Modulation of HBV infection and replication by cell-derived factors

[ACCESS RESTRICTED TO THE UNIVERSITY OF MISSOURI AT REQUEST OF AUTHOR.] Hepatitis B virus (HBV) infection is one of the most serious health problems. HBV infection leads to serious morbidity and mortality due to the complications of chronic HBV infection. For example, HBV chronic infection is responsible for most of the cases of hepatocellular carcinomas. HBV is a small DNA virus and considered the prototype of family Hepadnaviridae. HBV genome is composed of partially double-stranded, relaxed-circular DNA (rcDNA), of 3.2 kb, in which the plus strand is incomplete, while the minus strand is complete, and the viral polymerase is attached to the 5' end of the minus strand. In the infected cells, HBV rcDNA genome is converted to a plasmid-like, covalently closed circular DNA (cccDNA) in the nucleus. CccDNA is considered the template for all HBV RNAs. Current chronic HBV infection treatments include pegylated interferon-[alpha], which has severe side effects, and nucleos(t)ide reverse transcriptase inhibitors which have no effect on cccDNA, which is highly stable, and consequently, fail to completely cure most patients. Thus, targeting cccDNA is the key element for curing chronic HBV infection. Studying host factors that have a role in HBV infection and replication may help develop new strategies to cure chronic HBV infection. Moloney leukemia virus 10 (MOV10) is an RNA helicase that has been shown to affect the replication of several viruses. The effect of MOV10 on HBV infection is not known and its role on replication of this virus is poorly understood. We investigated the effect of MOV10 down-regulation and MOV10 over-expression on HBV in a variety of cell lines, as well as in an infection system using a replication competent virus. We report that MOV10 down-regulation, using siRNA, shRNA, and CRISPR/Cas9 gene editing technology, resulted in increased levels of HBV DNA, HBV pre-genomic RNA and HBV core protein. In contrast, MOV10 over-expression reduced HBV DNA, HBV pre-genomic RNA, and HBV core protein. These effects were consistent in all tested cell lines providing strong evidence for the involvement of MOV10 in restricting HBV replication cycle. We demonstrated that MOV10 does not interact with HBV core. However, MOV10 binds HBV pgRNA and this interaction does not affect HBV pgRNA decay rate. Further studies are still needed to investigate the exact mechanism of which MOV10 restricts HBV. As mentioned earlier, HBV rcDNA is converted in the nucleus of infected cell to cccDNA. To be converted to cccDNA, rcDNA undergoes some modifications including the removal of HBV polymerase from the 5' end of the minus-strand DNA, the plus-strand DNA should be completed, and both strands should be covalently ligated to form the closed, plasmid-like form. The exact kinetics of cccDNA formation are not clear yet. We studied the kinetics of early events following HBV infection. Our results demonstrated that HBV DNA can be detected in the nuclei of infected cells within 2 hours post-infection by qPCR and microscopy. Also, we were able to detect HBV cccDNA within 6 hours post-infection using qPCR. Early detection of nuclear HBV DNA and cccDNA formation after infection by qPCR and microscopy will help study the early HBV infection events, and screen new cccDNA formation inhibitors with an easy and fast way.