Autonomous parvoviruses are known to infect a wide variety of vertebrate animals and are frequently highly pathogenic. Moreover, a human parvovirus, B19, has recently been identified which has been associated with several clinical disorders including """"""""fifth disease"""""""", aplastic crises associated with hemolytic anemias, post infection arthropathy and possibly hydrops fetalis. Each autonomous parvovirus has a restricted host range. As an approach to study the mechanism of host-range restriction for autonomous parvoviruses, we will investigate the initial steps of virus- cell interaction using canine parvovirus (CPV) as a model. Evidence for a specific receptor for this virus has been obtained in a polarized epithelial cell. To obtain preliminary information on the biochemical nature of the receptor, we will treat cells with enzymes (proteases, lipases, glycosidases) followed by a radiolabelled virus binding assay. If the receptor is a polypeptide, the polypeptide components involved in the receptor structure will be identified by using a solid-phase 125I-virus binding assay on western blots of the crude membrane preparation or the octyl-beta-D-glucopyranoside (OG) extract of the membrane proteins. Alternately, immunoprecipitation of the polypeptides using purified CPV, antiserum to CPV and protein-A-agarose should also identify the polypeptide components. Further studies of the receptor will be done by developing monoclonal antibodies against the specific cell membrane components recognized by the virion. These antibodies will be used to determine the number and distribution of receptors, both on cultured cells and in canine intestinal epithelium, as well as for further purification of the receptor by affinity chromatography. If lipids appear to be the receptor, liposomes containing various lipids will be used in competition for virus binding to the receptor. On the other hand if proteoglycans seem to be involved in the receptor structure, we will investigate the inhibition of virus binding by proteoglycan moieties. The second major objective is to examine the nature of the processes which are involved in the internalization of virus and transport of the virus or viral genome to the nucleus. The effect of various lysosomotropic agents on viral entry will be determined by infectious center assays and by quantitating viral DNA synthesis. Ultrastructural studies will be carried out to investigate the uptake and localization of the virion inside the cell in the presence of these agents. Transport of the virions within the cell will be also monitored by studying the uptake of biotinylated virus. The infectivity of virions introduced directly into the cell cytoplasm will be studied to determine whether the infective pathway for CPV is dependent on vacuolar uptake. The effect of cytoskeletal inhibitors on virus entry, and the fate of input virus in the presence or absence of inhibitors, will be determined. These studies should provide important information on the early stages of parvovirus-host cell interaction.
| Basak, S; Turner, H; Parr, S (1994) Identification of a 40- to 42-kDa attachment polypeptide for canine parvovirus in A72 cells. Virology 205:7-16 |
| Basak, S; Turner, H; Compans, R W (1992) Expression of SV40 receptors on apical surfaces of polarized epithelial cells. Virology 190:393-402 |
