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 (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.

FIG. 1

Construction of the baculovirus expression vectors that express soluble, six-histidine-tagged MHVR and Bgp glycoproteins. (A) cDNAs encoding MHVR and related Bgp glycoproteins illustrate the cloning strategy used to express the signal sequence (SS) and Ig-like extracellular domains (d1, etc.) as soluble proteins. The transmembrane domain (TM) and cytoplasmic domain (C) were not amplified. Pfu polymerase and sequence-specific primers (arrows) introduced restriction sites at the 5′ (PstI) and 3′ (XbaI) ends of PCR-amplified fragments to allow cloning into the modified baculovirus expression vector. (B) The baculovirus expression vector pAcMP2 was modified by adding a consensus thrombin cleavage (Th. Cl.) site, a six-histidine tail, and a stop codon. Amplified DNA fragments encoding the extracellular domains of MHVR and Bgp were ligated in frame, using PstI and XbaI. All constructs were confirmed by sequencing. Expression in Sf9 cells is driven by the major basic protein promoter (MBP).

FIG. 2

The soluble MHVR[1-4] expressed by a recombinant vaccinia virus neutralizes MHV-A59. MHV-A59 (10,000 PFU) was preincubated with the unpurified culture medium of vac-MHVR-infected Vero cells (open circles) or with highly purified sMHVR(vac) (closed circles) prior to inoculation of L2 cells. MHV-A59 replication was monitored by an MHV N-protein-specific MAb, and the percent neutralization was calculated as described in Materials and Methods. The ND₅₀ of sMHVR(vac) is estimated to be 20 nM.

FIG. 3

Characterization of the purified, soluble receptor glycoproteins. In each panel, 0.5-μg aliquots of purified sMHVR[1-4] (lane 1), sMHVR[1,2] (lane 2), sMHVR[1,4] (lane 3), sBgp1^b[1-4] (lane 4), sBgp1^b[1,2] (lane 5), sBgp1^b[1,4] (lane 6), and sBgp2[1,4] (lane 7) were separated by SDS-PAGE (12% gel) and stained with Coomassie blue (A) or transferred to Immobilon-P for further analysis (B to E). In panel B, the protein blot was probed for total carbohydrate. The immunoreactivities of the purified glycoproteins with anti-MHVR MAb CC1 and polyclonal anti-MHVR antiserum 655 are shown in panels C and D, respectively. Panel E shows the relative MHV-A59 binding activities of the purified glycoproteins determined by VOPBA. Sizes are indicated in kilodaltons.

FIG. 4

The soluble MHVR-related glycoproteins expressed by baculovirus differ in the ability to neutralize MHV-A59. MHV-A59 (5,000 PFU) virions were preincubated with serial dilutions of the five purified soluble receptor glycoproteins. The surviving virions were quantitated by plaque assays, and the percent neutralization is plotted against the molar concentration of the purified glycoproteins. These data are from a representative experiment. The ND₅₀s are summarized in Table 1.

FIG. 5

Soluble receptor glycoproteins show different neutralizing activities for MHV-A59 and MHV-JHM. The plaque assay described in the legend to Fig. 4 was used to determine the virus neutralizing activities of sMHVR[1-4] and sMHVR[1,4] against MHV-A59 (A) and MHV-JHM (B) and of sBgp1^b[1-4] and sBgp1^b[1,4] against MHV-A59 (C) and MHV-JHM (D). The ND₅₀s are reported in Table 1.