Virus progeny of murine cytomegalovirus bacterial artificial chromosome pSM3fr show reduced growth in salivary Glands due to a fixed mutation of MCK-2

Abstract

Murine cytomegalovirus (MCMV) Smith strain has been cloned as a bacterial artificial chromosome (BAC) named pSM3fr and used for analysis of virus gene functions in vitro and in vivo. When sequencing the complete BAC genome, we identified a frameshift mutation within the open reading frame (ORF) encoding MCMV chemokine homologue MCK-2. This mutation would result in a truncated MCK-2 protein. When mice were infected with pSM3fr-derived virus, we observed reduced virus production in salivary glands, which could be reverted by repair of the frameshift mutation. When looking for the source of the mutation, we consistently found that virus stocks of cell culture-passaged MCMV Smith strain are mixtures of viruses with or without the MCK-2 mutation. We conclude that the MCK-2 mutation in the pSM3fr BAC is the result of clonal selection during the BAC cloning procedure.

Fig. 1.

Schematic presentation of the deletion found in the MCK-2 ORF of pSM3fr. The nucleotide sequence of MCK-2 exons and introns is shown. The initiating methionine residues for the m131 and m129 ORFs and the intron sequence are circled and boxed, respectively. The inset indicates the position of the T·A deletion at MCK-2 position 486 and the resulting stop mutation in pSM3fr.

Fig. 2.

Comparison of growth of unrepaired and repaired pSM3fr-derived viruses. Multistep growth curves of virus derived from pSM3fr or pSM3fr-MCK-2fl clone 3.3 or 4.1 are shown. NIH 3T3 cells were infected at an MOI of 0.1, and supernatants were harvested daily and titrated. The titers of supernatants are expressed as TCID₅₀ per ml.

Fig. 3.

Infection of BALB/c mice with virus derived from pSM3fr or pSM3fr-MCK-2fl clone 3.3 or 4.1. (A) Virus titers in the spleen, liver, lungs, and salivary glands of BALB/c mice injected i.p. with 3 × 10⁵ TCID₅₀ of pSM3fr (pSM) (open circles) or pSM3fr-MCK-2fl clone 3.3 (gray circles) or 4.1 (black circles). The organs were harvested on days 3, 8, and 14 postinfection (p.i.). (B) Virus titers in salivary glands of BALB/c mice infected with pSM3fr (pSM) (open circles) or pSM3fr-MCK-2fl clone 3.3 (gray circles) or 4.1 (black circles) via the footpad or i.v. with 1.5 × 10⁶ and 3 × 10⁵ TCID₅₀, respectively. The organs were harvested on day 8 and 14 p.i. Each circle shows the value for an individual mouse, and mean values for the groups of mice are indicated by the short horizontal bars. The broken lines indicate the detection limit. nd, not determined.

Fig. 4.

Cell culture-passaged MCMV Smith strain exhibits mixtures of MCK-2 wild-type and mutant genomes. (A) The locus of the MCK-2 gene spanning the pSM3fr base pair deletion was amplified from two MCMV Smith virus stocks by PCR, and the PCR products were sequenced. The electropherograms indicate that with respect to nucleotide position 486 of MCK-2, the stocks are mixtures. For each stock, the more abundant sequence is depicted above the electropherogram and the less abundant sequence is indicated below the electropherogram. The 5′→3′ orientation respective to the MCK-2 ORF and nucleotide position 486 (Fig. 1) are indicated. The sequence resulting in a full-length MCK-2 ORF contains a T enclosed in a red box. The sequencing direction is indicated by the white open arrow. (B) Suppression of the mutant population by passage of cell culture-derived MCMV Smith strain in salivary glands. Three BALB/c mice (animals 1, 2, and 3) were injected i.p. with 2 × 10⁵ TCID₅₀ cell culture-passaged Smith strain, and the salivary glands were harvested on day 14 p.i. Then, salivary gland-derived virus was used for another round of infection (animals 1′, 2′, and 3′). Viral DNA from all salivary glands was amplified as described above for panel A and sequenced. The electropherograms of the mutant locus are shown. (C) Salivary gland-derived virus from mouse 1 was used to infect MEF cells and passaged twice. Viral DNA was amplified from supernatant virus and sequenced. The electropherogram of the mutant locus is shown. (D) BALB/c mice were infected as described in the legend to Fig. 3A with cell culture-passaged Smith strain (small white squares), pSM3fr-derived virus (pSM) (open circles), or pSM3fr-MCK-2fl clone 3.3 (small gray circles). The salivary glands were harvested on days 8 and 14 p.i. Each symbol shows the value for an individual mouse, and mean values for the groups of mice are indicated by the short horizontal bars. The broken lines indicate the detection limit.