Murine cytomegalovirus open reading frame M27 plays an important role in growth and virulence in mice

Abstract

Using a Tn3-based transposon mutagenesis approach, we have generated a pool of murine cytomegalovirus (MCMV) mutants. In this study, one of the mutants, RvM27, which contained the transposon sequence at open reading frame M27, was characterized both in tissue culture and in immunocompetent BALB/c mice and immunodeficient SCID mice. Our results suggest that the M27 carboxyl-terminal sequence is dispensable for viral replication in vitro. Compared to the wild-type strain and a rescued virus that restored the M27 region, RvM27 was attenuated in growth in both BALB/c and SCID mice that were intraperitoneally infected with the viruses. Specifically, the titers of RvM27 in the salivary glands, lungs, spleens, livers, and kidneys of the infected SCID mice at 21 days postinfection were 50- to 500-fold lower than those of the wild-type virus and the rescued virus. Moreover, the virulence of the mutant virus appeared to be attenuated, because no deaths occurred among SCID mice infected with RvM27 for up to 37 days postinfection, while all the animals infected with the wild-type and rescued viruses died within 27 days postinfection. Our observations provide the first direct evidence to suggest that a disruption of M27 expression results in reduced viral growth and attenuated viral virulence in vivo in infected animals. Moreover, these results suggest that M27 is a viral determinant required for optimal MCMV growth and virulence in vivo and provide insight into the functions of the M27 homologues found in other animal and human CMVs as well as in other betaherpesviruses.

FIG. 1

(A) Schematic representation of the structure of the transposon construct used for mutagenesis. Tet, tetracycline resistance gene; gpt gene that encodes guanine phosphoribosyltransferase (gpt); poly(A), transcription termination signal. (B) Location of the transposon insertion in the recombinant virus. The transposon sequence is shown as a solid bar, while the coding sequence of open reading frame M27 is represented by an open arrow. The orientation of the arrow represents the direction of translation and transcription predicted from the nucleotide sequence (38). The numbers represent 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 recombinant viruses. The DNA fractions were isolated from cells infected with the wild-type (WT) virus, RvM27, or RqM27. The DNA samples (10 μg) were digested with either HindIII or EcoRI, separated on 1% agarose gels, transferred to a Zeta-Probe membrane, and hybridized to a DNA probe. The probe used for the analyses was the plasmid that contained the MCMV DNA fragment carrying the transposon sequence.

FIG. 2

Northern analyses of RNA fractions isolated from cells that were mock infected (lanes 4 and 8) or infected with the wild-type (WT) virus (lanes 1 and 5), RqM27 (lanes 2 and 6), and RvM27 (lanes 3 and 7). A total of 5 × 10⁶ NIH 3T3 cells were infected with each virus at an MOI of 5 PFU per cell, and cells were harvested at 24 h postinfection. RNA samples (20 μg in lanes 1 to 4 and 10 μg in lanes 5 to 8) 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 M27 (M27 probe) (lanes 1 to 4) or M25 (M25 probe) (lanes 5 to 8). Sizes are shown in kilobase pairs.

FIG. 3

In vitro growth of MCMV mutants in tissue culture. Mouse NIH 3T3 cells were infected with each virus at an MOI of either 0.5 PFU (A) or 5 PFU (B) per cell. At 0, 1, 2, 4, and 7 days postinfection, cells and culture medium were harvested and sonicated. The viral titers were determined by plaque assays on NIH 3T3 cells. The titers represent the averages obtained from triplicate experiments. The standard deviation is indicated by the error bars.

FIG. 4

Titers of MCMV mutants in the salivary glands (A), lungs (B), spleens (C), livers (D), and kidneys (E) of infected BALB/c mice. BALB/c-Byj mice were infected intraperitoneally with 10⁴ PFU of each virus. At 1, 3, 7, 10, 14, and 21 days postinfection, the animals (three mice per group) were sacrificed. The salivary glands, lungs, spleens, livers, and kidneys were collected and sonicated. Moreover, the salivary glands were also collected from animals at 28 and 35 days postinfection. The viral titers in the tissue homogenates were determined by standard plaque assays in NIH 3T3 cells. The limit of detection was 10 PFU/ml of tissue homogenate. The titers represent the averages obtained from triplicate experiments. The error bars indicate the standard deviation. Some error bars are not evident because the standard deviation bar is less than or equal to the height of the symbols.

FIG. 5

Mortality of SCID mice infected with strain Smith, (WT), RvM27, and RqM27. CB17 SCID mice (five animals per group) were infected intraperitoneally with 10⁴ PFU of each virus. Mortality of mice was monitored for at least 41 days postinfection, and survival rates were determined.

FIG. 6

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

FIG. 7

Stability of the genome and the transposon mutation of RvM27 during replication in SCID mice. Viral DNAs were isolated from cells that were infected with RvM27 (MOI of <0.01) and allowed to grow in culture for 5 days (P0) (lane 3) or from cells that were infected with the virus recovered from either the salivary glands (SG, lane 1) or spleen (Sp, lane 2) of SCID mice at 21 days after intraperitoneal inoculation with 10⁴ PFU of RvM27. Southern analyses of the viral DNA fractions digested with EcoRI are shown. The DNA of the wild-type virus (WT) is shown in lane 4. The ³²P-radiolabeled probe was derived from the same plasmid which was used for Southern analyses of RvM27 in Fig. 1 and contained the transposon and M27 open reading frame sequence.