Murine cytomegalovirus with a transposon insertional mutation at open reading frame m155 is deficient in growth and virulence in mice

Abstract

A pool of murine cytomegalovirus (MCMV) mutants was previously generated by using a Tn3-based transposon mutagenesis approach (X. Zhan, M. Lee, J. Xiao, and F. Liu, J. Virol. 74:7411-7421, 2000). In this study, one of the MCMV mutants, Rvm155, which contained the transposon insertion in open reading frame m155, was characterized in vitro for its replication in tissue culture and in vivo for its growth and virulence in immunodeficient SCID mice. Compared to the wild-type strain and a rescued virus that restored the m155 region, the mutant is significantly deficient in growth in many organs of the infected animals. At 21 days postinfection the titers of Rvm155 in the salivary glands, lungs, spleens, livers, and kidneys of the intraperitoneally infected SCID mice were lower than the titers of the wild-type virus and the rescued virus by 50-, 1,000-, 500-, 100-, and 500-fold, respectively. Moreover, the viral mutant was attenuated in killing the SCID mice, as none of the SCID mice that were intraperitoneally infected with Rvm155 died until 38 days postinfection while all the animals infected with the wild-type and rescued viruses died at 27 days postinfection. Our results provide the first direct evidence that a disruption of m155 expression leads to attenuation of viral virulence and growth in animals. Moreover, these results suggest that m155 is a viral determinant for optimal MCMV growth and virulence in vivo.

FIG. 1.

(A) Structure of the transposon construct used for mutagenesis. TR, terminal repeat; Tet, tetracycline resistance gene; gpt, gene that encodes guanine phosphoribosyltransferase (gpt); poly(A), transcription termination signal. (B) Transposon insertion in the recombinant virus. The open arrow shows the coding sequence of open reading frame m155; the filled bar represents the transposon sequence. The orientation of the arrow represents the direction of translation and transcription as predicted from the nucleotide sequence (25). The numbers show the sizes of the DNA fragments of the viruses that were generated by digestion with HindIII (H) or EcoRI (E). (C) Southern analyses of the DNAs of the recombinant viruses. The DNA fractions were isolated from cells infected with the wild-type (WT) virus, Rvm155 (Rvm155), or Rqm155 (Rqm155). The DNA samples (20 μg) were digested with either HindIII (H) or EcoRI (E), separated on 0.8% agarose gels, transferred to a Zeta probe membrane, and hybridized to a ³²P-radiolabeled DNA probe that contained the MCMV DNA fragment that was inserted with the transposon sequence.

FIG. 2.

Northern analyses of the expression of the m155 transcript. At 24 h postinfection, RNA fractions were isolated from 10⁶ NIH 3T3 cells that were mock infected (lanes 1 and 5) or infected (MOI, 5) with the wild-type virus (WT) (lanes 4 and 8), Rvm155 (lanes 2 and 6), and Rqm155 (lanes 3 and 7). Equal amounts of RNA samples (25 μg) were separated on agarose gels that contained formaldehyde, transferred to a nitrocellulose membrane, and hybridized to a ³²P-radiolabeled probe that contained the sequence of M25 (M25 probe) (lanes 1 to 4) or m155 (m155 probe) (lanes 5 to 8).

FIG. 3.

Growth of MCMV mutants in cultured NIH 3T3 cells. NIH 3T3 cells were infected with each virus at a MOI of either 0.5 PFU (A) or 5 PFU per cell (B). At 0, 1, 2, 4, and 7 days postinfection, we harvested cells and culture media and determined the viral titers by plaque assays on NIH 3T3 cells. The error bars indicate the standard deviations. The values of the viral titer represent the average obtained from triplicate experiments.

FIG. 4.

Survival rate of the SCID mice infected with the Smith strain, Rvm155, and Rqm155. CB17 SCID mice (5 animals per group) were infected intraperitoneally with 10⁴ PFU of each virus. Survival of mice was monitored for at least 53 days postinfection, and survival rates were determined.

FIG. 5.

Titers of MCMV mutants in the salivary glands (A), lungs (B), spleens (C), livers (D), and kidneys (E) of the CB17 SCID mice that were infected intraperitoneally with 10⁴ PFU of each virus. At 1, 3, 7, 10, 14, and 21 days postinfection, we sacrificed the animals (3 mice per group); collected the salivary glands, lungs, spleens, livers, and kidneys; and sonicated these organs. We subsequently determined the viral titers in the tissue homogenates by standard plaque assays in NIH 3T3 cells. The limit of detection was 10 PFU/ml of the tissue homogenate. The viral titers represent the average obtained from triplicate experiments. The error bars indicate the standard deviation, and those that are not evident indicate that the standard deviation was less than or equal to the height of the symbols.

FIG. 6.

The stability of the transposon insertional mutation of Rvm155 in CB17 SCID mice. We isolated viral DNAs from cells that were infected with Rvm155 (MOI < 0.01) and allowed to grow in culture for 5 days (P0) (lane 3) or from cells that were infected with the virus collected from the lungs (LU, lane 1) and salivary glands (SG, lane 2) of the infected SCID mice 21 days after intraperitoneal inoculation with 10⁴ PFU of Rvm155. Southern analyses of the viral DNA fractions digested with HindIII are shown. The ³²P-radiolabeled probe was derived from the same plasmid that was used for Southern analyses of Rvm155 shown in Fig. 1 and contained the transposon and the m155 sequence. The DNA of the wild-type virus (WT) is shown in lane 4.