Wang, Kang-Sheng (1991) Molecular characterization of a receptor for the Togavirus Sindbis virus. Dissertation (Ph.D.), California Institute of Technology. http://resolver.caltech.edu/CaltechTHESIS:04112011-142633680
The first step in any virus entry process is binding to the plasma membrane of the host cell. The nature of this obligatory step depends upon both the viral and cellular components and may be quite diverse among viruses. The entry of Sindbis virus into a host cell is reported to occur via receptor-mediated endocytosis. We have used several approaches to isolate and characterize the receptor(s) for Sindbis virus. In one such approach, we searched for monoclonal antibodies (mAb) that could interfere with Sindbis virus attachment to and infection of baby hamster kidney (BHK) cells. Mice were immunized with multiple injections of whole BHK cells or with BHK cell membranes. Hybridomas were prepared and supernatants from approximately 3600 hybridoma clones were screened by a plaque reduction assay for their ability to interfere with virus infection. One IgM mAb from a mouse immunized with whole BHK cells inhibited Sindbis virus attachment to BHK cells by 80% at 20 µg/ml, and immunoprecipitated a 68 kDal membrane protein from BHK cells. This mAb also inhibits virus attachment to two other mammalian cell lines tested, Vero cells (monkey) and SW13 cells (human), and also immunoprecipitates a 68 Kd protein from these cells. The mAb does not interfere with virus infection of chicken cells but did immunoprecipitate a 71 kDal protein from chicken cells. This mAb was used to screen 10^6 plaques from a λgt11 cDNA library from BHK cells, and 15 reactive phages were found. Six of the fifteen were shown by sequence analysis to react with overlapping regions of a protein that was identical in sequence to the mouse high affinity laminin receptor. By rescreening with a probe from one of these reactive phages, other lambda phages containing the remaining regions of the gene were found. The complete sequence of this protein was deduced by sequence analysis of the cDNA clones and was identical to that of the mouse laminin receptor and 99% identical to the human laminin receptor. A full length cDNA clone of the gene was constructed and inserted into a high efficiency expression vector. BHK cell lines stably transfected with vector expressing the plus sense BHK laminin receptor cDNA are 3-5 fold more susceptible to infection by Sindbis virus as measured by plaque assay, and overexpress the receptor protein on their surface as assayed by flow cytometry analysis. Conversely, cell lines transfected with vector expressing antisense laminin receptor cDNA are only about one half as susceptible to infection by Sindbis virus as the nontransformed BHK cells, and expression of laminin receptor on the cell surface is reduced as measured by flow cytometry analysis. In a second study we looked for Sindbis virus receptors on the surface of chicken cells, using specific molecular mimicry to identify receptor molecules. It has been postulated that viral receptors may share structural features (idiotypes) with antibodies directed against the cell attachment protein of virus. Using antiidiotypic antibodies directed against Sindbis-specific neutralization antibodies, we have demonstrated that an antiidiotypic antibody to a neutralizing mAb reactive with the E2 glycoprotein of Sindbis virus specifically interferes with the binding of wild type Sindbis virus to chicken cells. This antiidiotypic antibody also immunoprecipitates a 63 kDal protein from chicken cells and binds to the surface of these cells. This 63 kDal protein is presumably a receptor for Sindbis virus in chicken cells. The relationship between this protein and the laminin receptor used as a Sindbis receptor in mammalian cells remains to be determined. We also wished to determine the domains of the virus envelope proteins that are responsible for attachment to the cell membrane. The Sindbis virus envelope contains two species of integral membrane glycoproteins, El and E2, which assemble into heterodimers. Each spike on the surface of the virion is a trimer of these dimeric units. We attempted to map the neutralization epitopes on the surface of the virus, including epitopes implicated in virus binding to cells by the antiidiotypic antibody results described above. A λgt11 expression library was constructed containing cDNA inserts 100-300 nucleotides in length obtained by randomly primed synthesis on Sindbis genomic RNA. This library was probed with several neutralizing monoclonal antibodies specific for E2 and one neutralizing antibody specific for El. Four positive clones, all of which contained inserts from the region of the Sindbis genome that encodes amino acids 173 to 220 of glycoprotein E2, were found from the screening with mAb 23. No reactive clones could be identified using any of the other antibodies. We hypothesize that this domain of E2 centered at residue 200 forms part of the virus binding site for attachment to the cell to initiate infection.
|Item Type:||Thesis (Dissertation (Ph.D.))|
|Degree Grantor:||California Institute of Technology|
|Thesis Availability:||Restricted to Caltech community only|
|Defense Date:||22 May 1991|
|Default Usage Policy:||No commercial reproduction, distribution, display or performance rights in this work are provided.|
|Deposited By:||Tony Diaz|
|Deposited On:||11 Apr 2011 21:57|
|Last Modified:||26 Dec 2012 04:33|
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