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.



Parvovirus


These viruses are 26nm in diameter, non-enveloped, T=1 icosahedral viruses that package a ssDNA genome. The goal of the parvovirus research is to define virus and host protein interactions and to explore host adaptation and species jumping.



Canine Parvovirus (CPV) + antibody


Canine parvovirus (CPV) CPV + FabEis a highly contagious pathogen that causes severe disease in dogs and wildlife. Previously, a panel of neutralizing monoclonal antibodies (MAb) raised against CPV was characterized. An antibody fragment (Fab) of MAb E was found to neutralize the virus at low molar ratios. Using recent advances in cryo-electron microscopy we determined the structure of CPV in complex with Fab E to 4.1 Å resolution, which allowed de novo building of the Fab structure. The footprint identified was significantly different than the footprint obtained previously from models fitted into lower resolution maps. The near atomic structure also revealed that Fab binding had caused capsid destabilization in regions containing key residues conferring receptor binding and tropism. This finding suggests a mechanism for efficient antibody neutralization of virus. Furthermore, a general technical approach for solving the structures of small molecules is demonstrated, as binding the Fab to the capsid allowed us to determine the 50kDa Fab structure by cryo-EM.



Picornavirus


Viruses that are classified in the family Picornaviridae are small (~30 nm diameter), icosahedral, non-enveloped viruses that contain a (+)-sense single-stranded RNA genome. Historically, the most well studied picornavirus has been poliovirus, the causative agent of poliomyelitis.


Coxsackievirus B3 (CVB3)


CVB3 Complex with CAR at 4 DegCoxsackievirus B3 (CVB3) is a member of the Picornavirdae family and is a causative agent of myocarditis, mengioencephilitis, and pancreatitis. In order to gain entry into the cell, the virus must bind to a receptor located on the cell membrane. CVB3 utilizes two receptors for entry into the cell: coxsackie and adenovirus receptor (CAR) and decay accelerating factor (DAF). CAR is the primary entry receptor is essential for viral infection. However, only certain stains of CVB3 are able to bind to DAF and it is not required for infection. Our lab studies the binding of these two receptors to CVB3 in order to determine their importance in the life cycle of the virus.

CVB3 Entry



Virus attachment to DAF activates cell-signaling cascades that allow the virus to be presented to CAR. The virus-CAR interaction causes the transformation of the virus into an A-particle. The N-terminus of VP1 forms an amphipathic helix, which inserts into the membrane6 and VP4 forms a pore through which the virus genome passes. The A-particle no longer binds CAR; however, DAF may remain bound and aid in later stages of entry.


Previous studies of picornavirus indicate that the depression surrounding the five-fold axis of symmetry is the location of entry receptor binding. A high resolution cryoEM reconstruction of virus-receptor complex was solved in order to refine the previously published receptor footprint for CVB3. A pseudo-atomic model was created by fitting the four available crystal structures of CAR individually into the electron density of the map. By doing so, we were able to create a consensus footprint for CAR. The footprint revealed two sites on the capsid where receptor bound: the elevated “puff” region and the Northern rim of the canyon. A comparison of the CAR binding residues of CVB3 to the receptor binding residues of other picornaviruses found the binding sites are conserved across species even though they utilize different molecules. Major antigenic sites of rhinovirus and poliovirus overlap with all but one binding site.



CAR induced A particleCAR induces changes in the native virus that prepares the virus for the release of genome and viral replication. The resulting particle is referred to as the altered or A-particle. A 6.1Å cryo-EM reconstruction of A-particle was solved for CVB3. The A-particle is expanded compared to native particle and has a pore at the two-fold axis of symmetry.


CVB3 + Car + NanodiscUntil recently the entry mechanism of the picornavirus has been studied by using different forms of entry intermediates (or A-particles), that were induced by global- or local-stimulation. The globally-stimulated A-particles (classical A-particles) have generated high-resolution 3D structures by imposing icosahedral symmetry. Until recently, the 3D structures of the locally-stimulated A-particles had remained at low resolution although this form may represent a physiologically truer entry intermediate. A cryo-EM reconstruction for coxsackievirus B3 incubated with nano discs embedded with full-length receptors demonstrated how locally-stimulated A-particle differs from the classical A-particle. The icosahedrally averaged map was solved at 3.9 Å and enabled a de novo building of the atomic model. Breaking the symmetry imposed from the icosahedral reconstruction allowed us to examine the asymmetric features of the novel A-particle at atomic resolution.


Picornavirus Entry Model

A bunch of random text filler.



Enterovirus 71 (EV71)


Recently, much research focus has shifted to another member of this virus family, Enterovirus 71 (EV71), which is causing seasonal epidemics in the Asia-Pacific region. EV71 infections are most commonly mild and self-limiting causing hand, foot, and mouth disease. In a small subset of infected individuals the virus can progress through other disease stages that involve CNS infection, compromise of cardio-pulmonary function, convalescence, and even death. Alarmingly, the most severe cases of EV71 infection occur in young children and there is no vaccine or treatment available.


EV71 Drug + Peptide


EV71Enterovirus 71 (EV71) causes hand, foot, and mouth disease (HFMD) and sometimes polio-like symptoms. P-selectin glycoprotein ligand-1 (PSGL-1) is an initial attachment receptor of EV71 and has been linked with severe disease of the virus infection. Although some of the receptor binding viral residues have been identified, the exact PSGL-1 binding site on the capsid and molecular mechanism of the attachment were unknown. The atomic resolution cryo-EM structure combined with localized-reconstruction method and molecular docking simulation mapped the binding site on the 5-fold vertex and revealed the binding mechanism with fast on-/off-rates. The method also identified the binding mode of NF449, a "ready-to-go" drug for EV71 infection, that competes for a binding site with PSGL-1.



Human Paillomavirus (HPV)


Human papillomaviruses (HPVs) can cause warts on cutaneous epithelium, while in the anogenital region these viruses can cause both genital warts and various forms of cancer in men or woman. There are over 100 distinct HPV genotypes being identified so far, some of which are carcinogenic, for example, HPV16, 18, 31, 33 and 35. Although the recently developed vaccines protect younger people from two major carcinogenic HPV types (HPV16 and 18), there is still a big demand on broad-spectrum vaccines to protect people from full range of cancer-causing HPVs. Development of such vaccines will be facilitated by deeper understanding of infectious mechanics together with the conformational changes in virus structure. Neutralizing antibodies are one of most important tools to do this job. Identification of neutralization-sensitive epitopes on the capsid protein structures (conformational epitopes) may promote the understanding of the entry pathway and development of improved recombinant vaccines.