Construction and characterization of murine cytomegaloviruses that contain transposon insertions at open reading frames m09 and M83 - PubMed (original) (raw)

Construction and characterization of murine cytomegaloviruses that contain transposon insertions at open reading frames m09 and M83

X Zhan et al. J Virol. 2000 Aug.

Abstract

A transposon derived from Escherichia coli Tn3 was introduced into the genome of murine cytomegalovirus (MCMV) to generate a pool of viral mutants, including two recombinant viruses that contained the transposon sequence within open reading frames m09 and M83. Our studies provide the first direct evidence to suggest that m09 is not essential for viral replication in mouse NIH 3T3 cells. Studies in cultured cells and in both BALB/c-Byj and CB17 severe combined immunodeficient (SCID) mice indicated that the transposon insertion is stable during viral propagation both in vitro and in vivo. Moreover, the virus that contained the insertion mutation in m09 exhibited a titer similar to that of the wild-type virus in the salivary glands, lungs, livers, spleens, and kidneys of both the BALB/c and SCID mice and was as virulent as the wild-type virus in killing the SCID mice when these animals were intraperitoneally infected with these viruses. These results suggest that m09 is dispensable for viral growth in these organs and that the presence of the transposon sequence in the viral genome does not significantly affect viral replication in vivo. In contrast, the virus that contained the insertion mutation in M83 exhibited a titer of at least 60-fold lower than that of the wild-type virus in the organs of the SCID mice and was attenuated in killing the SCID mice. These results demonstrate the utility of using the Tn3-based system as a mutagenesis approach for studying the function of MCMV genes in both immunocompetent and immunodeficient animals.

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Figures

FIG. 1

Schematic representation of the structure of the transposon construct used for mutagenesis (A) and the procedure for the construction of MCMV mutants that contained random transposon insertions (B). TR, terminal repeat; Tet, tetracycline resistance gene; gpt, gene that encodes guanine phosphoribosyltransferase (gpt); poly(A), transcription termination signal.

FIG. 2

(A) The locations of the transposon insertions in the recombinant viruses. The transposon sequence is shown as a filled bar, while the coding sequence of each open reading frame is represented by an open arrow. The orientation of the arrow represents the direction of the translation and transcription predicted based on the nucleotide sequence (32). The numbers represent the sizes of the DNA fragments of the mutant viruses that contained the transposon sequence and were generated by digestion with _Hin_dIII (H), _Not_I (N), or _Eco_RI (E). (B) Southern blot analyses of the viral mutants. The DNA fractions were isolated from cells infected with the wild-type (WT) virus and different MCMV mutants. The DNA samples (20 μg) were digested with _Hin_dIII (H), _Not_I (N), or _Eco_RI (E); separated on 0.8% agarose gels; transferred to a Zeta-Probe membrane; and hybridized to a DNA probe. The probes used for the analyses were the plasmids that contained the MCMV DNA fragments inserted with the transposon sequence.

FIG. 3

Northern blot analyses of the RNA fractions isolated from cells that were mock infected (lanes 1, 4, and 7) or infected with the wild-type virus (WT) (lanes 2, 5, and 8) and the mutant viruses (lanes 3, 6, and 9). A total of 107 NIH 3T3 cells were infected with each virus at an MOI of 10 PFU/cell, and cells were harvested 24 h postinfection. Equal amounts of RNA samples (30 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 m09 (lanes 1 to 3), M83 (lanes 4 to 6), or M25 (lanes 7 to 9).

FIG. 4

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 media were harvested and sonicated. The viral titers were determined by plaque assays on NIH 3T3 cells. The values of the viral titer represent the average obtained from triplicate experiments. The standard deviation is indicated by the error bars.

FIG. 5

Titers of MCMV mutants in salivary glands (A), lungs (B), spleens (C), livers (D), and kidneys (E) of the infected BALB/c mice. BALB/c-Byj mice were infected intraperitoneally with 103 PFU of each virus. At 1, 3, 7, and 14 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 the tissue homogenate. The viral titers represent the average obtained from triplicate experiments. The error bars indicate the standard deviation. Error bars that are not evident indicate that the standard deviation was less than or equal to the height of the symbols.

FIG. 6

Mortality of the SCID mice infected with the Smith strain, Rvm09, and RvM83. CB17 SCID mice (five animals per group) were infected intraperitoneally with 104 PFU of each virus. Mortality of mice was monitored for 46 days postinfection, and survival rates were determined.

FIG. 7

Titers of MCMV mutants in the salivary glands (A), lungs (B), spleens (C), livers (D), and kidneys (E) of the 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 the tissue homogenate. The viral titers represent the average obtained from triplicate experiments. The error bars indicate the standard deviation. Error bars that are not evident indicate that the standard deviation was less than or equal to the height of the symbols.

FIG. 8

The stability of the transposon mutations in tissue cultured cells and in BALB/c and SCID mice. Viral DNAs were either isolated from cells that were infected with Rvm09 (MOI = <0.01) and allowed to grow in culture for 5 days (P0) (lane 2) or five generations (60 days) (P5) (lane 3) or from cells that were infected with the virus collected from the salivary glands (SG, lanes 4 and 6) and lungs (LU, lanes 5 and 7) of either BALB/c (BALB/c, lanes 4 and 5) or SCID mice (SCID, lanes 6 and 7) 14 days after intraperitoneal inoculation with 104 PFU of Rvm09. Southern blot analyses of the viral DNA fractions digested with _Hin_dIII are shown. The DNA of the wild-type virus (WT) is shown in lane 1. The 32P-radiolabeled probe was derived from the same plasmid which was used for Southern analyses of Rvm09 in Fig. 2 and contained the transposon and the m09 open reading frame sequence.

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Construction and characterization of murine cytomegaloviruses that contain transposon insertions at open reading frames m09 and M83 - PubMed (original) (raw)