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Replication and Pathogenesis of RNA Viruses
Summary: Michael Lai's laboratory studies the replication and pathogenesis of several human and animal RNA viruses, including hepatitis C virus, hepatitis delta virus, and coronavirus.
The persistence of old viruses and continuing emergence of new viruses that threaten human health have defied the best efforts of medical research. These threats challenge us to redouble our efforts to understand better the fundamental properties of viruses. Our laboratory has a long-standing interest in viruses, particularly RNA viruses, in which RNA, rather than DNA, is the genetic material. We study how these viruses replicate and cause diseases. Currently, we are focused on three different human and animal viruses.
Hepatitis C Virus
Our main research interest in recent years has been hepatitis C virus (HCV), a major cause of chronic hepatitis, liver cirrhosis, and liver cancer. HCV afflicts approximately 4 million people in the United States and 100 million people worldwide and is likely to become a major medical threat in the next two decades if no new treatments are developed soon. Our laboratory is interested in understanding the mechanism of viral replication and pathogenesis, with the goal of developing effective treatments for hepatitis C.
The prime challenge in the study of HCV currently is the lack of a robust tissue culture system for growing the virus. Based on the observation that HCV infection is frequently associated with lymphocyte anomaly, including the development of B cell lymphoma, my laboratory recently developed a B cell lymphoma cell line that is persistently infected with HCV. This cell line continues to produce HCV particles, even after prolonged culture in vitro. The released virus can infect primary human hepatocytes and other established B cell lines. Thus we now have access to an HCV cell culture system that supports the complete viral replication cycle. These cell lines will be of enormous help in screening potential antiviral agents and evaluating new antiviral therapies. For example, we have begun to understand that interferon, which is the primary drug for hepatitis C, is not very effective in directly inhibiting HCV replication, and that interferon causes the death of virus-infected cells.
These cell lines also enable studies of the biology and pathology of HCV infection. For example, we now know that HCV infection causes apoptosis (death) of the infected cells and changes in the cell surface molecules, which likely has profound effects on the immunological properties of the cells. Importantly, these cell lines have enabled us to prove that HCV infects not only liver cells but also B lymphocytes in patients. This is an important parameter in the disease manifestation and treatment of HCV infection, as B cells serve as a reservoir of viral infection. These cell lines have thus opened up new avenues of HCV research.
Another critical issue in HCV research is how HCV causes hepatitis. We approached this issue by studying how HCV proteins affect cellular functions. We found several such examples, most notably the binding of the viral core protein, which makes up the internal structure of virus particles, to members of the tumor necrosis factor (TNF) receptor family. This family includes TNF receptor I and lymphotoxin-b receptor, which are important components of inflammatory responses and the body's defense mechanism against viral infections. These interactions may lead to perturbation of the normal functions of these receptors, thus contributing to the occurrence of hepatitis. Other interactions between HCV proteins and host proteins, such as between viral nonstructural protein 5A and a cellular protein apolipoprotein A1, and between the viral envelope protein E2 and cellular protein kinase PKR, may contribute to the occurrence of fatty degeneration and the development of liver cancers associated with hepatitis C and account for the resistance of HCV to interferon treatment. We are studying these properties. (These studies are partially supported by the National Institutes of Health.)
Hepatitis Delta Virus
The second virus of interest to our laboratory is hepatitis delta virus (HDV), which is associated with hepatitis B virus infections and frequently causes fulminant hepatitis. The virus contains a small, circular RNA genome; this structure is unique among human and animal viruses. The RNA has a ribozyme activity, i.e., it cleaves itself without the help of a protein. The virus makes only one protein, hepatitis delta antigen (HDAg), which plays a key role in viral RNA replication.
The most unusual aspect in the HDV life cycle is that viral RNA synthesis, which uses RNA as a template, must rely on cellular transcription machineries, which normally utilize DNA as a template. How the template specificity of these machineries is switched is an issue of fundamental importance. The previous hypothesis stated that cellular RNA polymerase II, which normally makes cellular messenger RNA, carries out the entire process of HDV RNA replication, but this hypothesis has not been verified. To aid in the resolution of these questions, we recently developed a new RNA transfection procedure for studying HDV RNA synthesis. This method has led to the rethinking of many aspects of the old HDV replication model. We found that while HDV messenger RNA (for making the delta antigen) is synthesized by cellular polymerase II, the replication of the HDV RNA genome is carried out by other known DNA-dependent RNA polymerases or a yet-to-be-discovered polymerase. These findings suggest that mammalian cells have a previously unrecognized ability to replicate RNA. The nature of these enzymes is being actively studied. Thanks to progress in medical intervention, the incidence of HDV infection has dropped significantly in recent years. Nevertheless, the HDV replication cycle highlights many as yet unrecognized features, the understanding of which will likely open up new frontiers of the molecular biology of mammalian cells.
Coronavirus
Another virus of long-standing interest to our laboratory is coronavirus, which causes respiratory and gastrointestinal diseases in humans and animals. It also causes neurological symptoms similar to those of multiple sclerosis. The virus has an RNA genome of 31,000 nucleotides, which is the longest known viral RNA. Our laboratory has shown that coronavirus uses a novel RNA synthesis mechanism, a discontinuous process that fuses two distant RNA elements from different RNA molecules into a single molecule, a process not seen in other organisms. Using this virus as a model, we proposed that viral RNA synthesis, in general, requires the formation of a transcription complex involving cellular and viral proteins and viral RNA, in much the same way as the formation of DNA-dependent transcription complexes is involved in the transcription of cellular genes. We have identified the first two major proteins in this complex, namely, heterogeneous nuclear ribonucleoprotein A1 and polypyrimidine tract–binding protein, which are normally involved in cellular RNA transport and regulation of RNA splicing. The coronavirus apparently usurps these cellular proteins for viral RNA synthesis. Other components of this complex are being characterized. (These studies are partially supported by grants from the National Institutes of Health.)
Our laboratory is studying a wide spectrum of RNA viruses that use different principles for viral replication and employ different mechanisms for causing diseases. We hope to shed increasing light on the basic biology of viral infection and aid the design of strategies to control viral diseases.
Last updated June 27, 2001
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