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

Abstract

The murine coronavirus [murine hepatitis virus (MHV)] is limited to infection of susceptible mice and murine cell lines by the specificity of the spike glycoprotein (S) for its receptor, murine carcinoembryonic antigen cell adhesion molecule 1a (mCEACAM1a). We have recently shown that 21 aa substitutions and a 7-aa insert in the N-terminal region of S are associated with the extended host range of a virus variant derived from murine cells persistently infected with the A59 strain of MHV (MHV-A59). We used targeted RNA recombination (TRR) to generate isogenic viruses that differ from MHV-A59 by the 21 aa substitutions or the 7-aa insert in S. Only viruses with both the 21 aa substitutions and the 7-aa insert in S infected hamster, feline, and monkey cells. These viruses also infected murine cells in the presence of blocking anti-mCEACAM1a antibodies. Thus, relatively few changes in the N-terminal region of S1 are sufficient to permit MHV-A59 to interact with alternative receptors on murine and non-murine cells.

Fig. 1

Composition of S constructs used to introduce mutations into MHV-A59 using targeted RNA recombination. Mutations in the S gene of MHV/BHK were engineered into the S/pBC SK+ plasmid. These mutations result in 21 aa substitutions (Table 1) in the regions shaded in black, a 7-aa insert, TRTKKVL, (black triangles), and a R496L aa substitution (white star). The minimal receptor binding domain (RBD) for murine CEACAM1a is shaded in gray. The restriction enzyme sites HindIII (H) and KpnI (K) used to screen recombinant viruses are also indicated.

Fig. 2

Restriction enzyme digestion analysis of recombinant viruses. The 5′ of the S genes of MHV/BHK, the recombinant viruses (A, B, and C), and the pMH54 plasmid were amplified using primers S(AvrII)+ and A59.C7. The amplification products were incubated with the restriction enzymes HindIII and KpnI, and the DNA fragments were separated on a 4% agarose gel. Mutations derived from MHV/BHK generate a 501-bp fragment when cut with HindIII and a 205-bp fragment when cut with KpnI.

Fig. 3

Plaque phenotypes of recombinant viruses on murine 17 Cl 1 cell monolayers. Neutral red-stained plaques of MHV-A59, MHV/BHK, or the recombinant viruses at 72 h post inoculation (p.i).

Fig. 4

Growth of recombinant viruses in murine 17 Cl 1 cells. (A) Expression of viral nucleocapsid protein (N) of MHV-A59, MHV/BHK, or the recombinant viruses was detected at 8 h p.i. with anti-N MAb. Magnification ×400. (B) Yields of viruses released into tissue culture supernatants. Average virus yields ± SEM of two independent experiments are shown.

Fig. 5

Neutralization of recombinant viruses by soluble, murine CEACAM1a. MHV-A59, MHV/BHK, or the recombinant viruses (5000 PFU) were pre-incubated with serial dilutions of recombinant mCEACAM1a[1,4]. Virus survival was determined and percentage of neutralization calculated as described in Materials and methods. Percentage of neutralization shown is representative of two independent experiments.

Fig. 6

Binding of recombinant S proteins to soluble, murine CEACAM1a. Immobilized mCEACAM1a[1,4] was incubated with 0.05 mg/ml of anti-mCEACAM1a MAb-CC1 or an isotype-matched MAb-Ctrl, and then incubated with full-length S proteins. Binding of S proteins was detected by immunoperoxidase labeling using polyclonal anti-S AO4 serum. Binding was calculated from the absorbance of mCEACAM1a incubated with S proteins minus the absorbance of mCEACAM1a alone. Average binding ± SEM of two independent experiments is shown.

Fig. 7

Growth of recombinant viruses during anti-receptor antibody blockade of murine 17 Cl 1 cells. (A) Yields of MHV-A59, MHV/BHK, or the recombinant viruses at 24 h p.i. from cells treated with 0.05 mg/ml of anti-mCEACAM1a MAb-CC1 or an isotype-matched MAb-Ctrl before, during, and after inoculation. Average virus yields ± SEM of two independent experiments are shown. (B) Yields of viruses from cells treated with 0.5 mg/ml of MAb-CC1 or MAb-Ctrl, or with polyclonal rabbit anti-mCEACAM1a serum (649) or normal rabbit serum (NRS) before, during, and after inoculation. Virus yields shown are representative of two independent experiments.

Fig. 8

Infection of hamster cells by recombinant viruses. Expression of viral N in hamster (BHK) cells and hamster cells stably transfected with mCEACAM1a[1–4] (BHK+mCEACAM1a) inoculated with MHV-A59, MHV/BHK, or the recombinant viruses were detected at 24 h p.i. as described in Fig. 4. Magnification ×200.

Fig. 9

Growth of mutant viruses in hamster cells. (A) Yields of MHV/BHK or S21BHK+i A from hamster (BHK) cells or hamster cells stably transfected with mCEACAM1a[1–4] (BHK+mCEACAM1a). Virus titers were determined using murine 17 Cl 1 cell monolayers. Average virus yields ± SEM of two independent experiments are shown. (B) Spread of MHV/BHK or S21BHK+i A in BHK cell monolayers. Expression of viral N was detected as described in Fig. 4. Magnification ×200.