Three herpes simplex virus type 1 latency-associated transcript mutants with distinct and asymmetric effects on virulence in mice compared with rabbits - PubMed (original) (raw)
Comparative Study
Three herpes simplex virus type 1 latency-associated transcript mutants with distinct and asymmetric effects on virulence in mice compared with rabbits
G C Perng et al. J Virol. 2001 Oct.
Abstract
Herpes simplex virus type 1 latency-associated transcript (LAT)-null mutants have decreased reactivation but normal virulence in rabbits and mice. We report here on dLAT1.5, a mutant with LAT nucleotides 76 to 1667 deleted. Following ocular infection of rabbits, dLAT1.5 reactivated at a lower rate than its wild-type parent McKrae (6.1 versus 11.8%; P = 0.0025 [chi-square test]). Reactivation was restored in the marker-rescued virus dLAT1.5R (12.6%; P = 0.53 versus wild type), confirming the importance of the deleted region in spontaneous reactivation. Compared with wild-type or marker-rescued virus, dLAT1.5 had similar or slightly reduced virulence in rabbits (based on survival following ocular infection). In contrast, in mice, dLAT1.5 had increased virulence (P < 0.0001). Thus, deletion of LAT nucleotides 76 to 1667 increased viral virulence in mice but not in rabbits. In contrast, we also report here that LAT2.9A, a LAT mutant that we previously reported to have increased virulence in rabbits (G. C. Perng, S. M. Slanina, A. Yuhkt, B. S. Drolet, W. J. Keleher, H. Ghiasi, A. B. Nesburn, and S. L. Wechsler, J. Virol. 73:920-929, 1999), had decreased virulence in mice (P = 0.03). In addition, we also found that dLAT371, a LAT mutant that we previously reported to have wild-type virulence in rabbits (G. C. Perng, S. M. Slanina, H. Ghiasi, A. B. Nesburn, and S. L. Wechsler, J. Virol. 70:2014-2018, 1996), had decreased virulence in mice (P < 0.05). Thus, these three mutants, each of which encodes a different LAT RNA, have different virulence phenotypes. dLAT1.5 had wild-type virulence in rabbits but increased virulence in mice. In contrast, LAT2.9A had increased virulence in rabbits but decreased virulence in mice, and dLAT371 had wild-type virulence in rabbits but decreased virulence in mice. Taken together, these results suggest that (i) the 5' end of LAT and/or a gene that overlaps part of this region is involved in viral virulence, (ii) this virulence appears to have species-specific effects, and (iii) regulation of this virulence may be complex.
Figures
FIG. 1
Genomic structures of the viruses used in this study. (A) Prototypic genomic structure of parental wild-type McKrae and marker-rescued viruses _d_LAT1.5R, _d_LAT2903R, and _d_LAT371R. TRL and IRL, terminal and internal long repeats, respectively; TRS and IRS, terminal and internal short repeats, respectively; UL and US, long and short unique regions, respectively. The LAT gene is located in the viral long repeats and is thus present in two copies. The primary 8.3-kb LAT transcript is shown expanded below the genome. The stable 2-kb LAT is indicated by a black rectangle. The relative locations of the ICP0 and ICP34.5 transcripts are shown for reference. (B) _d_LAT2903 (20) has an _Eco_RV-to-_Hpa_I restriction fragment deletion from LAT nucleotide −161 to +1667 relative to the start of LAT transcription at +1 (XXXX). This deletion removes the core LAT promoter, including the TATA box, and a second proposed promoter near the 5′ end of the 2-kb LAT. No LAT is transcribed, as indicated by the dashed lines. (C) _d_LAT371 has a 371-nucleotide _Sty_I-_Sty_I restriction fragment deletion from LAT nucleotide 76 to 447 (X) in both copies of LAT (23). The primary LAT promoter is intact, and the remaining portion of the primary 8.3-kb LAT is transcribed. (D)_d_LAT1.5 has a _Sty_I-to-_Hpa_I restriction fragment deletion from LAT nucleotide 76 to 1667 (XXX). The primary LAT promoter is intact, and the remaining portion of the primary 8.3-kb LAT is transcribed. (E) LAT3.3A (22) contains the LAT promoter driving the first 1,499 nucleotides of LAT inserted into an ectopic location between the UL37 and UL38 viral genes of _d_LAT2903. This virus transcribes only the first 1.5 kb of LAT yet has wild-type spontaneous reactivation. LAT2.9AR (24) was marker rescued from LAT2.9A and has the same genomic structure as LAT3.3A. (F) LAT2.9A (24) has the same genomic structure as LAT3.3A except for a _Sty_I-_Sty_I restriction fragment deletion from LAT nucleotide 76 to 447 in the ectopic LAT insert.
FIG. 2
Spontaneous reactivation of _d_LAT1.5. Rabbits were ocularly infected with _d_LAT1.5 (_d_L1.5), marker-rescued _d_LAT1.5R (_d_L1.5R), or parental wild-type (wt) McKrae. Beginning on day 31 p.i., at which time latency had been established, tear films were collected daily for 26 days, plated on RS cells, and observed for the presence of CPE. Randomly selected positive cultures were confirmed by passage and Southern analysis. (A) The y axis represents the cumulative number of HSV-1-positive cultures for each virus group divided by the number of eyes in the group. (B) The percentage of tear films containing spontaneously reactivated virus was calculated for each individual eye. The results are shown as a scattergram. In the _d_LAT1.5 column, some of the zero points are plotted at 0.7 so that all of the zero points can be visualized. Similarly, in the _d_LAT1.5R column, two points with an actual value of 7.7 are plotted at 8.4. The P values shown were determined by one-way ANOVA and Dunnet's posttest. Results for the wild type and _d_LAT1.5R were similar (P > 0.05).
FIG. 3
Survival of rabbits (A) and mice (B) infected with _d_LAT1.5. (A) Survival of the rabbits used in the experiments shown in Fig. 2 was determined on day 21 p.i. The numbers above each bar indicate the number of surviving rabbits/total number of rabbits infected. P was determined by the chi-square test for all pairs of rabbits. _d_L1.5, _d_LAT1.5; _d_L1.5R, _d_LAT1.5R; wt, parental wild-type McKrae. (B) Mice were bilaterally ocularly infected with 106 PFU/eye without scarification, and survival was determined at 21 days p.i.
FIG. 4
Replication of _d_LAT1.5 in tissue culture, eyes, TG, and brains. (A) Semiconfluent monolayers of CV-1 cells were infected with 0.01 PFU of _d_LAT1.5, _d_LAT1.5R, the LAT-null mutant _d_LAT2903, or wild-type (wt) McKrae. At the indicated times, the infected-cell monolayers were harvested by freeze-thawing, and the amount of virus was determined by plaque assay on RS cells. The result for each time point is the average of two determinations. (B) Rabbits were ocularly infected with 2 × 105 PFU of the indicated virus per eye as described in Materials and Methods. In each group tear films were collected from five individual eyes (each from a different rabbit) on days 3, 5, 7, and 10, and virus titers were determined by standard plaque assays. There was no statistical difference between the groups on any day (by ANOVA, P = 0.69 for day 3, P = 0.055 for day 5, P = 0.76 for day 7, and P = 0.84 for day 10). (C) Mice were ocularly infected as described above, and virus replication in the eye was determined as for rabbits in panel B. Results from two independent experiments are shown. Experiment 1 (six eyes per group) compared _d_LAT1.5 to _d_LAT1.5R. Experiment 2 (five eyes per group) compared the wild type to _d_LAT1.5R. There was no statistical difference between the viruses in each experiment on any day (P > 0.2 for experiment 1; P > 0.5 for experiment 2 [Mann-Whitney test]). (D) Mice were infected as described above. Both TG were removed from five mice per group at necropsy on days 2, 3, 5, 7, and 10 p.i., and the amount of infectious virus in each individual TG was determine by plaque assay as described in Materials and Methods. There was no statistical difference on any day (P > 0.1 on each day [ANOVA]). (E) Mice were infected as described above, and brains (five per group per time point) were harvested and virus titers were determined on the days indicated. The asterisk indicates the only day for which statistical significance was obtained (day 5, P = 0.013 [ANOVA]; _d_LAT1.5 versus _d_LAT1.5R, P = 0.02 [Mann-Whitney test]; _d_LAT1.5 versus wild type, P = 0.008 [Mann-Whitney test]). (F) The experiment in panel E was repeated for _d_LAT1.5 and _d_LAT1.5R using 10 mice per group on day 5 p.i. Each data point represents the virus titer from an individual brain on this day. The horizontal lines indicate the medians. The P value was determined by the Mann-Whitney rank sum test. (G) The experiment in panel E was repeated for _d_LAT1.5 (9 mice) and _d_LAT1.5R (10 mice) on day 7 p.i., and the results are plotted as in panel F. Error bars indicate standard deviations.
FIG. 5
Virulence of LAT2.9A in mice. Mice were ocularly infected as described for Fig. 4, and survival was determined at 21 days p.i. Numbers above each bar indicate the number of surviving mice/total number of mice. P was determined by the chi-square test. wt, wild type.
FIG. 6
Replication of LAT2.9A in mouse eyes, TG, and brains. (A, B, and C) Experimental procedures and analyses were as described for Fig. 4C, D, and E, respectively, except that 10 samples were used per group at each time point. The P value was >0.05 at all time points. (D, E, and F) Scatterplots showing the virus titers for individual eyes, TG, and brains on the day of peak virus titer for the samples shown in panels A, B, and C, respectively. The horizontal lines indicate the median (as opposed to the means shown in panels A, B, and C). Error bars indicate standard deviations.
FIG. 7
Virulence of _d_LAT371 in mice. Mice were ocularly infected and survival was determined as described for Fig. 4. P was determined by the chi-square test. _d_L371, _d_LAT371; _d_LAT, _d_LAT2903; _d_LATR, _d_LAT2903R.
FIG. 8
Replication of _d_LAT371 (d371) in mouse eyes, TG, and brains. Experimental procedures and analyses were done as described in the legend to Fig. 6. The P value was >0.05 by the Mann-Whitney rank sum test at all time points except for mouse eyes on day 3. By the Student t test, the P value was 0.13 for this time point. wt, wild type. Error bars indicate standard deviations.
FIG. 9
Virulence of marker-rescued viruses in mice. Mice were ocularly infected as described for Fig. 4, and survival was determined at 21 days p.i. The numbers above each bar represent the number of surviving mice/total number of mice. The P value in the box was determined by the chi-square test using a 5-by-2 contingency table. The P values above individual bars were determined by the chi-square test compared with the wild type. wt, wild-type McKrae; _d_L15R, marker-rescued _d_LAT1.5; _d_LATR, marker-rescued _d_LAT2903; _d_L371R, marker-rescued _d_LAT371; LAT2.9R, marker-rescued LAT2.9A.
References
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