Request ID: cfg_rRequest_2224

Status : Approved
Project Description : Continued studies RR#1875 - to screen mutants at position 399, which appears to have a role in host tropism.
CFG Member : Member
Requester First Name : Mavi
Requester Last Name : Agbandje-McKenna
Head of Lab First Name : Mavis
Head of Lab Last Name : Agbandje-McKenna
Assigned First Name : Mavis
Assigned Last Name : Agbandje-McKenna
Requester Email : mckenna@ufl.edu
Requester Interest : This laboratory focuses on structure-function correlations for virus capsids, particularly, ssDNA capsids, with a T=1 icosahedral symmetry. The goal is to develop strategies for the treatments of diseases of that afflict plants, animals and humans in the form assembly disrupters, vaccines, and antigen and gene delivery vectors.
Request Date [yyyy-mm-dd] : 2010-12-18
Institution : University of Florida
Shipping Address : Information not entered/not applicable.
Comments : This is a follow on to cfg_rRequest_1875.
CFG Core : H
Resource Type : Glycoarray
Amount Requested : Information not entered/not applicable.
Date that your RNA/GBP samples will be sent to the core [yyyy-mm-dd] : 2011-01-04
Experiment to be conducted : We are requesting the screening of the binding specificity and affinity of Minute Virus of Mice (MVM) capsids and its mutants on the Consortium for Functional Glycomic’s (CFG’s) printed glycan array through core H. MVM belongs to the Parvovirus genus of Parvoviridae which includes several autonomously replicating members. Two strains of MVM, MVMp (the prototype strain) and MVMi (the immunosuppressive strain), have different cellular tropisms and are non-pathogenic and pathogenic, respectively, in vivo. These properties have been mapped to one or two amino acids (out of 587) that differ between these two virus strains. We have shown that differential glycan array likely plays a role in the tropism and pathogenicity disparities between MVMi and MVMp and have mapped the glycan binding sites to capsid surface adjacent to the residues controlling these phenotypes. We previously screened wild type (wt) and mutant MVM capsids on an old version of the CFG array, the mammalian plate array version 3 (Nam et al, 2006, JBC, 281:25670-25677) and more recently on the printed array version 4.1 through request cfg_rRequest_1875. Through collaboration with CFG core D we have obtained glycans identified in the arrays for further structural characterization of the receptor binding site(s) in the MVM capsid. We have also been able to correlate the results obtained from the glycan array screens with the distribution of the glycans on cells permissive for MVM infection through collaboration with CFG core C’s glycan profiling of three different cell lines. The wealth of information available for MVM’s glycan interactions and our ability to correlate these with the virus’ natural host infection and tropism has resulted in its selection as one of the CFG’s paradigm GBPs for non-enveloped virus capsids. Towards completing the milestones set forth by the CFG to fully define the role of glycans in cellular communication it’s selected GBPs we wish to determine the glycan binding specificity of a number of MVM capsid mutants with minor changes in non-structural as well as structural proteins which play a role in host tropism and pathogenicity. Specifically, we wish to screen mutations (8 in total) at capsid viral protein position 399 which appears to coordinate with non-coding changes in the viral gene to changes in host tropism alongside the wt viruses. The 1st screening of two mutants in CFG array v4.1 indicated that changes at this capsid position eliminated recognition of multi-sialylated glycans by MVM. We wish to confirm these results and pinpoint the combination of changes conferring this phenotype. In addition, we are interested in determining whether or not there is a difference between the recognition and affinity of binding between MVM capsids with a packaged genome (virions), those that are empty (and contain the wt VP1 and VP2) and those that are virus-like particles (VLPs) containing only VP2 produced in insect cells. This information is important for verification that our structural studies using VP2 VLPs complex with glycans identified in the CFG arrays recapitulate the interactions of infectious virions with host cells. This study was a component of cfg_rRequest_1875 but was not completed because at the time these studies were conducted we did not have enough virion samples.
Within Scope of Consortium : Y
If yes, indicate the person responsible for inputing data into core B : David Smith
GBP being Addressed : The samples will be detected with antibodies which we will supply.
Specifc aims being addressed : Define the specificity and affinity for carbohydrate ligands. Identify the ligand(s) that mediate GBP binding. Determine how GBP-ligand interactions mediate cell communication.