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Cloning, Molecular↗
Biomedical subjects
D R Wessner
Publications and source records attributed to D R Wessner.
Neutralization of MHV-A59 by soluble recombinant receptor glycoproteins.
The interaction of viruses with specific receptors is an important determinant of viral tissue tropism and species specificity. Our goals are to understand how mouse hepatitis virus (MHV) recognizes its cellular receptor, MHVR, and how post-binding interactions with this receptor influence viral fusion and entry. Murine cells express a variety of cell surface molecule in the biliary glycoprotein (Bgp) family that are closely related to the MHVR. When these proteins are expressed at high levels in cell culture, they function as MHV receptors. We used a baculovirus expression system to produce soluble recombinant murine Bgp receptors in which the transmembrane and cytoplasmic domains have been replaced with a six-histidine tag. The soluble glycoproteins were purified to apparent homogeneity and shown to react with antisera to the native receptor. We compared the virus neutralizing activities of various soluble receptor glycoproteins. Soluble MHVR [sMHVR(1-4)] had 10-20 fold more virus neutralizing activity the soluble protein derived from the Bgp1b glycoprotein [sBgp1b(1-4)], from MHV-resistant SJL mice. The sMHVR(1-4) glycoprotein was 60-100 fold more active than a truncated receptor molecule containing only the first two immunoglobulin-like domains, sMHVR(1,2). The observation that sMHVR lacking domains 3 and 4 neutralizes MHV-A59 very poorly suggests that these domains may influence virus binding or subsequent steps associated with neutralization.
Animals↗
Mutational analysis of the virus and monoclonal antibody binding sites in MHVR, the cellular receptor of the murine coronavirus mouse hepatitis virus strain A59.
The primary cellular receptor for mouse hepatitis virus (MHV), a murine coronavirus, is MHVR (also referred to as Bgp1a or C-CAM), a transmembrane glycoprotein with four immunoglobulin-like domains in the murine biliary glycoprotein (Bgp) subfamily of the carcinoembryonic antigen (CEA) family. Other murine glycoproteins in the Bgp subfamily, including Bgp1b and Bgp2, also can serve as MHV receptors when transfected into MHV-resistant cells. Previous studies have shown that the 108-amino-acid N-terminal domain of MHVR is essential for virus receptor activity and is the binding site for monoclonal antibody (MAb) CC1, an antireceptor MAb that blocks MHV infection in vivo and in vitro. To further elucidate the regions of MHVR required for virus receptor activity and MAb CC1 binding, we constructed chimeras between MHVR and other members of the CEA family and tested them for MHV strain A59 (MHV-A59) receptor activity and MAb CC1 binding activity. In addition, we used site-directed mutagenesis to introduce selected amino acid changes into the N-terminal domains of MHVR and these chimeras and tested the abilities of these mutant glycoproteins to bind MAb CC1 and to function as MHV receptors. Several recombinant glycoproteins exhibited virus receptor activity but did not bind MAb CC1, indicating that the virus and MAb binding sites on the N-terminal domain of MHVR are not identical. Analysis of the recombinant glycoproteins showed that a short region of MHVR, between amino acids 34 and 52, is critical for MHV-A59 receptor activity. Additional regions of the N-terminal variable domain and the constant domains, however, greatly affected receptor activity. Thus, the molecular context in which the amino acids critical for MHV-A59 receptor activity are found profoundly influences the virus receptor activity of the glycoprotein.
Amino Acid Sequence↗
Purified, soluble recombinant mouse hepatitis virus receptor, Bgp1(b), and Bgp2 murine coronavirus receptors differ in mouse hepatitis virus binding and neutralizing activities.
Mouse hepatitis virus receptor (MHVR) is a murine biliary glycoprotein (Bgp1(a)). Purified, soluble MHVR expressed from a recombinant vaccinia virus neutralized the infectivity of the A59 strain of mouse hepatitis virus (MHV-A59) in a concentration-dependent manner. Several anchored murine Bgps in addition to MHVR can also function as MHV-A59 receptors when expressed at high levels in nonmurine cells. To investigate the interactions of these alternative MHVR glycoproteins with MHV, we expressed and purified to apparent homogeneity the extracellular domains of several murine Bgps as soluble, six-histidine-tagged glycoproteins, using a baculovirus expression system. These include MHVR isoforms containing four or two extracellular domains and the corresponding Bgp1(b) glycoproteins from MHV-resistant SJL/J mice, as well as Bgp2 and truncation mutants of MHVR and Bgp1(b) comprised of the first two immunoglobulin-like domains. The soluble four-domain MHVR glycoprotein (sMHVR[1-4]) had fourfold more MHV-A59 neutralizing activity than the corresponding soluble Bgp1(b) (sBgp1(b)) glycoprotein and at least 1,000-fold more neutralizing activity than sBgp2. Although virus binds to the N-terminal domain (domain 1), soluble truncation mutants of MHVR and Bgp1(b) containing only domains 1 and 2 bound virus poorly and had 10- and 300-fold less MHV-A59 neutralizing activity than the corresponding four-domain glycoproteins. In contrast, the soluble MHVR glycoprotein containing domains 1 and 4 (sMHVR[1,4]) had as much neutralizing activity as the four-domain glycoprotein, sMHVR[1-4]. Thus, the virus neutralizing activity of MHVR domain 1 appears to be enhanced by domain 4. The sBgp1(b)[1-4] glycoprotein had 500-fold less neutralizing activity for MHV-JHM than for MHV-A59. Thus, MHV strains with differences in S-glycoprotein sequence, tissue tropism, and virulence can differ in the ability to utilize the various murine Bgps as receptors.
3T3 Cells↗
A common-source outbreak of callitrichid hepatitis in captive tamarins and marmosets.
Callitrichid hepatitis (CH) is a highly fatal, emerging arenavirus disease of captive South American marmosets and tamarins (Callitrichidae), including the endangered golden lion tamarin. A common-source outbreak of CH in golden lion tamarins and pygmy marmosets at a US zoo resulted from a single feeding of the primates with newborn mice in apparently infected with lymphocytic choriomeningitis virus (LCMV). Isolates from livers of mice and primates were related to isolates from previous CH outbreaks and to laboratory strains of LCMV by serology and nucleic acid hybridization, and 2 surviving animals developed antibody to other LCMVCH isolates and to laboratory strains of LCMV. Thus, LCMV, an arenavirus prevalent in wild mice in the US, can cause sporadic fatal hepatic disease in primates. Exposure of humans to wild or laboratory mice or to marmosets and tamarins that are infected with wild-type strains of LCMV poses the danger of serious disease.
Animals↗
Isolation and genetic characterization of ethanol-resistant reovirus mutants.
To better understand the mechanism(s) by which viruses respond to chemical or physical treatments, we isolated a series of mutant strains of reovirus type 3 Dearing that exhibit increased ethanol resistance. Following exposure to 33% ethanol for 20 min, the parental strain exhibited a 5 log10 decrease in infectivity. The mutant strains, however, exhibited a 2 to 3 log10 decrease in titer following identical treatment. Through the use of reassortant viruses, we mapped this increased ethanol resistance mutation to the M2 gene segment, which encodes a major outer capsid protein, mu1C. Sequence analysis of mutant M2 genes revealed that six of seven unique mutants possessed single-point mutations in this gene. In addition, the change in six of seven mutants caused a predicted amino acid change in a 35-amino-acid region of the gene product between amino acids 425 and 459. The identification of ethanol resistance mutations within a discrete region of this outer capsid protein identifies that portion of the protein as important in reovirus stability. The presence of viral particles possessing altered stability also suggests that subpopulations of viruses may possess altered environmental stability, which, in turn, could affect viral transmission.
Animals↗