Comparison of expression patterns and cell adhesion properties of the mouse biliary glycoproteins Bbgp1 and Bbgp2.

Biliary glycoproteins are members of the carcinoembryonic antigen (CEA) family and behave as cell adhesion molecules. The mouse genome contains two very similar Bgp genes, Bgp1 and Bgp2, whereas the human and rat genomes contain only one BGP gene. A Bgp2 isoform was previously identified as an alternative receptor for the mouse coronavirus mouse hepatitis virus. This isoform consists of two extracellular immunoglobulin domains, a transmembrane domain and a cytoplasmic tail of five amino acids. In this report, we have examined whether the Bgp2 gene can express other isoforms in different mouse tissues. We found only one other isoform, which has a long cytoplasmic tail of 73 amino acids. The long cytodomain of the Bgp2 protein is highly similar to that of the Bgp1/4L isoform. The Bgp2 protein is expressed in low amounts in kidney and in a rectal carcinoma cell line. Antibodies specific to Bgp2 detected a 42-kDa protein, which is expressed at the cell surface of these samples. Bgp2 was found by immunocytochemistry in smooth muscle layers of the kidney, the uterus, in gut mononuclear cells and in the crypt epithelia of intestinal tissues. Transfection studies showed that, in contrast with Bgp1, the Bgp2 glycoprotein was not directly involved in intercellular adhesion. However, this protein is found in the proliferative compartment of the intestinal crypts and in cells involved in immune recognition. This suggests that the Bgp2 protein represents a distinctive member of the CEA family; its unusual expression patterns in mouse tissues and the unique functions it may be fulfilling may provide novel clues about the multiple functions mediated by a common BGP protein in humans and rats.

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.

Selection in persistently infected murine cells of an MHV-A59 variant with extended host range.

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.

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.

The murine coronavirus mouse hepatitis virus strain A59 from persistently infected murine cells exhibits an extended host range.

In murine 17 Cl 1 cells persistently infected with murine coronavirus mouse hepatitis virus strain A59 (MHV-A59), expression of the virus receptor glycoprotein MHVR was markedly reduced (S. G. Sawicki, J. H. Lu, and K. V. Holmes, J. Virol. 69:5535-5543, 1995). Virus isolated from passage 600 of the persistently infected cells made smaller plaques on 17 Cl 1 cells than did MHV-A59. Unlike the parental MHV-A59, this variant virus also infected the BHK-21 (BHK) line of hamster cells. Virus plaque purified on BHK cells (MHV/BHK) grew more slowly in murine cells than did MHV-A59, and the rate of viral RNA synthesis was lower and the development of the viral nucleocapsid (N) protein was slower than those of MHV-A59. MHV/BHK was 100-fold more resistant to neutralization with the purified soluble recombinant MHV receptor glycoprotein (sMHVR) than was MHV-A59. Pretreatment of 17 Cl 1 cells with anti-MHVR monoclonal antibody CC1 protected the cells from infection with MHV-A59 but only partially protected them from infection with MHV/BHK. Thus, although MHV/BHK could still utilize MHVR as a receptor, its interactions with the receptor were significantly different from those of MHV-A59. To determine whether a hemagglutinin esterase (HE) glycoprotein that could bind the virions to 9-O-acetylated neuraminic acid moieties on the cell surface was expressed by MHV/BHK, an in situ esterase assay was used. No expression of HE activity was detected in 17 Cl 1 cells infected with MHV/BHK, suggesting that this virus, like MHV-A59, bound to cell membranes via its S glycoprotein. MHV/BHK was able to infect cell lines from many mammalian species, including murine (17 Cl 1), hamster (BHK), feline (Fcwf), bovine (MDBK), rat (RIE), monkey (Vero), and human (L132 and HeLa) cell lines. MHV/BHK could not infect dog kidney (MDCK I) or swine testis (ST) cell lines. Thus, in persistently infected murine cell lines that express very low levels of virus receptor MHVR and which also have and may express alternative virus receptors of lesser efficiency, there is a strong selective advantage for virus with altered interactions with receptor (D. S. Chen, M. Asanaka, F. S. Chen, J. E. Shively, and M. M. C. Lai, J. Virol. 71:1688-1691, 1997; D. S. Chen, M. Asanaka, K. Yokomori, F.-I. Wang, S. B. Hwang, H.-P. Li, and M. M. C. Lai, Proc. Natl. Acad. Sci. USA 92:12095-12099, 1995; P. Nedellec, G. S. Dveksler, E. Daniels, C. Turbide, B. Chow, A. A. Basile, K. V. Holmes, and N. Beauchemin, J. Virol. 68:4525-4537, 1994). Possibly, in coronavirus-infected animals, replication of the virus in tissues that express low levels of receptor might also select viruses with altered receptor recognition and extended host range.

Virus-receptor interactions in the enteric tract. Virus-receptor interactions.

Expression of specific virus receptors on the surface of intestinal epithelial cells or M cells can determine whether or not a animal is susceptible to infection with an enterotropic virus. Receptors for many animal viruses have been identified. The specificity of virus-receptor interactions clearly affects the species specificity of virus infection, and in some instances may be an important determinant of viral tissue tropism. In this paper, the specificity of coronavirus-receptor interactions is summarized. Porcine and human coronaviruses utilize aminopeptidase N as their receptors, but in a species-specific manner. Mouse hepatitis virus uses several rodent glycoproteins in the carcinoembryonic antigen family as receptors. In addition, some coronaviruses can interact with carbohydrate moieties on the cell surface. Understanding the molecular mechanisms of virus-receptor interactions may lead to development of novel strategies for the control of enteric viral diseases.

The virion host shutoff protein of herpes simplex virus type 1: messenger ribonucleolytic activity in vitro.

Shortly after tissue culture cells are infected with herpes simplex virus (HSV) type 1 or 2, the rate of host protein synthesis decreases 5- to 10-fold and most host mRNAs are degraded. mRNA destabilization is triggered by the virion host shutoff (vhs) protein, a virus encoded, 58-kDa protein located in the virion tegument. To determine whether it can function as a messenger RNase (mRNase), the capacity of vhs protein to degrade RNA in vitro in absence of host cell components was assessed. Two sources of vhs protein were used in these assays: crude extract from virions or protein translated in a reticulocyte-free system. In each case, wild-type but not mutant vhs protein degraded various RNA substrates. Preincubation with anti-vhs antibody blocked RNase activity. These studies do not prove that vhs protein on its own is an mRNase but do demonstrate that the protein, either on its own or in conjunction with another factor(s), has the biochemical property of an mRNase, consistent with its role in infected cells.

Organogenesis-stage cytochrome P450 isoforms: utilization of PCR for detection of CYP1A1 mRNA in rat conceptal tissues.

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.

Identification of a calcium-dependent calmodulin-binding domain in Xenopus membrane skeleton protein 4.1.

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.

High concentrations of a cGMP-stimulated phosphodiesterase mediate ANP-induced decreases in cAMP and steroidogenesis in adrenal glomerulosa cells.

An adrenal cGMP-stimulated phosphodiesterase (cGS-PDE) has been shown to mediate atrial natriuretic peptide (ANP)-induced reductions in aldosterone secretion and cAMP levels in primary bovine glomerulosa cells. High concentrations of cGS-PDE have been localized to the zona glomerulosa cell layer of the adrenal cortex using biochemical and immunological techniques. Immunoblot analysis using an affinity-purified, isozyme-specific antiserum revealed a single band that comigrated with a purified cGS-PDE (105 kDa) (1) and that was most highly concentrated in the outermost 1-2 mm of the cortex, representing the capsule and zona glomerulosa regions. Greater than 90% of the overall phosphodiesterase activity present in tissue extracts prepared from these regions was immunoprecipitated using a solid-phase monoclonal antibody reagent, indicating the cGS-PDE as the predominant phosphodiesterase isozyme. Immunohistochemical staining experiments of frozen thin sections of intact adrenal tissue revealed that the cGS-PDE present in this region was localized in the glomerulosa cells themselves. The role of this isozyme as a mediator of ANP-induced decreases in intracellular cAMP concentrations and aldosterone production was tested in primary cultures of bovine adrenal glomerulosa cells. In cells stimulated by ACTH, ANP treatment produced dose-dependent reductions in aldosterone secretion and cellular cAMP content over the same concentration range. Increases in aldosterone production elicited by three cell-permeable cAMP derivatives (8-bromo-cAMP, 8-p-chlorophenylthio-cAMP, and N6-2'-O-dibutyryl-cAMP) were antagonized by ANP, indicating a site of action distal to adenylate cyclase for this hormone. Because the relative magnitude of the ANP effect differed depending upon the derivative used, the three derivatives were compared with respect to their relative rates of in vitro hydrolysis by adrenal cGS-PDE. A positive correlation between their rates of hydrolysis and the degree to which the steroidogenic response produced by these derivatives was antagonized by ANP was demonstrated, further suggesting an ANP-induced activation of the cGS-PDE as being responsible for this effect. The possible contribution of an additional pathway mediated by an inhibitory guanine nucleotide binding regulatory protein (Gi) acting on adenylate cyclase was tested by pretreatment of primary glomerulosa cells with pertussis toxin. Levels of pertussis toxin sufficient to inhibit subsequent in vitro ribosylation did not significantly alter the ANP effect on aldosterone production, although a partial reduction in the ANP effect on cAMP levels was observed.(ABSTRACT TRUNCATED AT 400 WORDS)

Expression of the poly(A)-binding protein during development of Xenopus laevis.

We have isolated and sequenced cDNA clones encoding the poly(A)-binding protein of Xenopus laevis oocytes. Polyclonal antiserum was raised against a fusion protein encoding 185 amino acids of the Xenopus poly(A)-binding protein. This antiserum localizes the poly(A)-binding protein to subcellular sites associated with protein synthesis; in the retina, immunoreactive protein is detected in the synthetically active inner segment of the photoreceptor but not in the transductive outer segment. Transcripts encoding the poly(A)-binding protein are present in oocytes, although no protein is detected on protein blots. In contrast, the levels of both transcripts and protein increase in development, which correlates with the observed increase in total poly(A) during Xenopus embryogenesis (N. Sagata, K. Shiokawa, and K. Yamana, Dev. Biol. 77:431-448, 1980).

Changes in the expression of alpha-fodrin during embryonic development of Xenopus laevis.

Fodrin (nonerythroid spectrin) and its associated proteins have been previously implicated in the establishment of specialized membrane-cytoskeletal domains in differentiating cells. Using antiserum which is monospecific for the alpha-subunit of fodrin, we demonstrate that alpha-fodrin is present in oocytes and adult tissues of Xenopus laevis. Analyses of the de novo synthesis of alpha-fodrin during embryonic development reveal that alpha-fodrin is synthesized in oocytes, but not during early development. To investigate the level of control of alpha-fodrin expression, we isolated two cDNA clones for oocyte alpha-fodrin. The oocyte cDNA clones were identified as encoding portions of alpha-fodrin based on DNA sequence analysis and on the comparison of the predicted amino acid sequence of the cDNAs with the known sequence of human erythrocyte alpha-spectrin. The Xenopus alpha-fodrin cDNAs hybridize to a transcript of approximately 9 kb on RNA blots, and probably to a single gene type on genomic DNA blots. Both RNA blot analyses and S1 nuclease protection assays with the Xenopus alpha-fodrin cDNAs demonstrate that the observed decline in the de novo synthesis of alpha-fodrin polypeptides is controlled by a dramatic decrease in the abundance of alpha-fodrin transcripts after fertilization. In contrast, levels of actin transcripts do not decrease during this period. Inasmuch as steady-state levels of alpha-fodrin transcripts rise by the neurula stage of development, these results suggest that the synthesis of alpha-fodrin polypeptides during embryonic development of Xenopus is regulated, rather than constitutive, and that the primary level of control is the steady-state abundance of mRNA.