Research Overview

Viruses recognize a large variety of cell-surface receptors displayed on the host, from commonly found sugars to specific proteins. Receptor and co-receptor usage affect the specific cell type that viruses may enter as well as virus pathogenicity, penetration, and uncoating. Although a myriad of viral receptors have been identified the molecular mechanisms governing receptor recognition remain obscure for most viruses. The long-term goal of our research is to define virus structures and virus-host cell receptor interactions on a structural level, using cryoEM 3-D reconstruction techniques and X-ray crystallography.

Canine parvovirus and feline panleukopenia virus (CPV and FPV)

The goal of the parvovirus research is to define virus and host cell receptor interactions and to explore host adaptation and species jumping.

CPV + TfR

CryoEM reconstruction of a parvovirus complexed with the specific receptor, TfR to define the interacting surfaces of the TfR and capsid: CPV + TfR. Both cryoEM and biochemical analyses showed that TfR binds to only one or a few of the 60 icosahedrally-equivalent sites on the virion, indicating that the virus may have inherent asymmetry. Possibly, asymmetry of the virus has not been visualized previously due to the averaging methods employed in structural determinations. Alternatively, the interaction of the receptor may trigger a conformational change that induces asymmetry. Such a change could be a step to further the infection, perhaps mediating the release of the genome from the parvovirus capsid.

CPV + canine antibody

The properties of viral antigens and the effects of antibody binding vary widely; some viruses are efficiently neutralized, whereas others show varying degrees of escape from antibody binding or inactivation. The parvovirus capsid is a strong multivalent antigen, which stimulates strong antibody responses. We have recently mapped the binding footprints of 8 separate antibody Fabs on the virion surface structure using cryoEM. Those antibodies covered a total of ~70% of the capsid surface, and could be divided into two major clusters of overlapping binding sites (termed "A" and "B"). The structural study identified two separate mechanisms of antibody neutralization. Although all 8 of the antibodies studied were neutralizing, the corresponding FAbs of only two of the antibodies showed efficient neutralization. Competition between the Fabs and the feline TfR was much more efficient for the highly neutralizing Fabs suggesting there is a different mechanism of blocking receptor attachment. The antigenic study is continuing using canine antibodies to assess neutralization, competition, and structure.

Minute virus of mice (MVM)

Capsids of MVM are comprised of approximately 90% VP2 and an additional 10 copies of VP1, which is an N-terminal extension of VP2. A five-fold portal through which these N-termini are externalized is formed by the juxtaposition of ten anti-parallel β-strands from five symmetry related capsid proteins. Genetic evidence supports the idea that the pore may also serve as the portal for packaging and expulsion of the viral genome. The tightest constriction in the fivefold pore is formed by VP2 leucine 172, located at the base of the pore on the interior. Analysis of MVM mutants showed only two viable mutants (L172I and L172V). All other substitutions at 172 resulted a defective mutant that was not able to establish an infection. The structure of L172W has shown a blockage of the pore, indicating that the channel along one of the fivefold symmetry axes is the packaging portal for the parvovirus genome. We are continuing the study of these "gate keeper" mutants by solving the structure of L172T.

The threonine substitution produces an infectious particle after transfection at 37°C, but these can initiate infection only at 32°C. Furthermore the process can be blocked by exposing virions to a cellular factor(s) at 37°C during the first 8 h after entry. At 32°C, the mutant particle is highly infectious, and it remains stable prior to VP2 cleavage or following cleavage at pH 5.5 or below. However, upon exposure to neutral pH following VP2 cleavage, its VP1-specific sequences and genome are extruded even at room temperature, underscoring the significance of the VP2 cleavage step for MVM particle dynamics.

Enterovirus

Little is known about the genetic determinants of pathogenicity of enteroviruses and to what extent the structural genes contribute, either by mediating receptor interaction or by directing some aspect of viral uncoating or disassembly. For instance it has been known since the 1950's that the B group of coxsackieviruses cause cardiomyopathy, but it is still unclear how much of the pathogenic phenotype is contributed by receptor usage. Coxsackieviruses have also been implicated in the onset of juvenile diabetes. Although there have been detailed studies with escape mutants, genotypic comparisons of clinical isolates, and very comprehensive binding assays more structural studies are needed to compliment the ongoing investigation of these pathogenic viruses.

Coxsackieviruses belong to the family Picornaviridae that contains some of the most common viral pathogens of vertebrates, causing human disease, ranging from mild upper respiratory illness to severe myocarditis and aseptic meningitis. All picornaviruses are small, icosahedral, nonenveloped animal viruses for which detailed atomic structures have been determined. Capsids have 60 copies each of four viral proteins, VP1, VP2, VP3 and VP4 that form an icosahedral shell ~300 Å in diameter filled with a positive-sense, single-stranded RNA genome. Prominent features of the capsid surface include a narrow depression around the five-fold axes of symmetry called the 'canyon'. The results of both genetic and structural studies have shown that the canyon is the site of receptor binding for many of these viruses including coxsackieviruses, which utilize coxsackievirus-adenovirus receptor (CAR). When these receptor molecules bind into the canyon, they dislodge a "pocket factor" from within a pocket immediately below the surface of the canyon. Absence of the hydrophobic pocket factor destabilizes the virus and initiates a global conformational change. This transition to altered "A" particles is a necessary prelude to uncoating of the virion.

Some picornaviruses, including certain coxsackie- and echoviruses, utilize decay-accelerating factor (DAF, or CD55) as a cellular co-receptor. For a confirmed DAF binding strain of CVB3 (rhabdomyosarcoma, or RD, strain) the major site of receptor interaction involves the 'puff', a region comprised of the largest and most variable loops on the surface of the virus. The DAF molecule interacts with the virus surface below the puff on one asymmetric unit, stretching across the virus surface to interact with the top of the puff in the adjacent asymmetric unit.

The cryoEM reconstruction of CVB3-RD complexed with full-length DAF showed that the receptor does not bind into the canyon as does CAR, but rather, lies across the surface of the virus. Because each DAF molecule has a C-terminal 6-His tag, there is also a somewhat artificial interaction at each three-fold of the coxsackievirus where calcium ions are usually coordinated. This interaction would not take place in vivo.

Related viruses have adapted to bind to DAF at different sites on the receptor surface, demonstrating that the use of DAF as a receptor or co-receptor may be a convergent process representing alternative evolutionary pathways among the same family members to interact with DAF and it implies that there is some advantage for the viruses to bind DAF. Furthermore, coxsackievirus adaptation to bind DAF alters pathogenicity of the virus. Mutations that have been linked to changes in pathogenicity map to the virus surface in clusters in the vicinity of the known DAF binding sites. There are many contributing factors to viral pathogenicity, including age of the host, the effect of the 5' non-translated region NTR, and the host immune response. The exact genetic determinants for virulence and the extent to which the structural genes may play a part, requires further investigation. We are continuing the structural study by solving the structures of CVB1 + DAF, and CVB3RD + CAR. The CVB3RD + CAR complex will be studied at different temperatures.

RSV Assembly

The Parent Lab has made significant progress in dissecting the biochemical basis of Gag-RNA interactions and the mechanism of nuclear trafficking of the RSV Gag protein. The Gag-RNA complexes we have obtained form dimers, tetramers, and hexamers in vitro that are well suited for analysis using cryo-electron microscopy. As a proof-of-principle for further structural analyses, we have optimized conditions to produce hexamers on EM grids, collected negatively stained data at Penn State Hershey, which have been reconstructed to provide low resolution reconstruction of hexemeric assembly subunits. EMAN was used to generate a starting model directly from the data. Reconstructions and control reconstructions were performed with and without imposing 6-fold symmetry. With these successful demonstrations of feasibility, we are ready to proceed to cryo-electron microscopy for data collection.

Ocean Sediment - In Collaboration with Christopher House

The ocean drilling programs (DSDP, ODP, and IODP) have yielded many successful studies of deeply buried microbes. To date, we do not fully understand the metabolic role of the marine subsurface bacteria or archaea. Because of the abundance and diversity in deeply-buried marine sediment, the metabolic characteristics of members of these groups are of wide interest for understanding the Earth's global microbial biosphere and the recycling of biomass that is deposited on the seafloor and buried. We are examining core samples for these presumably important Bacteria and looking for microbial cell bodies or inclusions that will allow us to infer their possible similarity to surface bacteria and archaea. We are also surveying the buried ultramicrobacteria of these environments to search for nanowire biostructures, which have recently been linked to microbial metal reduction, and for virus structures.