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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.

Murine coronavirus MHV-A59 normally infects only murine cells in vitro and causes transmissible infection only in mice. In the 17 C1 1 line of murine cells, the receptor for MHV-A59 is MHVR, a biliary glycoprotein in the carcinoembryonic antigen (CEA) family of glycoproteins. We found that virus released from the 600th passage of 17 C1 1 cells persistently infected with MHV-A59 (MHV/pi600) replicated in hamster (BHK-21) cells. The virus was passaged and plaque-purified in BHK-21 cells, yielding the MHV/BHK strain. Because murine cells persistently infected with MHV-A59 express a markedly reduced level of MHVR (Sawicki, et al., 1995), we tested whether virus with altered receptor interactions was selected in the persistently infected culture. Infection of 17 C1 1 cells by MHV-A59 can be blocked by treating the cells with anti-MHVR MAb-CC1, while infection by MHV/BHK was only partially blocked by MAb-CC1. MHV/BHK virus was also more resistant than wild-type MHV-A59 to neutralization by purified, recombinant, soluble MHVR glycoprotein (sMHVR). Cells in the persistently infected culture may also express reduced levels of and have altered interactions with some of the Bgp-related glycoproteins that can serve as alternative receptors for MHV-A59. Unlike the parental MHV-A59 which only infects murine cells, MHV/BHK virus was able to infect cell lines derived from mice, hamsters, rats, cats, cows, monkeys and humans. However, MHV/BHK was not able to infect all mammalian species, because a pig (ST) cell line and a dog cell line (MDCK I) were not susceptible to infection. MHV/pi600 and MHV/BHK replicated in murine cells more slowly than MHV-A59 and formed smaller plaques. Thus, in the persistently infected murine cells which expressed a markedly reduced level of MHVR, virus variants were selected that have altered interactions with MHVR and an extended host range. In vivo, in mice infected with coronavirus, virus variants with altered receptor recognition and extended host range might be selected in tissues that have low levels of receptors. Depending upon the tissue in which such a virus variant was selected, it might be shed from the infected animal or eaten by a predator, thus presenting a possible means for initiating the transition of a variant virus into a new host as a model for an emerging virus disease.

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.

Utilizing the reverse transcriptase-linked polymerase chain reaction, we analyzed the capacity of three groups of rat conceptal tissues to express cytochrome P4501A1 (CYP1A1) mRNA during the dysmorphogenesis-sensitive stage of organogenesis. The visceral yolk sac, ectoplacental cone and embryo proper each were investigated on day 12 of gestation with and without prior exposure in utero to 3-methylcholanthrene as inducing agent. With two sets of discriminating oligonucleotide primers, definitive, reproducible signals were detectable only in tissues from 3-methylcholanthrene preexposed conceptuses. Signals of highest intensity were observed with visceral yolk sac tissues and signals of lowest intensity were observed with tissues of the embryo per se. Specificities of the amplified cDNAs were verified using Southern blotting with hybridization to an internal oligonucleotide probe. The results indicate that organogenesis-stage conceptual tissues of the rat will express CYP1A1 mRNA in response to environmental transregulating agents.

Xenopus membrane skeleton protein 4.1 is expressed constitutively during embryonic development and accumulates to high levels within the retina during normal morphogenesis. There exists a high degree of amino acid identity between Xenopus protein 4.1 and human protein 4.1, suggesting that the mechanisms known to modulate the function(s) of human protein 4.1 may also serve to regulate Xenopus protein 4.1. Calmodulin (CaM) is one regulatory protein known to affect membrane-cytoskeletal interactions. An in vitro binding assay was used to test the ability of Xenopus protein 4.1 to bind CaM. Two independent approaches, involving protein 4.1 synthesized in vitro from synthetic RNA or a partial length protein 4.1 fusion protein expressed in Escherichia coli, demonstrate calcium-dependent, CaM binding. Both approaches demonstrate that the CaM-binding site is within the amino-terminal region of Xenopus protein 4.1. Results of this calmodulin binding activity suggest a possible regulatory mechanism by which calcium and calmodulin may affect the function of protein 4.1 during development.