Molecular and Structural Basis of Cell Entry by Emerging and Zoonotic RNA Viruses

Emerging zoonotic viruses cross the species barrier from animals to humans or vice-versa. Their ability to propagate in human cells is dependent on a variety of factors, most importantly, their ability to target particular cell types by binding to receptor molecules displayed on the surface of the host cell. Despite the severity of disease caused in both animals and humans and threat these viruses pose to global health and economy, little is known of how they infect their host, information which has proven to be extremely valuable for the development of antiviral drugs or antibody-based medicines for better-studied viruses such as flu and HIV-1. Using techniques in structural biology, immunology, and cell biology, I aim to show in atomic detail the mechanism by which these viruses attach to and infect human cells. This will involve the combination of high-resolution structural studies on isolated molecules with lower resolution analyses of intact viral prototypes. The information derived from these combined techniques will ultimately aid in our ability to combat these pathogens.

I will elucidate the molecular mechanism by which by emergent single-stranded viruses undergo attachment and fusion with host cells. This will be addressed in the context of both intact virus and the individual structural glycoproteins responsible for viral entry. This investigation is limited to emergent viruses from three families: Paramyxoviridae (e.g. African Henipaviruses), Arenaviridae (e.g. Venezuelan Hemorrhagic fever virus), and Bunyaviridae (e.g. Hantavirus Pulmonary Syndrome associated diseases; a list of targets is provided in the Case for Support, Table 1).

There are four complementary goals:

1. Recombinant expression and purification of viral attachment glycoproteins, preparation of model non-pathogenic viruses, and the acquisition/development of neutralising antibodies (nAbs) against these glycoproteins.

2. Crystallographic analysis of viral glycoproteins alone and in complex with cellular receptors and nAbs to provide atomic descriptions in pre- and post-fusion assemblies, and illustrate the morphological changes required for viral entry.

3. Cryo-electron microscopic analysis of intact, non-pathogenic viral orthologues to visualise the ultrastructure of these viruses, the assembly of their subunit glycoproteins, and to provide a structural basis for -receptor and -antibody interactions for entire virions.

4. Mutagenesis of the viral genome to generate modified viruses to complement structure predicted determinants for virus host cell entry.

Aims: I will elucidate the molecular mechanism by which by emergent negative-sense, single-stranded viruses undergo attachment and fusion with host cells. This investigation is limited to emergent viruses from three families: Paramyxoviridae (e.g. African Henipaviruses), Arenaviridae (e.g. Guanarito virus), and Bunyaviridae (e.g. Sin nombre virus; a list of targets is provided in the Case for Support, Table 1).

Objectives: This will be addressed in the context of both intact virus and the individual structural glycoproteins responsible for viral entry. As a result, this work will integrate techniques in (1) protein chemistry and virology, (2) X-ray crystallography, (3) electron microscopy, and (4) molecular biology.

These techniques are detailed below:

1. Recombinant expression and purification of viral attachment glycoproteins alone in complex with functional cellular receptors, preparation of model non-pathogenic viral orthologues and VLPs, and the acquisition/development of neutralising antibodies (nAbs) against these glycoproteins.

2. Crystallographic analysis of viral glycoproteins alone and in complex with cellular receptors and nAbs to define the molecular specificity which underlies virus-host interactions.

3. Cryo-electron microscopic analysis of intact, non-pathogenic viral orthologues into visualise the ultrastructure of these viruses and the higher order organization of their subunit glycoproteins as they extend from the virus envelope.

4. Mutagenesis of the viral genome by reverse genetics to complement structure predicted determinants for virus host cell entry.

Scientific and Medical opportunities:

Through the concerted effort from my laboratory, this multidisciplinary work will offer a rational description of host cell entry and enhance our ability to design therapeutics against the targeted emergent pathogens.

Human encroachment upon previously untouched ecological niches has led to an increased emergence of zoonotic and highly virulent negative-sense single-stranded RNA viruses. Paramyxoviruses, Arenaviruses, and Bunyaviruses, constitute three families of viruses containing such zoonotic and highly virulent pathogens. The clinical progression of viral pathogenesis upon human infection is often extremely rapid and associated with high mortality rates. Currently there are no approved vaccines or specific antiviral treatments to treat infection. The potential of infection by human-to-human contact or aerosol by many of these viruses (please see Table 1 in Case for Support for list of viral targets) has resulted in their classification by the NIAID as high priority Category A biothreat agents.

In this research I will study tropical and exotic viruses for which there are limited vaccines or treatments and which are often underrepresented in terms of academic and pharmaceutical interest. My research will aim to answer the two following questions: (1) What are the molecular mechanisms by which these zoonotic viruses attach to human host cells? (2) Can broadly conserved epitopes for neutralizing antibodies be identified on the virion surface?

The design of this project is based upon the premise that emerging pathogens can be rationally targeted following structural and functional characterization. Success in applying basic biomedical knowledge towards the design of antiviral therapeutics is with precedence (e.g. design of antivirals against HIV-I and influenza virus).

This research will contribute to the following long-term impact: A. Greater readiness to predict and respond to the emergence of new, related viruses, B. Improved protection of human health in endemic areas, and C. An improved readiness to face bio-weapon threats. Details who will benefit from this long-term impact are summarized below:

A) As reported by Jones, et al (Nature 451, 990-93, 2008) the number of emerging infectious disease events are increasing each year. By structural and functional characterization of the prototypical viral glycoproteins listed in this work, it will be possible to predict, with confidence, the molecular determinants of host-cell entry of novel but related emerging zoonotic viruses. This will thus provide epidemiological clues of virus spread as well be informative for antiviral and vaccine strategies.

B) Although the proposed research is strongly rooted in basic science, the findings are expected make a positive impact on human health. Arena-, Bunya-, and Paramyxoviruses affect human populations in some of the World's poorest regions. Basic research, as outlined in this proposal, will contribute key knowledge required for antiviral and vaccine development. Thus, the mid-term beneficiaries will be in research and development teams in the pharmaceutical industry and governmental organisations. If drug and vaccine development is successful, the long term beneficiaries will be people affected by viral emergence in the endemic areas. Furthermore, vaccination would protect hospital personnel and protect populations in high risk areas.

C) As listed by the CDC, many of the viruses studied in this work could be used as a biological weapon against any nation, including the UK. Successful development of vaccines and antivirals (e.g. neutralizing antibody cocktails) would improve the nation's readiness to for treatment in face such threats.