Characterization of a spontaneous 9.5-kilobase-deletion mutant of murine gammaherpesvirus 68 reveals tissue-specific genetic requirements for latency

Abstract

Murine gammaherpesvirus 68 (gammaHV68 [also known as MHV-68]) establishes a latent infection in mice, providing a small-animal model with which to identify host and viral factors that regulate gammaherpesvirus latency. While gammaHV68 establishes a latent infection in multiple tissues, including splenocytes and peritoneal cells, the requirements for latent infection within these tissues are poorly defined. Here we report the characterization of a spontaneous 9.5-kb-deletion mutant of gammaHV68 that lacks the M1, M2, M3, and M4 genes and eight viral tRNA-like genes. Previously, this locus has been shown to contain the latency-associated M2, M3, and viral tRNA-like genes. Through characterization of this mutant, we found that the M1, M2, M3, M4 genes and the viral tRNA-like genes are dispensable for (i) in vitro replication and (ii) the establishment and maintenance of latency in vivo and reactivation from latency following intraperitoneal infection. In contrast, following intranasal infection with this mutant, there was a defect in splenic latency at both early and late times, a phenotype not observed in peritoneal cells. These results indicate (i) that there are different genetic requirements for the establishment of latency in different latent reservoirs and (ii) that the genetic requirements for latency depend on the route of infection. While some of these phenotypes have been observed with specific mutations in the M1 and M2 genes, other phenotypes have never been observed with the available gammaHV68 mutants. These studies highlight the importance of loss-of-function mutations in defining the genetic requirements for the establishment and maintenance of herpesvirus latency.

FIG.1.

Identification of γHV68Δ9473, a spontaneous deletion mutant of γHV68. (A) Schematic of the wt γHV68 genome with candidate latency-associated transcripts indicated above the genome (see the text for details; tRNAs have been excluded for clarity) and corresponding genomic structures of wt γHV68 and γHV68Δ9473 at the left end of the viral genome. To define the structure of γHV68Δ9473, we sequenced across the junction of the γHV68Δ9473 deletion with two independent sequencing reactions and primers (primers A and B, as indicated). The sequence at this junction is indicated beneath the γHV68Δ9473 genome. Restriction sites: E5, EcoRV; E1, EcoRI; H, HindIII. TR, terminal repeat. Empty genes represent genes missing from γHV68Δ9473. The asterisks indicate that the wt γHV68 coordinate for the NotI site is based upon the available genomic sequence, with the γHV68Δ9473 coordinate for NotI based upon the sequencing of a genomic subclone. Both coordinates define the distance between the NotI site and the first base pair of the unique γHV68 sequence. (B) Southern blot analysis of wt γHV68 or γHV68Δ9473 genomic DNA restriction digested with either NotI-NsiI or EcoRV (E5), followed by hybridization with a biotinylated probe for the sequence surrounding and containing gene 6 (bp 11,100 to 14,026; indicated in Fig. 1A). Hybridization was detected with chemiluminescence detection. High-molecular-weight (MW) DNA standards (stds) (Gibco BRL) and BstEII-digested lambda DNA (New England Biolabs) were included on the blot and detected by hybridization with biotinylated lambda DNA, and fragment sizes (kilobases) are indicated to the left. (C) Comparison of restriction endonuclease digestion of 1.5 μg each of the wt γHV68 and γHV68Δ9473 genomic DNAs with BamHI (B), EcoRI (E), and HindIII (H). Digested viral DNA was electrophoresed on a 1.2% agarose gel and stained with ethidium bromide. Restriction fragments absent from γHV68Δ9473 are identified by asterisks on the agarose gel. MW, molecular weight markers.

FIG.1.

Identification of γHV68Δ9473, a spontaneous deletion mutant of γHV68. (A) Schematic of the wt γHV68 genome with candidate latency-associated transcripts indicated above the genome (see the text for details; tRNAs have been excluded for clarity) and corresponding genomic structures of wt γHV68 and γHV68Δ9473 at the left end of the viral genome. To define the structure of γHV68Δ9473, we sequenced across the junction of the γHV68Δ9473 deletion with two independent sequencing reactions and primers (primers A and B, as indicated). The sequence at this junction is indicated beneath the γHV68Δ9473 genome. Restriction sites: E5, EcoRV; E1, EcoRI; H, HindIII. TR, terminal repeat. Empty genes represent genes missing from γHV68Δ9473. The asterisks indicate that the wt γHV68 coordinate for the NotI site is based upon the available genomic sequence, with the γHV68Δ9473 coordinate for NotI based upon the sequencing of a genomic subclone. Both coordinates define the distance between the NotI site and the first base pair of the unique γHV68 sequence. (B) Southern blot analysis of wt γHV68 or γHV68Δ9473 genomic DNA restriction digested with either NotI-NsiI or EcoRV (E5), followed by hybridization with a biotinylated probe for the sequence surrounding and containing gene 6 (bp 11,100 to 14,026; indicated in Fig. 1A). Hybridization was detected with chemiluminescence detection. High-molecular-weight (MW) DNA standards (stds) (Gibco BRL) and BstEII-digested lambda DNA (New England Biolabs) were included on the blot and detected by hybridization with biotinylated lambda DNA, and fragment sizes (kilobases) are indicated to the left. (C) Comparison of restriction endonuclease digestion of 1.5 μg each of the wt γHV68 and γHV68Δ9473 genomic DNAs with BamHI (B), EcoRI (E), and HindIII (H). Digested viral DNA was electrophoresed on a 1.2% agarose gel and stained with ethidium bromide. Restriction fragments absent from γHV68Δ9473 are identified by asterisks on the agarose gel. MW, molecular weight markers.

FIG.1.

Identification of γHV68Δ9473, a spontaneous deletion mutant of γHV68. (A) Schematic of the wt γHV68 genome with candidate latency-associated transcripts indicated above the genome (see the text for details; tRNAs have been excluded for clarity) and corresponding genomic structures of wt γHV68 and γHV68Δ9473 at the left end of the viral genome. To define the structure of γHV68Δ9473, we sequenced across the junction of the γHV68Δ9473 deletion with two independent sequencing reactions and primers (primers A and B, as indicated). The sequence at this junction is indicated beneath the γHV68Δ9473 genome. Restriction sites: E5, EcoRV; E1, EcoRI; H, HindIII. TR, terminal repeat. Empty genes represent genes missing from γHV68Δ9473. The asterisks indicate that the wt γHV68 coordinate for the NotI site is based upon the available genomic sequence, with the γHV68Δ9473 coordinate for NotI based upon the sequencing of a genomic subclone. Both coordinates define the distance between the NotI site and the first base pair of the unique γHV68 sequence. (B) Southern blot analysis of wt γHV68 or γHV68Δ9473 genomic DNA restriction digested with either NotI-NsiI or EcoRV (E5), followed by hybridization with a biotinylated probe for the sequence surrounding and containing gene 6 (bp 11,100 to 14,026; indicated in Fig. 1A). Hybridization was detected with chemiluminescence detection. High-molecular-weight (MW) DNA standards (stds) (Gibco BRL) and BstEII-digested lambda DNA (New England Biolabs) were included on the blot and detected by hybridization with biotinylated lambda DNA, and fragment sizes (kilobases) are indicated to the left. (C) Comparison of restriction endonuclease digestion of 1.5 μg each of the wt γHV68 and γHV68Δ9473 genomic DNAs with BamHI (B), EcoRI (E), and HindIII (H). Digested viral DNA was electrophoresed on a 1.2% agarose gel and stained with ethidium bromide. Restriction fragments absent from γHV68Δ9473 are identified by asterisks on the agarose gel. MW, molecular weight markers.

FIG. 2.

γHV68Δ9473 replicates comparably to wt γHV68 in vitro but has decreased acute-phase titers in vivo. NIH 3T12 monolayers were infected with 0.05 PFU of wt γHV68 or γHV68Δ9473 per cell, and supernatant (A) or cells (B) were harvested at the indicated times and titers were determined by plaque assay. The data shown are from two independent experiments and are plotted as mean titers (log₁₀) ± SEM. Acute-phase titers were quantitated by plaque assay following infection of C57BL/6 mice with 4 × 10⁵ to 10⁶ PFU of either wt γHV68 (open squares) or γHV68Δ9473 (closed circles). Animals were harvested at the times indicated, and the graphs show spleen titers following i.p. infection (C) or lung titers following i.n. infection (D). Statistically significant differences in γHV68Δ9473 infection were detected at day 9 p.i. after i.p. infection and day 6 p.i. after i.n. infection, as indicated. The data shown were compiled from two or three independent experiments for each time point, with three to five mice per group per experiment. Each symbol represents the viral titer of an individual mouse. The horizontal dashed line indicates the limit of detection by this assay (50 PFU/ml, log₁₀ 1.7).

FIG. 3.

γHV68Δ9473 establishes and reactivates from latency comparably to wt γHV68 at early times following i.p. infection. C57BL/6 mice were infected with 10⁶ PFU of wt γHV68 or γHV68Δ9473 by i.p. injection. Splenocytes (A and B) and peritoneal cells (C and D) were harvested on days 17 and 18 p.i. and analyzed for the frequency of viral genome-positive cells (A and C) or ex vivo reactivation (B and D). Reactivation efficiency (defined as the fraction of γHV68 genome-positive cells that reactivated from latency) is indicated to the right of each set of data. The asterisk denotes that although reactivation efficiency cannot be higher than 1 in 1, these assays are limited in the ability to resolve two- to threefold differences. Frequency analysis was based on the Poisson distribution with the horizontal dashed line at 63.2%. Mechanically disrupted (Dis) cells were plated in parallel to identify the presence of preformed infectious virus. The data shown represent three independent experiments with cells pooled from three to five mice per experiment per group. Curve fit lines were derived by nonlinear-regression analysis, and the symbols represent mean percentages of wells positive for virus (viral DNA or a CPE) ± SEM. There were no statistically significant differences between wt γHV68 and γHV68Δ9473 in the frequency of viral genome-positive cells or reactivation or reactivation efficiency at this time. Rxns, reactions.

FIG. 4.

γHV68Δ9473 maintains latency and has increased reactivation efficiency from peritoneal cells relative to that of wt γHV68 at late times following i.p. infection. C57BL/6 mice were infected with 10⁶ PFU of wt γHV68 or γHV68Δ9473 by i.p. injection, and splenocytes (A and B) and peritoneal cells (C and D) were harvested between days 42 and 48 postinfection. Cells were analyzed for the frequency of viral genome-positive cells (A and C) or ex vivo reactivation (B and D). Reactivation efficiency (defined as the fraction of γHV68 genome-positive cells that reactivated from latency) is indicated to the right of each set of data. Frequency analysis was based on the Poisson distribution with the horizontal dashed line at 63.2%. Mechanically disrupted (Dis) cells were plated in parallel to identify the presence of preformed infectious virus. The data shown represent three independent experiments with cells pooled from three to five mice per experiment per group. Curve fit lines were derived by nonlinear-regression analysis, and the symbols represent mean percentages of wells positive for virus (viral DNA or a CPE) ± SEM. There were no statistically significant differences between wt γHV68 and γHV68Δ9473 in the frequency of viral genome-positive cells or reactivation or in reactivation efficiency. n.d., not determined. Rxns, reactions.

FIG. 5.

γHV68Δ9473 has a defect in splenic latency, not observed in peritoneal cells, relative to wt γHV68 at early times following i.n. infection. C57BL/6 mice were infected with 4 × 10⁵ PFU of wt γHV68 or γHV68Δ9473 by i.n. injection, and splenocytes (A and B) and peritoneal cells (C and D) were harvested at day 17 postinfection. Cells were analyzed for the frequency of viral genome-positive cells (A and C) or ex vivo reactivation (B and D). For clarity, disrupted samples are not included in reactivation data on splenocytes (Fig. 5B); these samples were uniformly negative. Reactivation efficiency (defined as the fraction of γHV68 genome-positive cells that reactivated from latency) is indicated to the right of each set of data. Frequency analysis was based on the Poisson distribution with the horizontal dashed line at 63.2%. Mechanically disrupted (Dis) cells were plated in parallel to identify the presence of preformed infectious virus. The data shown represent four independent experiments, with cells pooled from three to five mice per experiment per group. Curve fit lines were derived by nonlinear-regression analysis, and symbols represent mean percentages of wells positive for virus (viral DNA or a CPE) ± SEM. Statistically significant differences in γHV68Δ9473 latency were observed in the frequency of viral genome-positive splenocytes, reactivation from splenocytes, and efficiency of reactivation from peritoneal cells, as indicated. Rxns, reactions.

FIG. 6.

γHV68Δ9473 has a defect in splenic latency and increased reactivation from peritoneal cells relative to wt γHV68 at late times following i.n. infection. C57BL/6 mice were infected with 4 × 10⁵ PFU of wt γHV68 or γHV68Δ9473 by i.n. injection, and splenocytes (A and B) and peritoneal cells (C and D) were harvested between day 42 and 46 postinfection. Cells were analyzed for the frequency of viral genome-positive cells (A and C) or ex vivo reactivation (B and D). Reactivation efficiency (defined as the fraction of γHV68 genome-positive cells that reactivated from latency) is indicated to the right of each set of data. Frequency analysis was based on the Poisson distribution with the horizontal dashed line at 63.2%. Mechanically disrupted (Dis) cells were plated in parallel to identify the presence of preformed infectious virus. The data shown represent three independent experiments with cells pooled from three to five mice per experiment per group. Curve fit lines were derived by nonlinear-regression analysis, and symbols represent mean percentages of wells positive for virus (viral DNA or a CPE) ± SEM. Statistically significant differences with γHV68Δ9473 were observed in the frequency of viral genome-positive splenocytes and reactivation from peritoneal cells, as indicated. n.d., not determined. Rxns, reactions.