Gain of virulence caused by loss of a gene in murine cytomegalovirus

Abstract

Mouse strains are either resistant or susceptible to murine cytomegalovirus (MCMV). Resistance is determined by the Cmv1(r) (Ly49h) gene, which encodes the Ly49H NK cell activation receptor. The protein encoded by the m157 gene of MCMV has been defined as a ligand for Ly49H. To find out whether the m157 protein is the only Ly49H ligand encoded by MCMV, we constructed the m157 deletion mutant and a revertant virus. Viruses were tested for susceptibility to NK cell control in Ly49H+ and Ly49H- mouse strains. Deletion of the m157 gene abolished the viral activation of Ly49H+ NK cells, resulting in higher virus virulence in vivo. Thus, in the absence of m157, Ly49H+ mice react like susceptible strains. 129/SvJ mice lack the Ly49H activation NK cell receptor but express the inhibitory Ly49I NK cell receptor that binds to the m157 protein. The Deltam157 inhibitory phenotype was weak because MCMV encodes a number of proteins that mediate NK inhibition, whose contribution could be shown by another mutant.

FIG. 1.

Genome structure of recombinant MCMVs. The HindIII cleavage map of the MCMV (Smith strain) genome is shown at the top. Deletion and insertion of the m157 gene, respectively, were achieved by homologous recombination between the parental MCMV BACs and a linear DNA fragment containing the desired sequence, a kanamycin resistance gene (Kn), and flanking homologies to the target site in the viral genome. The deletion genome pΔm157 was generated by homologous recombination of the linear DNA fragment and w.t. MCMV BAC pSM3fr. An independent Δm157 genome was constructed as follows. First, a 48-bp FRT site (grey box) was inserted into the intergenic region between genes m16 and m17, generating pSM3fr-FRT, to prove that insertion of sequences at this positions does not interfere with virus replication. In a second mutagenesis step, the m157 gene was deleted from pSM3fr-FRT, generating the second independent m157 deletion genome, pΔm157-FRT. Finally, the m157 gene, including its native promoter, was reinserted into pΔm157-FRT at the ectopic positions between genes m16 and m17, thereby removing the FRT site. The names of the MCMV BACs are indicated on the left. Recombinant viruses were reconstituted by transfection of the recombinant MCMV BACs into permissive MEFs. The names of the corresponding reconstituted viruses are given on the right.

FIG. 2.

In vitro growth of recombinant viruses. BALB/c MEFs were infected with Δm157, m157Rev, or w.t. MCMV at 0.01 PFU per cell. Supernatants were harvested at the indicated time points after infection, and virus titers were determined.

FIG. 3.

Deletion of the m157 gene abolishes MCMV susceptibility to NK cell-mediated control in Ly49H⁺ mice. Undepleted (open circles) or NK1.1-depleted (shaded circles) C57BL/6 and congenic BALB.B6-Cmv1^r mice were injected intravenously with 5 × 10⁵ PFU of Δm157 or w.t. MCMV. Three days after infection, the virus titers in the spleen, lungs, and liver were determined by standard plaque assay. Titers in individual mice (circles) and median values (horizontal bars) are shown. Virus titers were calculated per organ in the spleen and lungs and per gram of liver. The differences in viral titers between undepleted and NK cell-depleted groups of mice are shown by shaded areas. There were significant differences (P < 0.025) in virus titers between the groups of undepleted mice infected with Δm157 and w.t. MCMV in the spleen and lungs.

FIG. 4.

NK cell activation and reversal of susceptibility to NK cell control by reintroduction of the m157 gene. (A) C57BL/6 RAG1^−/− splenocytes were incubated for 12 h (1 h without brefeldin A and 11 h with brefeldin A) with IC-21 cells that were either uninfected or infected (MOI = 5, 24 h) with Δm157, m157Rev, or w.t. MCMV. Cells were stained with biotinylated anti-Ly49H, followed by PE-streptavidin and allophycocyanin-anti-NK1.1. The cells were fixed, permeabilized, and stained with fluorescein isothiocyanate-anti-IFN-γ antibody. Data represent NK1.1⁺ cells. The values are the percentages of cells within the quadrants. (B) Undepleted (open circles) or NK1.1-depleted (shaded circles) C57BL/6 mice were injected intravenously with 5 × 10⁵ PFU of Δm157, m157Rev, or w.t. MCMV, and virus titers in the spleen and lungs were determined 3 days after infection. Titers in individual mice (circles) and median values (horizontal bars) are shown. The differences in viral titers between undepleted and NK cell-depleted groups of mice are shown by shaded areas. There were significant differences (P < 0.025) in virus titers between the groups of undepleted mice infected with Δm157 and m157Rev and between the groups of undepleted mice infected with Δm157 and w.t. MCMV.

FIG. 5.

Ly49H⁺ NK cells play no role in control of Δm157 virus. (A) C57BL/6 mice were depleted with MAb 5E6 (αLy49 C/I), 1F8 (αLy49 C/H/I), or PK136 (αNK1.1) and injected intravenously with 5 × 10⁵ PFU of Δm157 and w.t. MCMV. Virus titers in the lungs were determined 3 days after infection. Data represent the mean value ± the standard deviation of four or five mice. Depletion with MAb 1F8 or PK136 resulted in a significant increase (P < 0.025) in the w.t. MCMV titer. (B) Recombinant BXD-8 mice were injected with 2 × 10⁵ PFU of Δm157 and w.t. MCMV, and virus titers in the spleen and lungs were determined 3 days after infection. Titers in individual mice (circles) and median values (horizontal bars) are shown.

FIG. 6.

Control of Δm157 virus in BALB/c and 129/SvJ mice. BALB/c and 129/SvJ mice were injected with 2 × 10⁵ PFU of Δm157 and w.t. MCMV, and virus titers in the spleen and lungs were determined 3 days later. Titers in individual mice (circles) and median values (horizontal bars) are shown. There were significant differences (P < 0.025) in virus titers in spleen between groups of mice infected with Δm157 and w.t. MCMV.

FIG. 7.

NK cell response against Δm157 in C57BL/6 mice is prevented by viral evasion genes. Undepleted (open circles) or NK1.1-depleted (shaded circles) C57BL/6 mice were injected intravenously with 5 × 10⁵ PFU of w.t., Δm157, or ΔMS94.5 MCMV, and virus titers in the lungs were determined 3 days after infection. Titers in individual mice (circles) and median values (horizontal bars) are shown. The differences in viral titers between undepleted and NK cell-depleted groups of mice are shown by shaded areas. There were significant differences (P < 0.025) in virus titers between the groups of undepleted mice infected with Δm157 and ΔMS94.5 and between the groups of undepleted mice infected with Δm157 and w.t. MCMV.