Purified, Soluble Recombinant Mouse Hepatitis Virus Receptor, Bgp1^b, and Bgp2 Murine Coronavirus Receptors Differ in Mouse Hepatitis Virus Binding and Neutralizing Activities

Abstract

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.

Communication between S1N330 and a Region in S2 of Murine Coronavirus Spike Protein Is Important for Virus Entry into Cells Expressing CEACAM1b Receptor

“…This difference could be accounted for by the difference in virus-binding ability between MHVR1 and MHVR2. The former has more than at least 100-fold higher binding ability than the latter, as examined by virus overlay protein blot assay and neutralization tests using soluble forms of these MHV receptors (Ohtsuka et al, 1996;Zelus et al, 1998). Tight binding of MHV to the receptor could compensate for the combination undesirable for efficient entry into cells found in srr7 S protein.…”

Amino acid substitutions and an insertion in the spike glycoprotein extend the host range of the murine coronavirus MHV-A59

“…The neutralization of the mutant viruses by soluble mCEACAM1a [1,4] at 37 jC suggested that soluble mCEA-CAM1a bound S1 on the mutant virions and induced an irreversible conformational change in S2-like wild-type MHV-A59 (Gallagher, 1997;Matsuyama and Taguchi, 2002;Zelus et al, 1998Zelus et al, , 2003. To further characterize the interaction between the mutant viruses and mCEACAM1a, we examined the binding of recombinant S proteins to soluble mCEACAM1a [1,4].…”

Pathogenesis of neurotropic murine coronavirus is multifactorial

“…(The cDNA library was derived from mice from the outbred CD-1 strain [21] that were apparently heterozygous at the receptor locus.) Although the SJL/J allele acts as an MHV receptor if over-expressed in tissue culture, it fails to bind MHV virions in virus overlay protein blot assays, and the soluble form has fourfold less virus neutralizing activity than the functional allele [25]. This suggests that it is too weak a receptor to function at endogenous levels; in addition, it is not recognized by CC1 [24].…”

“…Independent work identified the contiguous six-amino-acid motif at position 38-43 as crucial for MHV binding [33], and it is noteworthy that CEACAM1b has no homology with CEACAM1a at this sequence ( Figure 2). Although the N domain is necessary and sufficient for neutralization and receptor activity [34], a truncated soluble protein containing only the N and A1 domains neutralizes less efficiently than either the two-or four-domain form, which suggests that the fourth Ig-like domain improves MHV binding [25]. The crystal structure of the soluble two-domain protein shows no interaction between the N and A2 domains; however, the critical MHV-binding residues 38-43 are prominently displayed Box 1.…”

“…CEACAM2 (previously Bgp2) is similar to CEACAM1a in overall structure, but the MHV-binding loop at amino acids 38-43 has more homology to CEA-CAM1b ( Figure 2). Like CEACAM1b, CEACAM2 can support MHV infection if it is overexpressed in nonpermissive cells [29], but the purified soluble form is less efficient at neutralizing MHV than is CEACAM1a [25]. Studies of the receptor efficiency of the two proteins have been hampered by an inability to control for receptor density, but it is generally believed that CEACAM2 is a less efficient receptor than CEACAM1a [15,29].…”