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Biomedical subjects

Michael J McGarvey

Publications and source records attributed to Michael J McGarvey.

7 recordsLinked to original sources
PubMed2006

Increased liver pathology in hepatitis C virus transgenic mice expressing the hepatitis B virus X protein.

Transgenic mice expressing the full-length HCV coding sequence were crossed with mice that express the HBV X gene-encoded regulatory protein HBx (ATX mice) to test the hypothesis that HBx expression accelerates HCV-induced liver pathogenesis. At 16 months (mo) of age, hepatocellular carcinoma was identified in 21% of HCV/ATX mice, but in none of the single transgenic animals. Analysis of 8-mo animals revealed that, relative to HCV/WT mice, HCV/ATX mice had more severe steatosis, greater liver-to-body weight ratios, and a significant increase in the percentage of hepatocytes staining for proliferating cell nuclear antigen. Furthermore, primary hepatocytes from HCV, ATX, and HCV/ATX transgenic mice were more resistant to fas-mediated apoptosis than hepatocytes from nontransgenic littermates. These results indicate that HBx expression contributes to increased liver pathogenesis in HCV transgenic mice by a mechanism that involves an imbalance in hepatocyte death and regeneration within the context of severe steatosis.

Animals↗
PubMed2005

Complement inhibition rescued mice allowing observation of transgene expression following intraportal delivery of baculovirus in mice.

BACKGROUND: The baculovirus Autographa californica nucleo-polyhedrosis virus (AcNPV) is an alternative to other viral vectors for hepatic gene delivery. A barrier to AcNPV being used in vivo is its susceptibility to inactivation by serum complement 1. In vivo utility has only been demonstrated using methods that avoid contact with serum 2-5. We have studied the complement pathways involved in baculovirus inactivation in vitro and the systemic administration of baculovirus vectors in vivo, with the co-administration of the complement inhibitor, soluble complement inhibitor 1 (sCR1). RESULTS: EDTA increased baculovirus survival in human serum more than EGTA showing that both the alternative and classical pathways of complement are activated. Depleting serum of IgM increased survival, whereas reconstitution with pooled IgM restored activity against baculovirus, suggesting naturally occurring IgM antibodies with affinity for baculovirus may be partially responsible for complement activation. Intraportal administration of baculovirus led to hepatic expression when the complement inhibitor sCR1 (soluble complement receptor type 1) was co-administered by tail vein injection; however, liver histology showed hepatic necrosis. Without co-administration of sCR1, intraportal infusion of baculovirus was fatal within 24 h. Histology demonstrated massive hepatic necrosis. Yolk sac vein injection of baculovirus was associated with fetal death. CONCLUSIONS: Transgene expression was demonstrated following intraportal infusion of recombinant baculovirus vectors in combination with sCR1; however, our experiments suggest a significant associated toxicity.

Animals↗
PubMed2005

Stable inhibition of hepatitis B virus proteins by small interfering RNA expressed from viral vectors.

BACKGROUND: There has been much research into the use of RNA interference (RNAi) for the treatment of human diseases. Many viruses, including hepatitis B virus (HBV), are susceptible to inhibition by this mechanism. However, for RNAi to be effective therapeutically, a suitable delivery system is required. METHODS: Here we identify an RNAi sequence active against the HBV surface antigen (HBsAg), and demonstrate its expression from a polymerase III expression cassette. The expression cassette was inserted into two different vector systems, based on either prototype foamy virus (PFV) or adeno-associated virus (AAV), both of which are non-pathogenic and capable of integration into cellular DNA. The vectors containing the HBV-targeted RNAi molecule were introduced into 293T.HBs cells, a cell line stably expressing HBsAg. The vectors were also assessed in HepG2.2.15 cells, which secrete infectious HBV virions. RESULTS: Seven days post-transduction, a knockdown of HBsAg by approximately 90%, compared with controls, was detected in 293T.HBs cells transduced by shRNA encoding PFV and AAV vectors. This reduction has been observed up to 5 months post-transduction in single cell clones. Both vectors successfully inhibited HBsAg expression from HepG2.2.15 cells even in the presence of HBV replication. RT-PCR of RNA extracted from these cells showed a reduction in the level of HBV pre-genomic RNA, an essential replication intermediate and messenger RNA for HBV core and polymerase proteins, as well as the HBsAg messenger RNA. CONCLUSIONS: This work is the first to demonstrate that delivery of RNAi by viral vectors has therapeutic potential for chronic HBV infection and establishes the ground work for the use of such vectors in vivo.

Base Sequence↗
PubMed2003

The importance of the Q motif in the ATPase activity of a viral helicase.

NS3 proteins of flaviviruses contain motifs which indicate that they possess protease and helicase activities. The helicases are members of the DExD/H box helicase superfamily and NS3 proteins from some flaviviruses have been shown to possess ATPase and helicase activities in vitro. The Q motif is a recently recognised cluster of nine amino acids common to most DExD/H box helicases which is proposed to regulate ATP binding and hydrolysis. In addition a conserved residue occurs 17 amino acids upstream of the Q motif ('+17'). We have analysed full-length and truncated NS3 proteins from Powassan virus (a tick-borne flavivirus) to investigate the role that the Q motif plays in the hydrolysis of ATP by a viral helicase. The Q motif appears to be essential for the activity of Powassan virus NS3 ATPase, however NS3 deletion mutants that contain the Q motif but lack the '+17' amino acid have ATPase activity albeit at a reduced level.

Adenosine Triphosphatases↗
PubMed2001

Intracellular localization of the hepatitis B virus HBx protein.

The hepatitis B virus (HBV) X protein (HBx) was originally suggested to be a viral transcriptional activator, but its functional mechanisms are still unclear. In this study we have analysed the intracellular localization of HBx in transfected cells and demonstrate that its compartmentalization is dependent on overall expression levels. HBx was exclusively or predominantly localized in the nuclei in weakly expressing cells. However, elevated cellular levels correlated with its accumulation in the cytoplasm, suggesting that the capacity of HBx for nuclear compartmentalization might be limited. Cytoplasmic HBx was detected either as punctate granular staining or in dispersed, finely granular patterns. We have further analysed the detailed cytoplasmic compartmentalization, using confocal microscopy, and show no association with the endoplasmic reticulum, plasma membrane or lysosomes, but a substantial association of HBx with mitochondria. However, a major fraction of cytoplasmic HBx did not localize in mitochondria, indicating the presence of two distinctly compartmentalized cytoplasmic populations. Furthermore, high levels of HBx expression led to an abnormal mitochondrial distribution, involving clumping and organelle aggregation, which was not observed at lower expression levels. The data presented here provide novel insights into the compartmentalization of HBx and may prove important for future evaluations of its functions, both in the viral life-cycle and in the pathology of HBV-related liver disease.

Amino Acid Sequence↗
PubMed1999

The 5' untranslated region of GB virus B shows functional similarity to the internal ribosome entry site of hepatitis C virus.

Since its characterization in 1995, there has been increasing interest in the significance of GB virus B (GBV-B) due to its close phylogenetic relationship to hepatitis C virus (HCV). The genome of GBV-B is similar in length and organization to that of HCV and the two viruses share sequence similarity in their 5' untranslated regions (5'UTR). A secondary structure model of the GBV-B 5'UTR has been proposed by comparative sequence analysis with HCV. The highly conserved secondary structure, present in HCV and the pestiviruses, is also present in the 5'UTR of GBV-B. Translation of the HCV polyprotein initiates via an internal ribosome entry site (IRES) and it is proposed that the GBV-B UTR may function in a similar manner. Dicistronic reporter constructs were made to investigate the function of the GBV-B 5'UTR. Mutational analysis and in vitro translation experiments demonstrate that GBV-B initiates translation via an IRES.

5' Untranslated Regions↗