In vivo replication, latency, and immunogenicity of murine cytomegalovirus mutants with deletions in the M83 and M84 genes, the putative homologs of human cytomegalovirus pp65 (UL83) - PubMed (original) (raw)

In vivo replication, latency, and immunogenicity of murine cytomegalovirus mutants with deletions in the M83 and M84 genes, the putative homologs of human cytomegalovirus pp65 (UL83)

C S Morello et al. J Virol. 1999 Sep.

Abstract

We previously identified two open reading frames (ORFs) of murine cytomegalovirus (MCMV), M83 and M84, which are putative homologs of the human cytomegalovirus (HCMV) UL83 tegument phosphoprotein pp65 (L. D. Cranmer, C. L. Clark, C. S. Morello, H. E. Farrell, W. D. Rawlinson, and D. H. Spector, J. Virol. 70:7929-7939, 1996). In this report, we show that unlike the M83 gene product, the M84 protein is expressed at early times in the infection and cannot be detected in the virion. To elucidate the functional differences between the two pp65 homologs in acute and latent MCMV infections, we constructed two MCMV K181 mutants in which either the M83 or M84 ORF was deleted. The resultant viruses, designated DeltaM83 and DeltaM84, respectively, were found to replicate in NIH 3T3 cells with kinetics identical to those of the parent strain. Western blot analysis demonstrated that except for the absence of M83 or M84 protein expression in the respective mutants, no global perturbations of protein expression were detected. When DeltaM83 and DeltaM84 were inoculated intraperitoneally (i.p.) into BALB/c mice, both viruses showed similar attenuated growth in the spleen, liver, and kidney. However, only DeltaM83 was severely growth restricted in the salivary glands, a phenotype that was abolished upon restoration of the M83 ORF. DeltaM83's growth was similarly restricted in the salivary glands of the resistant C3H/HeN or highly sensitive 129/J strain, as well as in the lungs of all three strains following intranasal inoculation. Using a nested-PCR assay, we found that both DeltaM83 and DeltaM84 established latency in BALB/c mice, with slightly decreased levels of DeltaM83 and DeltaM84 genomic DNAs, relative to K181, observed in the salivary glands and lungs. Immunization of BALB/c mice with 10(5) PFU of K181, DeltaM83, or DeltaM84 i.p. provided similar levels of protection against lethal challenge. Although immunization with 200 PFU of DeltaM83 also provided complete protection, this dose allowed both the immunizing and challenge viruses to establish latency in the spleen. Our results show that the two MCMV pp65 homologs differ in their expression kinetics, virion association, and influence on viral tropism and/or dissemination.

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Figures

FIG. 1

FIG. 1

Kinetics of M84 protein expression and absence of virion association. NIH 3T3 cells were infected with K181 at an MOI of 3, and at 8, 24, and 48 h p.i., cells were harvested and whole-cell lysates were prepared as described in Materials and Methods. Cells were also treated for the first 8 h of infection with CHX before the 12-h-p.i. harvest or were infected and incubated in the presence of PAA until the 48-h-p.i. harvest. Western blots of lysates and purified virions (Vir.) were probed with an affinity-purified rabbit antiserum to GST-M84 or a GST-M83-specific antiserum. Each panel depicts the same blot, and lanes are numbered at the bottom. Lanes 8 to 10 depict lanes 5 to 7 after prolonged exposure, and lanes 11 to 13 show lanes 5 to 7 after the blot was stripped and reprobed with the M83 antiserum. Un., uninfected. Positions of molecular mass markers are shown on the left.

FIG. 2

FIG. 2

Construction of the ΔM83, ΔM83-2, and ΔM84 deletion mutants of MCMV. (A) _Hin_dIII restriction map of MCMV K181 and location of ORFs M82 to M86 in the _Hin_dIII C region. Arrows indicate the transcription direction and sizes of the ORFs. All ORF lengths are to scale, with the scale (in base pairs) indicated on the right. Also shown are the positions of restriction sites important for plasmid construction. Abbreviations: Hind, _Hin_dIII; Eco, _Eco_RI; RV, _Eco_RV; Pst, _Pst_I; Not, _Not_I. (B) Construction of ΔM83 and ΔM83-2 by homologous recombination between either the pΔM83KO8 or pΔM83-2 insert, respectively, and the K181 genome. Identical shading and cross-hatching indicate homologous regions, while dashed lines indicate continuation of genome sequences. Indicated by a horizontal bar above pΔM83-2 is the 28 bp provided as a _Not_I-to-_Pst_I oligonucleotide adapter as described in Materials and Methods. (C) Construction of ΔM84 by recombination of homologous sequences of K181 and the insert from pΔM83KOT-5. The restriction sites at the borders of some homologous regions have been changed in the plasmid to facilitate subcloning.

FIG. 3

FIG. 3

Construction of the M83 revertant rΔM83, using the EGFP-puro cassette. (A) DNA sequences of the ΔM83 genome were recombined in NIH 3T3 cells with homologous sequences (indicated by identical shading and cross-hatching) in the rescue plasmid cassette pM83Res13.6 (B). Selected restriction sites used for subcloning or for reference are indicated with abbreviations as in Fig. 2. Bam, _Bam_HI. (C) Recombination between both homologous regions yields an rΔM83 intermediate virus in which the EGFP-puro cassette and full-length M83 ORF have been inserted into the ΔM83 genome as indicated. Intramolecular recombination between M84 direct-repeat sequences in the rΔM83 intermediate would be expected to delete intervening marker gene sequences (bracketed in panel C), resulting in an EGFP− LacZ− virus with a wild-type (wt) _Hin_dIII C region (D).

FIG. 4

FIG. 4

Genomic analysis of ΔM83, ΔM83-2, and rΔM83 viruses by restriction digestion and Southern blotting. (Left panels) The restriction maps of the portions of the _Hin_dIII C regions under analysis are shown for the K181, ΔM83, and ΔM83-2 viruses, with arrows indicating the positions, lengths, and directions of transcription of ORFs. B, _Bam_HI; St, _Stu_I; X, _Xho_I. Above each restriction map are shown the lengths (in kilobase pairs) and positions of restriction fragments detected by the genomic probe (indicated by the shaded regions). (Right panels) Genomic DNA was purified from uninfected cells or cells infected with the virus indicated. Genomic and _Hin_dIII C plasmid DNAs were digested with _Bam_HI, electrophoresed, and blotted. The 4.4-kbp _Stu_I-to-_Xho_I genomic probe was isolated from the _Hin_dIII C plasmid, 32P labeled by random priming, and used to probe the blot. The expected 0.52-kbp band in both panels was visible upon prolonged exposure of the blots.

FIG. 5

FIG. 5

Genomic analysis of the ΔM84 and rΔM83 viruses by restriction digestion and Southern blotting. Details of genomic maps and DNA preparation are as described in the legend to Fig. 4. Ss, _Sst_I; RV, _Eco_RV; N, _Not_I; X, _Xho_I. Genomic and _Hin_dIII C plasmid DNAs were digested with _Sst_I and _Xho_I prior to electrophoresis, blotting, and detection with the 7.6-kbp _Eco_RV-to-_Not_I genomic probe.

FIG. 6

FIG. 6

Growth of mutant and revertant MCMVs in NIH 3T3 cells. All titers presented are the means of the log10 PFU/ml of the extracellular tissue culture medium of triplicate cultures, with error bars indicating the standard deviations (SD). (A) Single-cycle growth of K181 and ΔM84 after infection at an MOI of 3. (B) Single-cycle growth of K181, ΔM83, and rΔM83 in NIH 3T3 cells as described for panel A. (C) Single-cycle growth of K181, ΔM83, ΔM83-2, and rΔM83 as described for panel A. (D) Multicycle growth of K181, ΔM83, and ΔM84 in NIH 3T3 cells infected at an MOI of 0.05.

FIG. 7

FIG. 7

Western blot analysis of mutant and revertant MCMVs. Whole-cell lysates were prepared from uninfected or MCMV-infected NIH 3T3 cells harvested at 8, 24, or 48 h p.i. Lysates were electrophoresed on 7.5% polyacrylamide–SDS gels, and separated proteins were electroblotted onto nitrocellulose. M83, M84, and M32 proteins were detected with rabbit polyclonal antisera as described in Materials and Methods. pp89 protein was detected with an antiserum from BALB/c mice immunized with intradermal injections of pcDNA3-pp89. Bound antibodies were detected with horseradish peroxidase-coupled anti-mouse or anti-rabbit antibodies as in Fig. 1. (A) Expression of M83, M84, M32, and pp89 proteins in NIH 3T3 cells after infection with K181, rΔM83, ΔM83, or ΔM84. (B) An independent experiment showing M83 and M84 expression 8 or 48 h after infection with K181, ΔM83, or ΔM83-2. uninf., uninfected. The positions of molecular mass markers are shown on the right.

FIG. 8

FIG. 8

Growth of mutant and revertant MCMVs in BALB/c mice following i.p. or i.n. inoculation. (A) Five BALB/c mice per group were inoculated i.p. with 5 × 105 PFU of tissue culture-derived virus, and resultant viral titers in the livers, kidneys, and spleens 4 days postinoculation, or in the salivary glands 10 days postinoculation, are shown. Titers represent the means of the log10 PFU/organ values, with the standard deviations (SD) being shown by error bars. Values above each bar indicate the fold reductions of the nonlogarithmic mean titers (i.e., PFU/organ) of that group relative to the corresponding K181-infected controls. The subscript 0 indicates that in at least one organ in that group, virus was undetectable, and that the titer of that organ was set to the limit of detection for calculation purposes. (B) Day 4 spleen and day 10 salivary gland titers of four mice per group inoculated i.p. with K181, rΔM83, ΔM83, or ΔM83-2 as described for panel A. (C) MCMV titers in the lungs and salivary glands of four mice per group 14 days following inoculation i.n. with 1.5 × 105 PFU of virus.

FIG. 9

FIG. 9

Growth of mutant MCMV in the salivary glands of C3H/HeN mice. Shown are MCMV titers 10 days following i.p. inoculation with 1.5 × 106 PFU of tissue culture-derived virus and 14 days following i.n. inoculation with 1.5 × 105 PFU of virus. Means, standard deviations (SD), and fold-reduction values above each bar are as described in the legend to Fig. 8.

FIG. 10

FIG. 10

Growth of mutant MCMV in 129/J mice following i.p. or i.n. inoculation. (A) MCMV titers of four 129/J mice per group following i.p. inoculation with 5 × 105 PFU of tissue culture-derived virus. Organ harvest days, titer value calculations, and fold reduction values above each bar are as described in the legend to Fig. 8. (B) MCMV titers in the lungs and salivary glands of four mice per group 14 days following i.n. inoculation with 2.5 × 105 PFU of virus. SD, standard deviation.

FIG. 11

FIG. 11

PCR amplification of MCMV DNA in latently infected BALB/c mice. (A) Control nested PCRs in which IE1-specific primer pairs were used to amplify serial dilutions of linearized _Hin_dIII L plasmid. Dilutions were made such that 25 to 0.2 copies of plasmid and 1 μg of uninfected mouse genomic DNA purified from the organs shown were amplified. The negative controls contained 1 μg of the organ DNA from uninfected mice, and PCR results for two to three individual uninfected mice are shown. All positive reactions produced only a single 310-bp product (shown) upon ethidium bromide staining, and representative results for the plasmid dilutions are shown (see Results). (B) Nested-PCR amplification of IE1 in the splenic stromal DNA isolated from four mice (no. 1 to 4) per group infected with 105 PFU of tissue culture-derived K181, ΔM83, or ΔM84. Serial 10-fold dilutions of organ DNA were amplified independently. (C) Nested PCR of serial dilutions of salivary gland DNAs from mice 1 to 4 in each group as described for panel B. (D) Nested PCR of serial dilutions of lung DNAs from mice 1 to 4 as described for panel B.

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