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, that precluded cleavage between S1 and S2 could also be triggered to undergo a conformational change at 37°C by soluble receptor at neutral pH or by pH 8 alone. A novel 120-kDa subunit was formed following incubation of the receptortriggered S A59 H716D virions with trypsin at 4°C. The data show that unlike class 1 fusion glycoproteins of other enveloped viruses, the murine coronavirus S protein can be triggered to a membrane-binding conformation at 37°C either by soluble receptor at neutral pH or by alkaline pH alone, without requiring previous activation by cleavage between S1 and S2.As an initial step in virus replication, specialized attachment proteins on virions bind to specific receptors on host cell membranes. The specificity of virus-receptor interactions frequently determines which cells, tissues, and species are susceptible to virus infection. On binding to its specific receptor under optimal conditions of pH and temperature, the viral attachment glycoprotein undergoes one or more programmed conformational changes that exposes a hydrophobic fusion peptide which mediates fusion of the viral envelope with host cell membranes, releasing the viral nucleocapsid into the cytoplasm (24, 61). Class I viral fusion glycoproteins have the general structure of the HA protein of influenza A virus, consisting of an N-terminal receptor binding domain followed by an exposed protease cleavage site, a fusion domain containing several heptad repeats, and transmembrane and cytoplasmic domains (23).So-called pH-independent fusion occurs when the viral envelope can fuse directly with the plasma membrane at neutral pH. Conformational changes in spike glycoproteins of some large minus-strand RNA viruses including paramyxoviruses such as simian virus 5 (SV5) and respiratory syncytial virus, and retroviruses including human immunodeficiency virus type 1 (HIV-1) and avian and murine leukemia viruses are triggered by binding at 37°C to specific receptors on the plasma membrane (1, 9, 24, 61). These pH-independent viruses cause cellto-cell fusion, forming multinucleated syncytia in infected cell cultures and tissues when viral spike proteins expressed on the membrane of a cell are triggered to undergo a fusion-inducing conformational change by binding to a specific virus receptor on an adjacent cell. In contrast, the spike glycoproteins of other large, enveloped RNA viruses including influenza A virus, rabies virus, Ebola virus, and bunyavirus require an acidic environment to trigger the conformational changes in the viral fusion protein that lead to membrane fusion (16,24,44,51,61). Therefore, fusion of these viral envelopes occurs not at the plasma membrane but within the cell at endosomal membranes once the pH drops to 5.5. These pH-dependent or acid-dependent viruses do not induce the formation of multinucleated syncytia in cells and tissues at neutral pH.This report describes conformational changes in the spike glycoprotein (S) of mouse hepatitis virus (MHV), a murine

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.

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).

b ; this is most probably due to spread via CEACAM1 receptor-independent cell-to-cell fusion, an activity displayed only by S 4 R among the recombinant viruses studied here. These data suggest that the RBD domain and the rest of the spike must coevolve to optimize function in viral entry and spread.Mouse hepatitis virus (MHV) is a murine coronavirus capable of infecting the liver, the central nervous system, and other internal organs, depending on the virus strain and the mouse strain. The observations that coronavirus infection is highly host species specific and MHV pathogenesis is virus strain specific have led to investigations of the viral and host determinants of pathogenesis. We have used two neurotropic strains in this study. MHV-4 is a highly neurovirulent isolate of the JHM strain that does not cause significant hepatitis; MHV-A59 is a less neurovirulent strain which causes moderate to severe hepatitis (29,30,50).Murine carcinoembryonic antigen-related cell adhesion molecules (mCEACAMs; CD66a, previously known as mmCGM or Bgp), serve as receptors that mediate MHV entry (3,14,36,54). Members of the CEA family are involved in intercellular adhesion and the development of hepatocellular, colorectal, and epithelial tumors (3). mCEACAMs are glycoproteins of two or four immunoglobulin-like extracellular domains followed by a transmembrane domain and a long or short cytoplasmic tail. MHV binding activity has been mapped to the N-terminal domain of CEACAM1 a (16, 41, 52). The MHV receptor glycoproteins are encoded by two murine ceacam genes, mceacam1 and mceacam2 (14, 36). Murine ceacam1 is expressed as allelic glycoproteins, mCEACAM1 a and mCEACAM1 b , which differ in 27 of the 108 amino acids in the N-terminal immunoglobulin-like domain(14). The proteins are expressed as four-domain and two-domain isoforms primarily on the epithelial and endothelial cells of the respiratory tract and the intestines and other tissues (21,42,45 (14,35,36,41,56). Although the biological requirements for an efficient MHV receptor are not known, these data are consistent with reports of the relative resistance to MHV infection of the SJL/J mouse, which contains only the mCEACAM1 b allele (14, 37, 54). The MHV surface glycoprotein spike (S) mediates many biological properties of MHV, including receptor attachment, fusion of viral and cell membranes during entry, cell-to-cell fusion during viral spread, and immune activation (5). Spike is a type I membrane protein of approximately 180 kDa. On the MHV-A59 and MHV-4 virions, S is cleaved posttranslationally into two approximately 90-kDa subunits, the amino-terminal S1 and the carboxy-terminal S2 (5): S1 binding to mCEACAM is thought to induce structural rearrangements within spike

Mouse hepatitis virus receptor (MHVR) is a murine biliary glycoprotein (Bgp1a). 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 Bgp1bglycoproteins from MHV-resistant SJL/J mice, as well as Bgp2 and truncation mutants of MHVR and Bgp1b 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 Bgp1b(sBgp1b) 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 Bgp1b 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 sBgp1b[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.

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 the 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.

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.