News and Publications

Molecular Pathogenesis of Viral Infections

J. Rempel, B. Neuman, E. Burke, J. Botten, A. Saunders, J. Meisner, A. Paulino, N. Benning, S. Murray, M. Buchmeier

STRUCTURE OF THE CORONAVIRUS SPIKE PROTEIN

Coronaviruses are a family of human and animal respiratory and enteric pathogens of great economic importance. According to estimates, human coronaviruses are responsible for up to half of the cases of the common cold, and they may be the etiologic agents in diseases such as multiple sclerosis, gastroenteritis, and myocarditis.

The virion spike of these viruses extends radially approximately 200 Å from the viral membrane, giving the virions a crownlike appearance (Fig. 1), and is composed of multiple copies of a 180-kD glycosylated protein. Receptor binding and fusion are activities of the spike. The spike is also a target for the host response. Infection with murine hepatitis virus (MHV) is a particularly useful model for understanding the pathogenesis of coronaviruses. Previously, we identified strains of MHV that contain mutations in heptad repeat regions of the spike protein and have altered fusion capacity. In a collaborative study with M. Yeager, Department of Cell Biology, we are using electron cryomicroscopy and image processing to examine the MHV membrane glycoprotein and construct a working model of its structure. Maps obtained at approximately 10- to 20-Å resolution will reveal the quaternary structure of the spike and its behavior upon fusion activation.

PATHOGENESIS OF VIRAL ENCEPHALITIS AND DEMYELINATION

The information on the MHV spike is helping us interpret recent biological data that indicate a direct role of the spike in the induction of early cytokine and chemokine responses. The ability of a virus to manipulate the immune system has a pivotal effect on the progression of disease and the fate of the infected host. Using infections with 2 murine coronaviruses, MHV-A59 and MHV-JHM, as models for virus-induced demyelination and lethal encephalitis, respectively, we evaluated the contribution of virally induced immune responses to disease.

We detected distinct glial cell cytokine and chemokine responses soon after infection (Fig. 2), and as the infections progressed and peripheral immune cells became increasingly involved, these responses became more polarized. Compared with mice infected with MHV-JHM, mice infected with MHV-A59 had early and sustained upregulation of IFN-g mRNA in the brain and a 3-fold increase in infiltrating CD8⁺ T cells. Mice infected with MHV-A59 also had increased numbers of IFN-g ­producing cells during acute encephalitis. In contrast, MHV-JHM infection induced strong and ongoing transcription of the innate immune products IFN-g , IL-6, and IL-1. Compared with mice infected with MHV-A59, mice infected with MHV-JHM had high levels of mRNA for macrophage inflammatory proteins 1 and 2 at the onset of infection that correlated with a marked elevation in the number of macrophages and neutrophils in the brain.

Taken together, our results indicate that early innate responses in brain cells influence the outcome of neurotropic viral infections. We are now using a genomic approach to evaluate the mechanisms of activation of these responses and their implications for acute encephalitis and chronic demyelinating diseases.

MODELING LASSA VIRUS GLYCOPROTEIN STRUCTURE AND FUNCTION

Lassa virus is an arenavirus endemic to West Africa that persists in the rodent host Mastomys natalensis and causes Lassa fever in humans. Molecular characterization of Lassa virus gene products has been difficult because of its negative-strand RNA genome and its classification as a biosafety level 4 pathogen. We are interested in the structure and function of viral glycoproteins. To investigate glycoprotein function, in a collaborative study with P Cannon, University of Southern California, Los Angeles, California, we used a retroviral pseudotype system in which the Lassa virus glycoprotein is packaged with a murine leukemia virus core. The pseudotype viruses acquire the host range of Lassa virus and can infect the human kidney epithelial cell line 293T. Pseudotype viruses expressing the glycoprotein of lymphocytic choriomeningitis virus were also made.

Both kinds of pseudotyped viruses were neutralized by their corresponding antibody but not by the heterologous antiviral antibody. This pattern of antibody recognition and the high degree of sequence conservation between the 2 proteins suggested that chimeric glycoproteins could be used to map key structural features of viral glycoprotein. We constructed a panel of chimeric glycoproteins and found that they had proper folding, cell-surface transport, and expression in the pseudotype system. We are using the Lassa virus­lymphocytic choriomeningitis virus chimeras to study biological activities, including antibody neutralization and receptor binding.

MOLECULAR BIOLOGY AND PATHOGENESIS OF LASSA VIRUS

The structure of the arenaviruses, including Lassa virus, is quite simple; the viruses consist of only 4 primary gene products encoded on 2 RNA strands. Despite this simplicity, these viruses cause infections in a variety of ways that result in a range of syndromes, from a lifelong persistent carrier state to lethal acute diseases such as the hemorrhagic fevers caused by Lassa virus and other arenaviruses. Lassa virus infects 100,000­300,000 persons annually in West Africa; of these, 5000­10,000 die, and many survivors have nerve deafness.

Lassa virus is a biosafety level 4 agent, and the security and biosafety constraints imposed by this classification have markedly impeded the ability to study the virus. In collaboration with L. Whitton, Department of Neuropharmacology, we are investigating the protective immune response to Lassa virus. We hope to use this information to develop a vaccine. The protective components of vaccine-induced immunity for infection caused by Lassa virus have not been identified. However, by drawing on our experience with lymphocytic choriomeningitis virus, we plan to evaluate the requirements for protection and to exploit recent advances in DNA vaccination to optimize the induction of CD8⁺ and CD4⁺ T cells and antibodies.

PUBLICATIONS

Bonthius, D.J., Mahoney, J., Buchmeier, M.J., Karacay, B., Taggard, D. Critical role for glial cells in the propagation and spread of lymphocytic choriomeningitis virus in the developing rat brain. J. Virol. 76:6618, 2002.

Buchmeier, M.J. Arenaviruses: protein structure and function. Curr. Top. Microbiol. Immunol. 262:159, 2002.

Kahn, S., de Giuli, R., Schmidtke, G., Bruns, M., Buchmeier, M., van den Broek, M., Groettrup, M. Cutting edge: neosynthesis is required for the presentation of a T cell epitope from a long-lived viral protein. J. Immunol. 167:4801, 2001.

Lane, T.E., Buchmeier, M.J. Chemokine responses in virus-induced neurologic disease: balancing host defense and neuropathology. In: Universes in Delicate Balance: Chemokines and the Nervous System. Ransohoff, R.M., et al. (Eds.). Elsevier Science, New York, 2002, p. 191.

Redwine, J.M., Buchmeier, M.J., Evans, C.F. In vivo expression of major histocompatibility complex molecules on oligodendrocytes and neurons during viral infection. Am. J. Pathol. 159:1219, 2001.

Rempel, J.D., Buchmeier, M.J. Analysis of CNS inflammatory responses to MHV: role of spike determinants in initiating chemokine and cytokine responses. Adv. Exp. Med. Biol. 494:77, 2001.