Three Herpes Simplex Virus Type 1 Latency-Associated Transcript Mutants with Distinct and Asymmetric Effects on Virulence in Mice Compared with Rabbits

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.

Herpes simplex virus type 1 (HSV-1) is a double-stranded neurotropic DNA virus. It is ubiquitous in the general population and establishes lifelong latent infections in host sensory neurons. Following ocular infection, the virus travels up nerves and establishes latent infection in neurons of the trigeminal ganglia (TG). Via a mechanism that remains to be fully elucidated, the latent virus can reactivate at various times throughout the life of the individual and produce recurrent disease. Recurrent HSV-1 infection in the eye can result in corneal scarring, leading to loss of vision. As a result, HSV-1 is one of the most common infectious causes of corneal blindness in the developed world.

During neuronal latency, the latency-associated transcript (LAT) is the only viral gene that is abundantly transcribed (4, 11, 27, 30, 32, 33). The primary LAT transcript is 8.3 kb long (35, 40) and overlaps two viral genes, ICP0 and ICP34.5, in an antisense direction (27, 33). A very stable intron, the 2-kb LAT, is spliced from the primary transcript (9) and is the major LAT RNA detected during latency (6, 30, 31, 36, 37, 39).

LAT is essential for wild-type levels of spontaneous and induced reactivation in the rabbit ocular model (12, 20). This function maps to within the first 1.5 kb of the primary LAT (22). This 1.5-kb region does not overlap ICP0 or ICP34.5, encompasses only the first 837 nucleotides of the stable 2-kb LAT, and does not retain the 2-kb LAT stability (22). Thus, LAT appears to enhance spontaneous reactivation in the rabbit by a mechanism that does not involve antisense regulation of ICP0, does not require production of the 2-kb LAT, and does not require the presence of a highly stable LAT RNA. We have recently shown that LAT can promote cell survival in vitro and block apoptosis in rabbit TG during acute infection (13, 19). This may increase the number of neurons that become latently infected (25, 28, 34), thereby increasing spontaneous reactivation by increasing the pool of latently infected neurons available for subsequent reactivation. In some situations, it might also affect viral virulence.

LAT-null mutants in which the LAT promoter has been deleted and no LAT RNA is detected appear to have wild-type virulence in animal models (1, 2, 12, 20, 29). This suggests that LAT does not play a role in viral virulence. However, we recently showed that deletion of LAT nucleotides 76 to 447 from a virus that could transcribe only the first 1.5 kb of LAT (producing a mutant designated LAT2.9A), significantly increases the death rate in the rabbit ocular model (24). This suggests that the first 1.5 kb of LAT is involved in virulence as well as latency. However, the potential role of the remainder of the primary 8.3-kb LAT transcript (the region of LAT from 1.5 to 8.3 kb) has not been addressed.

We describe here a LAT mutant designated dLAT1.5. This mutant has LAT nucleotides 76 to 1667 deleted but contains the entire LAT promoter and expresses the primary 8.3-kb LAT with the exception of the deleted region (i.e., it expresses the region of LAT from 1.67 to 8.3 kb). dLAT1.5 was wild type for ocular replication and eye disease. As expected, dLAT1.5 had reduced spontaneous reactivation compared with the parental McKrae wild-type virus or marker-rescued virus (dLAT1.5R). In rabbits dLAT1.5 had wild-type or slightly reduced virulence (as measured by survival). In contrast, in mice, dLAT1.5 was significantly more virulent than dLAT1.5R or wild-type virus.

We previously reported that LAT2.9A has increased virulence in the rabbit (24). In contrast, we found here that LAT2.9A had reduced virulence in mice. We also previously reported that a third LAT mutant, dLAT371, has wild-type virulence in rabbits. dLAT371 contains the same 371-nucleotide deletion in the 5′ end of LAT as does LAT2.9A. However, in contrast to LAT2.9A, dLAT371 expresses the remainder of both copies of LAT. Thus, dLAT371 is structurally similar to dLAT1.5 in that both mutants express both copies of LAT with the exception of a deletion in the 5′ end. Furthermore, both deletions begin at the same StyI restriction enzyme site. In contrast to the case for dLAT1.5, we found that dLAT371 has decreased (rather than increased) virulence in mice. Thus, compared with wild-type virus and their corresponding marker-rescued viruses, each of these three LAT mutants affected virulence differently in rabbits compared with mice. There were also differences among the mutants regarding in which animal model they were more virulent. These results suggest that LAT and/or a gene overlapping part of the 5′ end of LAT can affect virulence and that this effect can differ in rabbits compared with mice depending on the exact portion of LAT that is deleted.

MATERIALS AND METHODS

Cells and virus.

Rabbit skin (RS) cells were grown in Eagle's minimal essential medium (MEM) supplemented with 5% fetal calf serum (FCS). CV-1 cells were grown in MEM supplemented with 10% FCS. CV-1 cells were used for growth kinetic studies. RS cells were used for all other tissue culture procedures, including preparation of virus stocks. All mutants were derived from HSV-1 strain McKrae. The parental McKrae virus and all mutants were triple plaque purified and passaged only one or two times prior to use. The construction and properties of dLAT2903, dLAT371, LAT3.3A, and LAT2.9A have been previously described (20, 22, 24). LAT3.3A was originally designated LAT1.5a (22), but its designation was changed (7, 8, 24) to maintain nomenclature consistency with our other mutants (the number prior to the A indicates the size of the LAT region added between UL37 and UL38).

Construction of the mutant dLAT1.5.

The previously cloned BamHI B fragment from HSV-1 strain McKrae (21) was digested with MluI, the overhang was filled in using the Klenow fragment and digested with SwaI, and PacI linker was added. The fragment was then digested with PacI, and the products were separated by agarose gel electrophoresis. A resulting 3.6-kb band (SwaI to MluI; LAT nucleotides −798 to +2850) which includes the LAT promoter and the entire 2-kb LAT was isolated by electroelution and cloned into the PacI site of plasmid pNEB193 to produce the plasmid SMpNEB193. SMpNEB193 was digested with StyI and HpaI, the StyI overhang was filled in using the Klenow fragment, and the blunt ends were self-ligated to produce the plasmid pLAT1.5dl, which contains a deletion from LAT nucleotide 76 to 1667. pLAT1.5dl was amplified by transformation into Escherichia coli strain RR1λCI857 according to standard protocols (21).

dLAT1.5 was generated by homologous recombination of pLAT1.5dl with dLAT2903 as we previously described (20, 22, 23, 26). dLAT2903 is a mutant of HSV-1 strain McKrae containing a 1.8-kb (EcoRV-HpaI) deletion in both copies of LAT that removed 0.2 kb of the LAT promoter and 1.6 kb of the 5′ end of the primary 8.3-kb LAT transcript (20). Briefly, pLAT1.5dl was cotransfected with infectious dLAT2903 DNA by the calcium phosphate method. Viruses from the cotransfection were plated, and isolated plaques were picked and screened by Southern analysis. Selected plaques were triple plaque purified and reanalyzed by restriction digestion and Southern analysis to ensure that the LAT promoter had been restored and that the region from LAT nucleotide 76 to 1667 was deleted from both copies of LAT (one in each long repeat). A final plaque was purified and designated dLAT1.5. The marker-rescued virus, dLAT1.5R, was generated as described above by homologous recombination of dLAT1.5 DNA with the plasmid SMpNEB193. This restored the deletion in dLAT1.5 back to wild type.

Replication of virus in tissue culture.

CV-1 cell monolayers at approximately 70 to 80% confluency were infected with virus at 0.01 PFU/cell, and all monolayers were refed with exactly the same amount of MEM containing 10% FCS. Virus was harvested for titration at various times by two cycles of freeze-thawing of the monolayers plus medium (−80°C to room temperature). PFU per milliliter were determined by standard plaque assays on RS cells.

Animals.

Rabbits were 8- to 10-week-old female New Zealand White from Irish Farms. Mice were 6-week-old female Swiss Webster from Harlan. Rabbits and mice were treated in accordance with Association for Research in Vision and Ophthalmology, American Association for Laboratory Animal Care, and National Institutes of Health guidelines.

Rabbit model of ocular HSV-1 infection, latency, and spontaneous reactivation.

The McKrae strain of HSV-1 does not require corneal scarification in rabbits or mice for efficient ocular infection (20). Rabbits were bilaterally infected without scarification or anesthesia by placing 2 × 10⁵ PFU of HSV-1 per eye into the conjunctival cul-de-sac, closing the eye, and rubbing the lid gently against the eye for 30 s (20). At this dose of HSV-1 McKrae, virtually all of the surviving rabbits harbor a bilateral latent HSV infection in both TG, resulting in a high group rate of spontaneous reactivation with the McKrae strain of HSV-1. In each animal group, tear films were collected from five eyes, each from a different rabbit, on days 3, 5, 7, and 10 postinfection (p.i.) for acute ocular replication. Virulence (animal death) was monitored daily for up to 21 days p.i. Beginning on day 31 p.i., tear film specimens were collected daily from each eye for 26 days as previously described (20, 23, 26), using a nylon-tipped swab. The swab was then placed in 0.5 ml of tissue culture medium and squeezed, and the inoculated medium was used to infect RS cell monolayers. These cell monolayers were observed in a masked fashion by phase light microscopy for up to 5 days for HSV-1 cytopathic effects (CPE). All positive monolayers were blind passaged onto fresh cells to confirm the presence of virus. DNA was purified from positive cultures and analyzed by restriction enzyme digestion and Southern blotting to confirm that the CPE was due to reactivated HSV-1 and that the reactivated virus was identical to the input virus.

Ocular infection of mice.

Swiss Webster mice were bilaterally infected without scarification or anesthesia by placing 10⁶ PFU of HSV-1 per eye into the conjunctival cul-de-sac as described for rabbits. Tear films were collected on various days p.i. from one eye per animal. The amount of virus in each tear film was determined by standard plaque assays on RS cells. Virulence was determined by survival at 21 days after ocular infection.

Virus replication in mouse TG and brains.

Mice were infected as described above and euthanized at various times p.i. The brain and TG were removed and individually homogenized, debris was pelleted by low-speed centrifugation, and the amount of infectious virus in the supernatant was determined by standard plaque assays on RS cells.

Statistical analyses.

Statistical analyses were performed using GraphPad Prism version 3.00 for Windows (GraphPad Software, San Diego, Calif.). Results were considered statistically significant when the P value was <0.05.

RESULTS

Genomic structures of viruses.

dLAT1.5 was constructed as described in Materials and Methods. This mutant contains a deletion from LAT nucleotide 76 (a StyI restriction enzyme site) to 1667 (a HpaI restriction enzyme site) in both copies of the LAT gene (one in each long repeat) (Fig. 1D). This deletion does not alter the primary LAT promoter. Thus, dLAT1.5 transcribes the primary LAT with the exception of the deleted region. The marker-rescued virus, dLAT1.5R, is identical to the parental wild-type strain McKrae (Fig. 1A). Also employed in this study were the following previously described mutants. (i) dLAT2903 (20) (Fig. 1B) has LAT nucleotides −161 to 1667 deleted (a region including the primary LAT promoter and a putative second LAT promoter sometimes called LAP2). (ii) dLAT371 (17, 23) (Fig. 1C) contains a StyI-StyI restriction site fragment deletion in both copies of LAT (LAT nucleotides 76 to 447). The StyI-StyI deletion in dLAT371 and the StyI-HpaI deletion in dLAT1.5 begin at the same StyI site. Thus, the genomic structure of dLAT371 is identical to that of dLAT1.5 except for the size of the deletion in both copies of LAT. (iii) LAT2.9A (24) (Fig. 1F) contains the same LAT deletion as dLAT2903 (Fig. 1B) and therefore makes no LAT from the normal LAT genomic location. LAT2.9A contains an ectopic insert between UL37 and UL38 consisting of the entire LAT promoter and the first 1.5 kb of the primary LAT transcript from which the same StyI-StyI region deleted in dLAT371 (Fig. 1C) has been deleted. This virus therefore expresses only LAT nucleotides 1 to 76 plus 447 to 1499. (iv) LAT3.3A (22) (Fig. 1E), from which LAT2.9A was derived, is included here for clarity. This mutant, originally designated LAT1.5a, was renamed to maintain consistency with some of our other mutants. The 3.3 in the designation refers to the approximate size, in kilobases, of the ectopic insert.

FIG. 1.

Genomic structures of the viruses used in this study. (A) Prototypic genomic structure of parental wild-type McKrae and marker-rescued viruses dLAT1.5R, dLAT2903R, and dLAT371R. 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) dLAT2903 (20) has an EcoRV-to-HpaI 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) dLAT371 has a 371-nucleotide StyI-StyI 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)dLAT1.5 has a StyI-to-HpaI 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 dLAT2903. 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 StyI-StyI restriction fragment deletion from LAT nucleotide 76 to 447 in the ectopic LAT insert.

Spontaneous reactivation.

Rabbit eyes were infected with 2 × 10⁵ PFU of dLAT1.5, dLAT1.5R, or wild-type McKrae per eye as described in Materials and Methods. Starting at 31 days p.i., at which time latency had already been established, tear films were collected daily from all eyes and individually plated on indicator cells (RS cells) to detect spontaneously reactivated virus. Southern analysis of spontaneously reactivated viruses grown from tear films (not shown) confirmed that all cultures which scored positive based on the presence of viral CPE contained reactivated virus and that the spontaneously reactivated dLAT1.5 virus retained the deletion in both copies of LAT. The cumulative number of virus-positive tear film cultures per eye during 26 days for each virus are shown in Fig. 2A. Wild-type McKrae and dLAT1.5R appeared to have similar average cumulative spontaneous reactivation rates of approximately three positive cultures per eye on day 26 of collection. In contrast, dLAT1.5 appeared to have a lower average spontaneous reactivation rate of approximately 1.6 positive cultures per eye.

FIG. 2.

Spontaneous reactivation of dLAT1.5. Rabbits were ocularly infected with dLAT1.5 (dL1.5), marker-rescued dLAT1.5R (dL1.5R), 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 dLAT1.5 column, some of the zero points are plotted at 0.7 so that all of the zero points can be visualized. Similarly, in the dLAT1.5R 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 dLAT1.5R were similar (P > 0.05).

A statistical analysis of positive (spontaneously reactivated) tear film cultures versus total cultures is shown in Table 1. Approximately 6.1% of the tear films from rabbits latently infected with dLAT1.5 contained spontaneously reactivated virus. This was significantly less than the results for wild-type McKrae (11.8%) and dLAT1.5R (12.6%). Because the above analyses do not take into account the number of eyes in each group, we calculated the percentage of time that each eye in each group was positive for spontaneous reactivation (i.e., the percentage of virus- positive cultures) and plotted the results as scattergrams (Fig. 2B). This analysis is considered more stringent because it takes into account both the number of eyes and the period of time in a single calculation (22–24). By this analysis spontaneous reactivation of dLAT1.5 was also significantly reduced compared with those of the wild-type and marker-rescued viruses (P < 0.01). The above analyses indicated that spontaneous reactivation of dLAT1.5 was reduced compared with those of its marker-rescued virus and wild-type virus.

TABLE 1.

Spontaneous reactivation of dLAT1.5^a

Virus	No. of positive cultures/total (% positive)	P versus dLAT1.5b
dLAT1.5	38/624 (6.1)
dLAT1.5R	59/468 (12.6)	0.0002
McKrae	43/364 (11.8)	0.0025

^aNumerical presentation of the spontaneous reactivation data shown in Fig. 2. 

^bBy the chi-square test. 

Survival following ocular dLAT1.5 infection.

Survival in the rabbits used for the above-described spontaneous reactivation study was quantitated at 21 days p.i. (Fig. 3A). Although not statistically significant, there was a suggestion in this experiment of increased survival in the rabbits infected with dLAT1.5 (60%) compared with marker-rescued dLAT1.5R (45%) and wild-type McKrae (41%). Thus, it was of interest to determine if significant differences might be seen with larger numbers of animals. Due to difficulties with using large numbers of rabbits, we decided to examine survival in mice. Groups of 104 mice were infected with 10⁶ PFU of dLAT1.5 or dLAT1.5R per eye. Surprisingly, instead of the expected decrease in virulence, in mice dLAT1.5 was significantly more virulent than dLAT1.5R (Fig. 3B) (43% survival compared with 71% survival; P < 0.0001 [chi-square test]). In a separate experiment (see Fig. 9 below) the virulence of dLAT1.5R in mice was similar to that of the original wild-type parental virus (P = 0.39). Thus, as judged by survival of mice following ocular HSV-1 challenge, dLAT1.5 was more virulent than either wild-type virus or its marker-rescued virus.

FIG. 3.

Survival of rabbits (A) and mice (B) infected with dLAT1.5. (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. dL1.5, dLAT1.5; dL1.5R, dLAT1.5R; 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. 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; dL15R, marker-rescued dLAT1.5; dLATR, marker-rescued dLAT2903; dL371R, marker-rescued dLAT371; LAT2.9R, marker-rescued LAT2.9A.

Replication of dLAT1.5 in tissue culture, eyes, TG, and brains.

CV-1 cell monolayers were infected at a multiplicity of infection of 0.01 PFU/cell, and the kinetics of replication were determined as described in Materials and Methods. Replication of dLAT1.5 was indistinguishable from that of dLAT1.5R, wild-type virus, or the LAT-null mutant dLAT2903 (Fig. 4A). Rabbits were infected with 2 × 10⁵ PFU/eye. Tear films were collected from five rabbit eyes on days 3, 5, 7, and 10 p.i. Figure 4B shows the average amount of virus detected in the eye swabs at each time point. Although replication of dLAT1.5 appeared to be slightly less than that of dLAT1.5R or the wild type in rabbit eyes on day 5 p.i., the differences were not significant (P > 0.05 at all time points by analysis of variance [ANOVA]). In addition, the peak virus titers (day 5 p.i. for the wild type and dLAT1.5R; day 7 for dLAT1.5) appeared to be very similar for all three viruses (P = 0.8).

FIG. 4.

Replication of dLAT1.5 in tissue culture, eyes, TG, and brains. (A) Semiconfluent monolayers of CV-1 cells were infected with 0.01 PFU of dLAT1.5, dLAT1.5R, the LAT-null mutant dLAT2903, 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 dLAT1.5 to dLAT1.5R. Experiment 2 (five eyes per group) compared the wild type to dLAT1.5R. 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]; dLAT1.5 versus dLAT1.5R, P = 0.02 [Mann-Whitney test]; dLAT1.5 versus wild type, P = 0.008 [Mann-Whitney test]). (F) The experiment in panel E was repeated for dLAT1.5 and dLAT1.5R 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 dLAT1.5 (9 mice) and dLAT1.5R (10 mice) on day 7 p.i., and the results are plotted as in panel F. Error bars indicate standard deviations.

Mice were infected with 10⁶ PFU/eye, and replication in eyes was examined. Figure 4C shows the results of two independent experiments. In experiment 1, ocular replication of dLAT1.5 and dLAT1.5R were similar (P > 0.23 at each time point). In experiment 2, the wild type and dLAT1.5R were similar (P > 0.53 at each time point). Thus, replication of dLAT1.5 in mouse eyes was similar to that of its marker-rescued virus, which in turn was similar to that of the original parental wild-type virus. Thus, alterations in dLAT1.5 virulence (death rate) did not appear to be due to altered ocular replication.

For analysis of replication in mouse TG, five mice per group were ocularly infected for each time point shown (Fig. 4D), and the amount of infectious virus was determined individually for each TG at each time point. There was no significant difference among the dLAT1.5, dLAT1.5R, and wild-type viruses at any time point (P > 0.1 [ANOVA]). The amounts of virus in the brains of the same mice were similarly determined. On day 5 p.i. (Fig. 4E), the average amount of virus in the brains of mice infected with dLAT1.5 was significantly higher than that of either dLAT1.5R or the wild type (P = 0.005 [ANOVA]). On day 7 p.i., the day of peak virus titer in the brain, the amount of dLAT1.5 detected was 3.5- to 10-fold higher than that of dLAT1.5R or the wild type. However, this difference did not reach statistical significance (P = 0.1 [ANOVA]), perhaps because only five mice were used per time point. To increase the statistical power, additional experiments were performed using 9 or 10 mice per group. (Fig. 4F and G). On both days 5 and 7 p.i. significantly more virus was detected in the brains of dLAT1.5-infected mice than in those of dLAT1.5R-infected mice (P = 0.009 and P = 0.04, respectively [Mann-Whitney rank sum test]). Thus, it appears that dLAT1.5 grew more efficiently in mouse brains than its marker-rescued virus or wild-type virus. This was consistent with the increased virulence of dLAT1.5 in mice and suggests that the increased virulence in mice may have been due to increased virus load in the brain. Replication of dLAT1.5 in rabbit TG or brain was not examined both because this virus did not have significantly altered virulence in rabbits and because these analyses are highly variable in the rabbit.

Virulence of LAT2.9A in mice.

LAT3.3A (Fig. 1E) was constructed by inserting the LAT promoter and the first 1.5 kb of LAT into an ectopic location in the LAT-null mutant dLAT2903. This virus, which expresses just the first 1.5 kb of LAT, has wild-type virulence in rabbits (22) and mice (unpublished results). The LAT2.9A genome is structurally identical to that of LAT3.3A, with the exception of a 371-nucleotide deletion between LAT nucleotides 76 and 447 in the ectopic insert. We previously showed that LAT2.9A has increased virulence in rabbits (24). Because of the difference in virulence found above for dLAT1.5 in mice compared with rabbits, it was of interest to examine the virulence of LAT2.9A in mice, since to our knowledge, this is the only other LAT mutant reported to have altered virulence. Groups of 104 mice were therefore bilaterally ocularly infected with 10⁶ PFU/eye, and survival was determined on day 21 p.i. (Fig 5). LAT2.9A had significantly reduced virulence in mice compared with wild-type virus (P = 0.03). As shown below (see Fig. 9), the marker-rescued virus LAT2.9AR (24) and wild-type virus had similar virulence in mice (P = 0.19). Taken together, these two results indicate that LAT2.9A had reduced virulence in mice compared with both wild-type virus and its marker-rescued virus. Thus, as judged by the ability to kill animals following ocular infection, LAT2.9A had decreased virulence in mice and increased virulence in rabbits, while dLAT1.5 had increased virulence in mice but wild-type or slightly decreased virulence in rabbits.

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.

Replication of LAT2.9A.

We previously showed that replication of LAT2.9A in tissue culture and in rabbit eyes was similar to that of wild-type virus, suggesting that the increased virulence of LAT2.9A in rabbits is not due to enhanced replication (24). Replication in rabbit TG and brain was not examined because of problems with high variability, as mentioned above. To determine if the decreased virulence of LAT2.9A in mice might be related to decreased replication of the virus in mice, we examined replication in mouse eyes, TG, and brain. Mice were infected and replication was determined as described above for dLAT1.5, using either five eyes, five TG, or five brains per time point per group. The replication kinetics of LAT2.9A were indistinguishable from those of wild-type virus in the eyes, TG, and brains of mice (Fig. 6A, B, and C) (P > 0.05 at each time point). For further confirmation, 10 eyes, 10 TG, or 10 brains were similarly analyzed on the day of peak virus titer as determined from these results (Fig. 6D, E, and F). Again, no differences were detected (P > 0.05). Thus, the decreased virulence of LAT2.9A in mice did not appear to be a result of decreased virus load. Since replication of LAT2.9A was indistinguishable from that of wild-type virus, there was no need to compare replication of LAT2.9A with that of its marker-rescued virus.

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.

Virulence of dLAT371 in mice.

dLAT371 contains the same StyI-StyI restriction fragment deletion (LAT nucleotides 76 to 447) as LAT2.9A. Like dLAT1.5, dLAT371 contains a deletion starting at LAT nucleotide 76 in both copies of the otherwise unaltered LAT gene. The only difference between the structures of dLAT1.5 and dLAT371 is the size of the deletion (1,591 nucleotides for dLAT1.5 versus 371 nucleotides for dLAT371). Although LAT2.9A contains the same deletion as dLAT371, LAT2.9A contains only one copy of LAT, the LAT is in an ectopic location, and LAT2.9A transcribes LAT only up to LAT nucleotide 1499. Thus, although we have previously shown that dLAT371 has wild-type virulence in rabbits, it was of interest to examine the virulence of dLAT371 in mice.

As done above with dLAT1.5 and LAT2.9A, 104 mice per group were infected with 10⁶ PFU of dLAT371, dLAT2903, or dLAT2903R per eye. dLAT2903 is a LAT-null mutant, and dLAT2903R is its marker-rescued virus (Fig. 1A and B) (20). This particular combination of mutants was used because this experiment was originally aimed at comparing dLAT371 with the LAT-null mutant. dLAT371 had reduced virulence compared with either dLAT2903 or dLAT2903R (20) (Fig. 7) (P = 0.0004 and P = 0.008, respectively [chi-square] test). As shown below (see Fig. 9), in mice the virulences of dLAT2903R and dLAT371R (marker-rescued dLAT371) were both similar to that of the wild type (P = 0.19 and P = 0.68, respectively). Thus, consistent with LAT2.9A, but in contrast to dLAT1.5, in mice, dLAT371 was less virulent than wild-type virus or its marker-rescued virus.

FIG. 7.

Virulence of dLAT371 in mice. Mice were ocularly infected and survival was determined as described for Fig. 4. P was determined by the chi-square test. dL371, dLAT371; dLAT, dLAT2903; dLATR, dLAT2903R.

Replication of dLAT371.

We previously reported that replication of dLAT371 was indistinguishable from that of wild-type virus in tissue culture and in rabbit eyes. The kinetics of replication of dLAT371 in mouse eyes, TG, and brain were examined, using 10 samples per time point per group (Fig. 8A, B, and C). Scattergrams are also shown for the day of peak replication at each site (Fig. 8D, E, and F). Although dLAT371 appeared to grow slightly less well than wild-type virus in mouse eyes (P = 0.043 on day 3 p.i. [Mann-Whitney rank sum test]), replication of dLAT371 in mouse TG and brains was indistinguishable from that of wild-type virus (P > 0.05 for all times and P = 0.74 and P = 0.68, respectively, for peak titers). Thus, it appears unlikely that decreased replication of dLAT371 in mouse brains could account for the decreased virulence of this mutant in mice. Since replication of dLAT371 in TG and brains was wild type, comparisons with dLAT371R were not made.

FIG. 8.

Replication of dLAT371 (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.

Virulence of marker-rescued viruses.

Groups of mice were ocularly infected as described above, and virulence was determined based on survival on day 21 p.i. The virulences of dLAT1.5R, LAT2.9AR, dLAT371R, and dLAT2903R were all indistinguishable from that of wild-type McKrae (Fig. 9) (P > 0.05).

DISCUSSION

The virulence results presented here can perhaps be most simply summarized by separating the mouse and rabbit data. Thus, in mice, compared with wild-type virus and marker-rescued viruses (all of which had wild-type virulence), dLAT1.5 had increased virulence, while LAT2.9A and dLAT371 had decreased virulence. In rabbits, compared with wild-type and marker-rescued viruses, LAT2.9A had increased virulence, dLAT371 had wild-type virulence, and dLAT1.5 had wild-type or slightly reduced virulence.

LAT is essential for wild-type levels of spontaneous reactivation in the rabbit ocular model. We previously showed that expression of just the first 1.5 kb of the 8.3-kb LAT, as found in LAT3.3A, is sufficient for wild-type levels of spontaneous reactivation (22). Thus, a major function involved in spontaneous reactivation in the rabbit maps to within the first 1.5 kb of LAT, a region representing less than 20% of the primary LAT transcript. dLAT1.5 was constructed and studied in this work because it represents a mutant complementary to LAT3.3A. Thus, while LAT3.3A expresses only the first 1.5 kb of LAT, dLAT1.5 expresses all of LAT except for a similar region (LAT nucleotides 76 to 1667). As expected, like the LAT-null mutant dLAT2903, dLAT1.5 had reduced spontaneous reactivation. This demonstrated that in the rabbit ocular model, not only is the first 1.5 kb of LAT sufficient for wild-type spontaneous reactivation, but part or all of the first 1.5 kb is also required for wild-type levels of spontaneous reactivation. In addition, these results suggest that LAT does not contain a region outside of the first 1.67 kb of LAT that is independently able to support wild-type levels of spontaneous reactivation

We previously showed that compared with wild-type virus, LAT2.9A (Fig. 1E) has increased virulence in rabbits (i.e., increased death rate following ocular infection) compared with marker-rescued or wild-type virus (24). In contrast, as reported here, in mice LAT2.9A was significantly less virulent than marker-rescued or wild-type virus. Thus, the virulence phenotype for LAT2.9A not only appeared to be very different in mice compared with rabbits, it appeared to be asymmetric in rabbits compared with mice (i.e., more virulent than the wild type in one species and less virulent than the wild type in another species).

Note that in this report we use the term virulence to mean death following ocular infection. We are not distinguishing between neuroinvasiveness (the ability of the virus to get to the brain following infection at a peripheral site) and neurovirulence (the ability of the virus to kill the animal once it reaches the brain).

We also showed in this report that following ocular infection of rabbits, dLAT1.5 had virulence similar to or slightly less than that of wild-type virus or marker-rescued dLAT1.5R. In contrast, in mice, dLAT1.5 was significantly more virulent than the wild type or dLAT1.5R. Thus, the virulence phenotype for dLAT1.5 also appeared to differ in mice compared with rabbits. The asymmetric virulence results in mice versus rabbits with LAT2.9A and dLAT1.5 were unexpected. Although mutations that alter the virulence of HSV-1 in mice but not rabbits have been reported (38), we are not aware of any previous report of a mutant that is more virulent than its marker-rescued and parental wild-type viruses in one animal species and less virulent than its marker-rescued and parental wild-type viruses in a second species.

Also surprising was that dLAT1.5 and LAT2.9A had opposite phenotypes. That is, compared with wild-type virus and the mutant-specific marker-rescued virus, one mutant had decreased virulence in mice while the other mutant had increased virulence in mice, and vice versa in rabbits. This is of particular interest, because both of these viruses contain deletions of part of the LAT gene. The most direct conclusion to draw from these results is that different LAT deletion mutants can affect virulence differently and that the direction of virulence (i.e., increased or decreased compared with the wild type) can differ in mice versus rabbits. This in turn strongly suggests that in addition to being involved in the HSV-1 latency cycle of establishment of and reactivation from latency, LAT is also involved in viral virulence.

Because dLAT1.5 and LAT2.9A have significant genomic structural differences in addition to the size of the LAT deletion beginning at LAT nucleotide 76, it was of interest to also examine the virulence of a third LAT mutant, dLAT371 (23). All three of these mutants (dLAT1.5, LAT2.9A, and dLAT371) contain a deletion starting at LAT nucleotide 76 (Fig. 1). dLAT371 and dLAT1.5 have identical genomic structures except for the size of the deletion (371 versus 1,591 nucleotides). In contrast, LAT2.9A and dLAT371 have significantly different genomic structures (Fig. 1C and F) but contain identical 371-nucleotide deletions in LAT. Consistent with LAT2.9A, but in contrast to dLAT1.5, dLAT371 had reduced virulence in mice. Thus, in mice, the larger 1,591-nucleotide deletion in dLAT1.5 resulted in increased virulence, while the smaller 371-nucleotide deletion in dLAT371 and LAT2.9A resulted in decreased virulence.

We previously reported that dLAT371 has wild-type virulence and wild-type spontaneous reactivation in rabbits (23). This is in contrast to LAT2.9A, which has increased virulence and decreased spontaneous reactivation in rabbits. Since these mutants have identical deletions in the 5′ end of LAT, the phenotypic differences in rabbits appear to be due to either (i) the lack of LAT nucleotides 1500 to 8324 in LAT2.9A, (ii) undetected differences in LAT expression or stability in LAT2.9A compared with dLAT371, (iii) the single copy of LAT in LAT2.9A, or (iv) the ectopic location of LAT in LAT2.9A. However, we think that the ectopic location and the single LAT copy are unlikely possibilities, since LAT3.3A and LAT2.9AR, which contain the same single-copy ectopic insert but without the 371-nucleotide deletion, both have wild-type virulence and wild-type spontaneous reactivation.

HSV-1 kills rabbits and mice by causing encephalitis. In this study, virulence refers to this encephalitic death following ocular infection. In a simple model, the relative level of HSV-1 virulence would be expected to be directly related to the virus load in the brain. That is, if there was more virus in the brain, there would be more encephalitic death. For dLAT1.5 in mice, this situation appeared to hold. dLAT1.5 was more virulent in mice than its marker-rescued virus, and the amount of dLAT1.5 in mouse brains was significantly increased compared with that of its marker-rescued virus. In contrast, this model did not appear to hold for LAT2.9A or dLAT371 in mice. These mutants had reduced virulence in mice, yet the viral load in the brain was the same as that of wild-type virus. This strongly suggests, and supports a previous report (15), that the virus load in the brain is not always a good predictor of neurovirulence for HSV-1.

LAT may partially inhibit productive gene expression (3, 10, 18). In addition, it has been proposed that a 0.7-kb transcript beginning and terminating just upstream of the LAT promoter affects viral virulence (38). Both of these findings suggest that deletion of the LAT promoter in LAT-null mutants might alter virulence. However, we are not aware of any reports of a LAT-null mutant with altered virulence (1, 5, 14, 16, 20). We have, however, previously reported that LAT2.9A has increased virulence in rabbits (24). Thus, although complete lack of LAT expression (see Fig. 1B) does not appear to alter virulence, expression of just specific portions of LAT, as in LAT2.9A (Fig. 1F), dLAT371 (Fig. 1C), and dLAT1.5 (Fig. 1D), can alter virulence. Thus, we have a situation in which a large deletion (i.e., LAT-null mutants, which have no LAT promoter and which are therefore equivalent to deletion of the entire gene) has no impact on virulence, while smaller deletions which are encompassed by the larger deletion (those in LAT2.9A, dLAT371, and dLAT1.5) do alter virulence. As we previously speculated in regard to LAT2.9A (24), this situation is similar to that seen in promoter-mapping studies. Because promoters usually have multiple functional elements that may work either in concert or antagonistically, deletion of increasingly larger portions of the 5′ end of a promoter often produces multiple increases and decreases in promoter activity. The situation with LAT2.9A, dLAT371, and dLAT1.5 is analogous. These different deletions produced different increases and decreases in virulence, suggesting that LAT may have multiple functional elements. Since LAT may not encode a protein (8), it is possible that the LAT RNA may directly or indirectly regulate the expression or function of viral and/or cellular genes that in turn influence virulence. In this case, the different deletions may alter the LAT RNA structure or stability, which in turn affects the LAT RNA function.

Finally, the results presented here strongly suggest that LAT has multiple functions, since it affects both virulence and spontaneous reactivation.