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Virus Replication and Virus-Host Interactions
Summary: Paul Ahlquist is interested in the mechanisms of virus replication, gene expression, evolution, pathogenicity, and virus-host interactions. His research combines studies of selected RNA, DNA, and reverse-transcribing viruses, including several human tumor viruses.
Viruses remain serious threats to public health because of the lack of effective controls for most viruses and the continuing emergence of new, highly pathogenic viruses such as HIV and the SARS (severe acute respiratory syndrome) virus. In addition to debilitating and life-threatening acute infections, viruses cause at least 15–20 percent of human cancers. Viruses also are potential bioterrorism threats that are relatively easy to produce, engineer, and disseminate. Developing more effective virus controls requires better understanding of virus replication and virus-host interactions. Such virus studies also provide insights into biochemistry and cell biology, new biomedical tools, and other advances.
Our research integrates studies of selected RNA, DNA, and reverse-transcribing viruses. The results have provided new approaches to study viruses, defined molecular pathways and host interactions by which viruses replicate and cause disease, and revealed functional and likely evolutionary links uniting a surprisingly wide range of viruses.
Three-Dimensional Organization of a Viral Mini-organelle for RNA Replication
Positive-strand RNA [(+)RNA] viruses, the largest genetic class of viruses, include many serious human pathogens. All (+)RNA viruses replicate their genomes in association with intracellular membrane rearrangements such as single- or double-membrane vesicles, but little has been understood about how and why these viruses use membranes in RNA replication.
Recently we found multiple parallels between the intracellular, membrane-associated replication complexes of some (+)RNA viruses and the extracellular, membrane-enveloped infectious particles (virions) of two other virus classes, reverse-transcribing viruses such as HIV and double-strand RNA (dsRNA) viruses (Figure 1). To further define the nature and generality of these parallels, we expanded these studies to include a second, highly distinct (+)RNA virus family and used electron microscope tomography and complementary approaches to provide the first three-dimensional analysis of an RNA replication complex.
We found that viral RNA synthesis localizes inside virus-induced, ~50-nm, vesicular membrane invaginations, with striking similarities to budding retrovirus virions (Movie 1). The interior of each vesicle is connected to the cytoplasm by a necked channel of ~10 nm diameter, large enough to import ribonucleotide substrates and to export product RNA (Movie 2). Tomographic, biochemical, and other results showed that each vesicular complex contains, on average, three RNA replication intermediates and an interior shell of ~100 copies of a single, self-interacting viral RNA replication protein.
Thus, viral RNA replication occurs in a virus-induced mini-organelle that organizes replication factors and protects replicating RNAs from competing processes and host defenses such as RNA interference. The results appear relevant to many other (+)RNA viruses, and support and extend the earlier parallels that we found with reverse-transcribing and dsRNA virus virions. Thus, three of the six major classes of viruses share similarities in the form and function of their replication complexes and may have evolved from common precursors, supporting a significant unification across virology. Other results suggest that the dependence of these processes on membranes, on high-level multimerization of viral replication proteins, and on other features may provide useful targets for developing potentially broad-spectrum antivirals.
Host Functions in Hepatitis B Virus Virion Release from Cells
Hepatitis B virus (HBV) is a reverse-transcribing virus that is clinically and molecularly distinct from HIV. HBV chronically infects ~400 million people (>6 percent of humanity), causing liver disease and most cases of liver cancer, the third leading cause of cancer death worldwide. As noted above, formation of the infectious virion particles of HIV and HBV is similar to (+)RNA replication complex assembly, involving the capture of a viral RNA genome and a viral polymerase inside a protein shell. This protein shell pushes through or is wrapped by a cellular membrane to create a vesicle-like membrane pocket (Figure 1). However, although (+)RNA replication complexes remain attached to the membrane as intracellular RNA factories, HIV and HBV have evolved to sever the neck of the membrane invagination, releasing or "budding" off the resulting particle as an infectious virion that can infect a new cell.
Budding of HIV and some other enveloped viruses from the cell surface requires host functions involved in sorting proteins into subcellular structures called multivesicular bodies (MVBs). We found that budding of HBV virions, which occurs at an intracellular secretory compartment, also involves MVB functions. The HBV virion membrane or "envelope" protein colocalizes by immunofluorescence with host cell MVB proteins AIP1/ALIX and VPS4B in human hepatoma cells. Moreover, a dominant-negative AIP1 mutant inhibited production and/or release of membrane-enveloped virions, without significant effects on intracellular assembly of the nucleocapsid cores of these virions. Dominant-negative VPS4B inhibited both nucleocapsid production and virion budding. Thus, host MVB functions are required for efficient budding and release of enveloped HBV virions and may be a valuable target for HBV control. Dominant-negative AIP1 and VPS4 had no effect on the release of enveloped, nucleocapsid-lacking HBV subviral particles, which are produced in vast excess over virions, apparently as an immunological decoy. Thus, these subviral particles must be produced and released from cells by a distinct pathway with different host factor requirements.
Papillomavirus Replication and Oncogenesis
Human papillomaviruses (HPVs) cause essentially all cervical cancers (the leading cause of cancer death for women in developing countries), 20–25 percent of head/neck cancers (the fourth most common cancer in men worldwide), and other cancers. The HPV life cycle is tightly linked to epithelial cell differentiation, which has greatly restricted experimental production of HPV virions. To overcome these restrictions, we recently developed a system that allows high-yield, differentiation-independent generation of infectious particles containing wild-type, mutant, or chimeric HPV genomes, which we are using to study selected aspects of HPV replication, virus-host interaction, and control.
To better understand the role of HPV in tumor induction, progression, and maintenance, we and our collaborators are comparing diverse molecular characteristics of HPV-positive cervical cancers, HPV-positive head/neck cancers, and HPV-negative head/neck cancers. Comparing global gene expression in dozens of tumor and normal tissue samples showed that, despite their differing anatomical sites, HPV-positive cervical and head/neck cancers share many gene expression patterns distinct from HPV-negative head/neck cancers. Notably, HPV-positive cervical and head/neck cancers up-regulate the expression of a distinct and much larger subset of cell cycle genes than HPV-negative head/neck cancers. Moreover, HPV-positive cancers overexpress certain germline-specific genes that are normally expressed only in meiotic cells. We showed that many, though not all, of these hallmark differences between HPV-positive and -negative head/neck cancers are a direct consequence of HPV and in particular the viral E6 and E7 oncogenes. These findings in primary human tumors provide novel biomarkers for early detection of HPV-positive and -negative cancers, emphasize the need to consider HPV status in treating head/neck cancers, and suggest some specific targets and strategies for treating HPV-positive cancers.
Grants from the National Institutes of Health provided partial support for these projects.
Last updated: July 17, 2008
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