple cellular functions. It is involved in the development of the reproductive system, nervous system, and prostate cancer as well as in the regulation of sphingolipid catabolism by activating several lysosomal hydrolases involved in the metabolism of various sphingolipids. In this research, it was found to be a novel interaction partner for Rhox5 using yeast two-hybrid screening. The interaction between Rhox5 and the full-length prosapsoin (the transcript without exon 8) as well as the C-terminal domain of prosaposin, was further confirmed in both yeast two hybrid analysis and in vitro assay. It suggested that the C-terminal domain of prosaposin may be critical for the Rhox5-prosaposin interaction. Given the important roles played by both Rhox5 and prosaposin in maintaining the differentiation of male reproductive organs, spermatogenesis, and fertilization, the interaction between Rhox5 and prosaposin might regulate the development of male reproductive organs dynamically.

Human cytomegalovirus UL23 inhibits transcription of interferon-γ stimulated genes and blocks antiviral interferon-γ responses by interacting with human N-myc interactor protein

Interferon-γ (IFN-γ) represents one of the most important innate immunity responses in a host to combat infections of many human viruses including human herpesviruses. Human N-myc interactor (Nmi) protein, which has been shown to interact with signal transducer and activator of transcription (STAT) proteins including STAT1, is important for the activation of IFN-γ induced STAT1-dependent transcription of many genes responsible for IFN-γ immune responses. However, no proteins encoded by herpesviruses have been reported to interact with Nmi and inhibit Nmi-mediated activation of IFN-γ immune responses to achieve immune evasion from IFN-γ responses. In this study, we show strong evidence that the UL23 protein of human cytomegalovirus (HCMV), a human herpesvirus, specifically interacts with Nmi. This interaction was identified through a yeast two-hybrid screen and co-immunoprecipitation in human cells. We observed that Nmi, when bound to UL23, was not associated with STAT1, suggesting that UL23 binding of Nmi disrupts the interaction of Nmi with STAT1. In cells overexpressing UL23, we observed (a) significantly reduced levels of Nmi and STAT1 in the nuclei, the sites where these proteins act to induce transcription of IFN-γ stimulated genes, and (b) decreased levels of the induction of the transcription of IFN-γ stimulated genes. UL23-deficient HCMV mutants induced higher transcription of IFN-γ stimulated genes and exhibited lower titers than parental and control revertant viruses expressing functional UL23 in IFN-γ treated cells. Thus, UL23 appears to interact directly with Nmi and inhibit nuclear translocation of Nmi and its associated protein STAT1, leading to a decrease of IFN-γ induced responses and an increase of viral resistance to IFN-γ. Our results further highlight the roles of UL23-Nmi interactions in facilitating viral immune escape from IFN-γ responses and enhancing viral resistance to IFN antiviral effects.

Human cytomegalovirus UL49 encodes an early, virion-associated protein essential for virus growth in human foreskin fibroblasts

Despite recent results of deletion experiments showing that open reading frame (ORF) UL49 of human cytomegalovirus (HCMV) is essential, the expression, function and functional location of its encoded protein remain unknown. We generated an antibody specific for pUL49 to investigate the protein product encoded by the UL49 ORF and identified its function in HCMV-infected host foreskin fibroblasts. A bacterial artificial chromosome (BAC) of HCMV strain Towne (pRV-Towne) and the UL49-deleted mutant pRV-delUL49Towne were used to observe virus growth by plaque assay. Using a UL49-protein-binding antibody, we located pUL49 in the fibroblast cytoplasm. pUL49 exhibited expression kinetics resembling those of the class β-2 proteins and was detected in the virion tegument. Following deletion of UL49 ORF, the virus failed to replicate, but it could be recovered by addition of pUL49 from pCDNA3.1 (+)-UL49. Our findings indicate that UL49 ORF is essential for HCMV replication in host foreskin fibroblasts.