Abstract
The herpes simplex virus type 1 (HSV-1) latency-associated transcript (LAT) gene is essential for efficient spontaneous reactivation of HSV-1 from latency. We previously reported that insertion of the LAT promoter and just the first 1.5 kb of the 8.3-kb LAT gene into an ectopic location in the virus restored wild-type spontaneous reactivation to a LAT null mutant. This mutant, LAT3.3A (previously designated LAT1.5a), thus showed that the expression of just the first 1.5 kb of LAT is sufficient for wild-type spontaneous reactivation. We also showed that in the context of the entire LAT gene, deletion of LAT nucleotides 76 to 447 (LAT mutant dLAT371) had no effect on spontaneous reactivation or virulence. We report here on a LAT mutant designated LAT2.9A. This mutant is similar to LAT3.3A, except that the ectopic LAT insert contains the same 371-nucleotide deletion found in dLAT371. We found that LAT2.9A had a significantly reduced rate of spontaneous reactivation compared to marker-rescued and wild-type viruses. This was unexpected, since the combined results of dLAT371 and LAT3.3A predicted that spontaneous reactivation of LAT2.9A would be wild type. We also found that LAT2.9A was more virulent than wild-type or marker-rescued viruses after ocular infection of rabbits. This was unexpected, since LAT null mutants and LAT3.3A have wild-type virulence. These results suggest for the first time (i) that regions past the first 1.5 kb of LAT can compensate for deletions in the first 1.5kb of LAT and may therefore play a role in LAT dependent spontaneous reactivation and (ii) that regions of LAT affect viral virulence.
After ocular herpes simplex virus type 1 (HSV-1) ocular infection, the virus travels up nerves to the trigeminal ganglia (TG), where it establishes a latent infection. Latency lasts for the life of the infected individual. HSV-1 can reactivate from latency and travel back to the eye, where it can be detected in tears and cause recurrent corneal disease. Recurrent ocular HSV-1 can lead to scarring of the cornea and loss of sight. In developed nations, HSV-1 is the most common cause of corneal blindness due to an infectious agent (12). How HSV-1 establishes, maintains, and reactivates from latency remains unknown.
The latency-associated transcript (LAT) is the only viral gene that is abundantly transcribed during latency (20). LAT is located in the long repeat region of the viral genome and thus is present in two copies per genome. LAT is initially transcribed as an 8.3-kb RNA (4, 26). This primary LAT transcript gives rise to a family of LAT RNAs, including the very stable 2-kb LAT (20, 24, 25), which appears to be an intron produced by splicing (6). LAT transcription-negative mutants have been shown to reactivate poorly by explant or induced reactivation in the mouse (9, 10, 21), by induced reactivation in the rabbit (1, 23), and by spontaneous reactivation in the rabbit (14, 17). Thus, LAT is essential for efficient, wild-type reactivation from sensory neurons.
The mechanism by which LAT functions remains unknown. No LAT-encoded protein has been detected during latency and there does not appear to be a LAT open reading frame that is well conserved among LAT genes capable of sustaining spontaneous reactivation (5). Thus, in the absence of undetected, atypical splicing, it is unlikely that LAT’s function is due to a LAT protein. LAT’s function also does not appear to be due to antisense downregulation of the important immediate-early gene ICP0, which LAT overlaps in an antisense direction. We recently showed that the first 1.5 kb of LAT alone is sufficient for wild-type levels of spontaneous reactivation (17). This region does not overlap any portion of any known HSV-1 gene.
Mapping a LAT spontaneous reactivation function to the first 1.5 kb of LAT was done by inserting the LAT promoter and the first 1.5 kb of LAT into an ectopic location in the genome of a LAT null mutant between HSV-1 genes UL37 and UL38 (17). This completely restored wild-type levels of spontaneous reactivation. Another LAT mutant, dLAT371, containing a StyI-StyI deletion that removed LAT nucleotides 76 to 447 was also wild type for spontaneous reactivation (18). Thus, this 371-nucleotide region within the first 1.5 kb of the primary LAT transcript did not appear to be essential for efficient spontaneous reactivation.
We report here on a mutant that is a combination of LAT3.3A and dLAT371. This virus, designated LAT2.9A, contains a LAT insert at the same ectopic location as LAT3.3A. This insert is identical to that in LAT3.3A (i.e., the LAT promoter and the first 1.5 kb of LAT), except that LAT nucleotides 76 to 447 are deleted. As with LAT3.3A, the LAT promoter and the first 1.67 kb of LAT are deleted from both copies of LAT in the long repeat and therefore the only LAT produced originates from the ectopic insert. We predicted that LAT2.9A, like LAT3.3A and dLAT371, would have wild-type spontaneous reactivation. Instead, LAT2.9A had a significantly reduced spontaneous reactivation rate that was comparable to that of the LAT null mutant dLAT2903. LAT2.9A also was significantly more virulent than wild-type virus. This was also unexpected, since dLAT2903, LAT3.3A, and dLAT371 all appeared to have wild-type virulence.
MATERIALS AND METHODS
Cells and virus.
Rabbit skin (RS) cells were grown in Eagle minimal essential media (MEM) supplemental 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 the 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, LAT3.3A (previously designated LAT1.5a), and dLAT371 have been previously described (14, 17, 18).
Construction of LAT2.9A.
The parental virus for this construct was dLAT2093, 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 (14). The previously cloned EcoRI A fragment from HSV-1 strain McKrae (15) was digested with BamHI, and the products were separated by agarose gel electrophoresis. A resulting 7.5-kb band containing the McKrae genomic region including UL37 and UL38 was isolated by electroelution and cloned into the BamHI site of plasmid pEV-vrf3 (3, 15) to produce the plasmid pV375. pV375 was digested with AflII, the overhang was filled in using the Klenow fragment, and the blunt ends were self-ligated to create a unique PacI site in the plasmid between the sequences for UL37 and UL38. The resulting plasmid, designated pV375Pac, was amplified by transformation into Escherichia coli RR1λCI857 according to standard protocol. An HpaI-HpaI restriction fragment consisting of 1.8 kb of the LAT promoter and the first 1.5 kb of the LAT RNA (17) was cloned into the PacI site of pNEB193 and further digested with StyI to remove a 371-nucleotide StyI-StyI region corresponding to LAT nucleotides 76 to 447. The plasmid was then self-ligated and digested with PacI, and the resulting 2.9-kb band was cloned into the PacI site of pV375Pac to produce pV375LAT2.9.
LAT2.9A was generated by homologous recombination as we previously described (14, 17–19). Briefly, pV375LAT2.9 was cotransfected with infectious dLAT2903 (the LAT deletion mutant described above) DNA by the calcium phosphate method. Viruses from the cotransfection were plated, and isolated plaques were picked and screened for insertion of the 2.9-kb LAT fragment between UL37 and UL38 by restriction digestion and Southern analysis. Selected plaques were triple plaque purified and reanalyzed by restriction digestion and Southern analysis to ensure that the 2.9-kb fragment was present between UL37 and UL38 and that both long repeats retained the original 1.8-kb LAT deletion of the promoter and first 1.6 kb of the 5′ end of the primary LAT transcript (see Fig. 1 and 2). A final plaque was purified and designated LAT2.9A (LAT2.9 indicated the 2.9-kb LAT fragment; A indicates addition). The marker rescued virus LAT2.9AR, was generated as described above by homologous recombination of LAT2.9A DNA with the plasmid pV375LAT3.3. This restored the 371-nucleotide StyI-StyI deletion in the ectopic LAT insert and rescued LAT2.9A back to a wild-type LAT3.3A structure.
FIG. 1.
Construction and structure of LAT2.9A. (A) The top panel (wt McKrae, dLAT2903R, and dLAT371R) shows a schematic representation of the genome of wild-type McKrae and the wild-type marker-rescued viruses dLAT2903R and dLAT371R, which are identical to wild-type HSV-1. The prototypic orientation of HSV-1 shown here contains a unique long region and a unique short region, each bounded by inverted repeats. The unique regions are shown as solid lines. The repeats are shown as open rectangles. UL, unique long; US, unique short; TRL, terminal repeat long; IRL, internal repeat long; IRS, internal repeat short; TRS, terminal repeat short. The lines with arrows under the genome indicate the locations and directions of the LAT, ICP34.5, and ICP0 transcripts. The solid rectangle within the primary 8.3-kb LAT transcript indicates the location of the stable 2-kb LAT. TATA indicates the location (in the genomic DNA) of the LAT promoter TATA box. The next panel shows that dLAT2903, the previously described LAT null mutant (14), has a deletion extending from LAT nucleotides −161 to +1667. This deletion encompasses the LAT promoter, and dLAT2903 therefore cannot transcribe any LAT RNA. The dashed rectangle and the preceding dashed line covered by “XXXXX” represent the portion of LAT that is deleted from both long repeats. Panel dLAT371 shows a previously described mutant containing a deletion of LAT nucleotides 767 to 447 in both copies of LAT (18). The blow-up in panel LAT3.3A and LAT2.9R shows the location of the 3.3-kb LAT fragment inserted between genes UL37 and UL38 in the unique long region of the LAT deletion mutant dLAT2903 to generate the virus LAT3.3A (17). The insert contains 1.8 kb of the LAT promoter and the first 1.5 kb of the primary LAT. The insertion site is outside the domains of the UL37 and UL38 promoters, and these genes are not affected (17). The structure of LAT2.9AR, a marker-rescued virus derived from LAT2.9A (see below) is identical to LAT3.3A. Panel LAT2.9A shows that LAT2.9A contains the same insert between UL37 and Ul38 as LAT3.3A, except that the StyI-StyI region (LAT nucleotides 76 to 447) has been deleted from the insert. This deletion corresponds to the deletion in dLAT371 described above. (B) The LAT RNAs made by each of the viruses shown in panel A are indicated. Wild-type McKrae, dLAT2903R, and dLAT371R all make the complete primary LAT transcript. The solid rectangle starting at LAT nucleotide 662 indicates the location of the stable 2-kb LAT. dLAT2903 makes no LAT RNA. dLAT371 transcribes all of the LAT except nucleotides 76 to 447. LAT3.3A and LAT2.9AR make no LAT from the original LAT region (one in each long repeat), but they do transcribe the first 1.5 kb of LAT from the LAT insert between UL37 and UL38. LAT2.9A also makes not LAT from the original LAT region. It transcribes LAT nucleotides 1 to 76 and 447 to 1499 (i.e., the first 1.5 kb of LAT minus the StyI-StyI region).
FIG. 2.
Southern analysis of LAT2.9A. (A) Viral DNAs were isolated, individually digested with PacI, and hybridized to a 32P-labeled probed corresponding to part of the LAT region deleted in dLAT2903 but present in wild-type McKrae and the LAT insert in LAT2.9A (see text). (B) The viral DNAs were digested with BamHI and probed with the BamHI-H restriction fragment that hybridizes to the UL37–UL38 region, but not to LAT (see text). (C) The viral DNAs were digested with BamHI and probed with a cloned HpaI-MluI restriction fragment corresponding to LAT nucleotides 1,667 to 2850 (see the text) that should hybridize to LAT DNA from the long repeats but not to the LAT inserts in the UL37–UL38 region (see text). Lanes: 1, wild-type McKrae; 2, dLAT2903; 3, LAT2.9A; 4, LAT2.9AR; 5, LAT3.3A.
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 the monolayers plus media (−80°C to room temperature). The PFU/milliliter values were determined by standard plaque assays on RS cells.
Rabbits.
Eight- to ten-week old New Zealand White female rabbits (Irish Farms) were used for all experiments. Rabbits were treated in accordance with ARVO (Association for Research in Vision and Ophthalmology), AALAC (American Association for Laboratory Animal Care), and NIH (National Institutes of Health) guidelines.
Rabbit model of ocular HSV-infection, latency, and spontaneous reactivation.
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 trigeminal ganglia, resulting in a high group rate of spontaneous reactivation with the McKrae strain of HSV-1. Latency is assumed to have been established by 28 days postinfection. Acute ocular infection of all eyes was confirmed by HSV-1 positive tear film cultures collected on days 3 and 4 postinfection.
Detection of spontaneous reactivation by ocular shedding.
Beginning on day 31 postinfection, tear film specimens were collected daily from each eye for 26 days as previously described (22), 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 primary rabbit kidney cell monolayers. These cell monolayers were observed in a masked fashion by phase light microscopy for up to 30 days to monitor HSV-1 cytopathic effects (CPE). All positive monolayers were blind passaged onto fresh cells to confirm the presence of virus. DNA was purified from randomly selected positive cultures derived from latently infected rabbits and analyzed by restriction enzyme digestion and Southern blots to confirm that the CPE was due to reactivated HSV-1 and that the reactivated virus was identical to the input virus.
Virus replication in rabbit eyes.
Tear films were collected as described above on various days postinfection. The amount of virus in each tear film was determined by standard plaque assays on RS cells.
RT-PCR.
Reverse transcriptase (RT)-PCR was done as we previously described (17) with minor modifications. Briefly, RNA was isolated with Trizol (Gibco-BRL, Grand Island, N.Y.) from individual TG from latently infected rabbits or from infected CV-1 cell monolayers and treated with DNase I (12 U, 37°C, 30 min; Stratagene, La Jolla, Calif.). RNA was isolated with an RNeasy Mini-Kit (Quiagen, Santa Clarita, Calif.). The purified RNAs were subjected to first-strand cDNA synthesis by Superscript II (Gibco-BRL) according to the manufacturer’s protocol. The primer for first-strand cDNA synthesis from the LAT RNA was 5′-CTTTGTTGAACGACACCGGGGCGCCCTCGA-3′. The cDNA product was then amplified by PCR with the primer 5′-CCACAACGGCCCGGCGCATGCGCTGTGGTT-3′ and the first-strand primer. These primers generate a 160-bp product specific for LAT nucleotides 471 to 631. The amplified products were fractionated by gel electrophoresis, transferred to a nylon membrane, and hybridized to the 32P-labeled internal probe 5′-TCTCCCCCCCCCCTTCTTCACCCCCAGTAC-3′ corresponding to LAT nucleotides 550 to 580.
Statistical analysis.
Statistical analyses were performed by using Instat, a personal computer software program. For analyses with either the Student t test, the Mann-Whitney rank sum test, the chi-square test, or the Fisher exact test the results were considered statistically significant when the P value was <0.05.
RESULTS
Structure of LAT2.9A.
The genomic structures of wild-type HSV-1 McKrae, LAT2.9A, and the other viruses used in this study are shown in Fig. 1A. All of the viruses were derived from HSV-1 strain McKrae. The construction and properties of dLAT2903 and its marker-rescued virus dLAT2903R, dLAT371 and its marker-rescued virus dLAT371R, and LAT3.3A (previously designated LAT1.5a) have been described previously (14, 17, 18). The LAT transcript(s) made by each virus are detailed in Fig. 1B. Wild-type McKrae and the marker-rescued viruses dLAT2903R and dLAT371R contain two copies of LAT, one in each viral long repeat (Fig. 1A, top). The viral long repeats are expanded (in dashed lines) to show the relative location and status of the LAT gene. In the topmost panel, the location of the ICP0 and ICP34.5 genes are shown for reference. The primary LAT transcript in the wild-type and the marker-rescued viruses is approximately 8.3 kb (Fig. 1A, top panel, and Fig. 1B) (24, 25). A very stable and easily detected 2-kb LAT (solid rectangle) appears to be an intron derived by splicing of the primary LAT (6). dLAT2903 contains a deletion in both copies of LAT from −161 to +1,667 relative to the start of the primary LAT transcript (Fig. 1A, indicated by “XXXXX”). This virus is missing key promoter elements, makes no LAT RNA (Fig. 1B), and is a true LAT null mutant. dLAT371 contains a 371-nucleotide deletion of LAT nucleotides 76 to 447, corresponding to a StyI-StyI region prior to the 2-kb LAT (Fig. 1A, third panel, indicated by “X”). This virus makes a normal primary LAT transcript except that it is missing LAT nucleotides 76 to 447 (Fig. 1B). LAT3.3A is derived from dLAT2903 by insertion of 1.8 kb of the LAT promoter and the first 1.5 kb of LAT into a unique PacI site that was constructed between UL37 and UL38 (Fig. 1A). LAT3.3A and LAT2.9AR make no LAT RNA from either copy of LAT in the long repeats, but they do make a 1.5-kb LAT RNA from the ectopic insert that corresponds to the first 1.5 kb of the primary LAT (Fig. 1B). LAT2.9A is identical to LAT3.3A, except that LAT nucleotides 76 to 447 have been deleted from the inserted 1.5-kb LAT region (Fig. 1A). LAT2.9A therefore transcribes an RNA of 1,128 nucleotides corresponding to LAT nucleotides 1 to 76 and 447 to 1,499. The construction of all the above mutants except LAT2.9A and LAT2.9AR have been previously described (14, 17, 18). Additional details of the construction of LAT2.9A and LAT2.9AR are given in Materials and Methods.
Southern analysis of the structure of LAT2.9A.
Southern analyses of viral DNAs were performed to confirm the structure of LAT2.9A (Fig. 2). DNAs were individually digested with PacI and probed with a 32P-labeled restriction fragment (StyI-HpaI; LAT nucleotides 447 to 1,499) that is completely within the LAT region deleted in dLAT2903 and that therefore can hybridize only with LAT sequences in the LAT2.9A insert (Fig. 2A). The wild-type virus (lane 1) produced a single large band of genome size as expected, since PacI does not cut wild-type HSV-1. The dLAT2903 DNA produced no band (lane 2), since the sequences corresponding to the probe are deleted from this virus. PacI should cut the PacI sites flanking the 2.9-kb LAT fragment inserted between UL37 and UL38 in LAT2.9A to produce a single band of 2.9 kb that hybridizes to the probe as is seen in lane 3. Similarly, PacI cuts the correspondingPacI sites in LAT2.9AR and LAT3.3A, producing bands of 3.3 kb (lanes 4 and 5). These results indicate that LAT2.9A contains the expected 2.9-kb LAT insert between UL37 and UL38 and that the corresponding region in LAT2.9AR has been rescued back to the wild-type size (lane 4) seen in LAT3.3A (lane 5).
The viral DNAs in Fig. 2B were digested with BamHI and probed with the BamHI H restriction fragment that hybridizes to the UL37–UL38 region but not to any of the LAT sequences (see top of Fig. 1 for location of BamHI H). Both wild type (lane 1) and dLAT2903 (lane 2) show a single band of 7.5 kb corresponding in size to the BamHI H restriction fragment containing UL37 and UL38. Lanes 3, 4, and 5 contain only a slower-migrating band of 10.4 or 10.8 kb, corresponding to BamHI H plus the 2.9- or 3.3-kb LAT insert. This again indicates that LAT2.9A contains the appropriate LAT fragment inserted between UL37 and UL38. The 371-nucleotide difference between the 10.4-kb LAT2.9A band and the 10.8-kb LAT2.9AR and LAT3.3A bands cannot be distinguished because of their relatively large size compared to the deletion.
The viral DNAs in Fig. 2C were also digested with BamHI but were hybridized to a probe specific for a region of LAT that is present in wild type and dLAT2903 but not in the LAT insert of LAT2.9A or LAT3.3A (a cloned HpaI-MluI restriction fragment corresponding to LAT nucleotides 1,667 to 2,850). This probe should hybridize only to the BamHI B and BamHI E restriction fragments generated from the long repeats (one from each repeat; locations shown at the top of Fig. 1) that contain the HSV-1 sequence corresponding to the probe. As seen in Fig. 2C, lane 1, these two bands are of different sizes because in both restriction fragments one BamHI cut is in the repeat and one is in the adjacent unique long region. The larger band contains LAT from the internal repeat, while the smaller band is from the terminal repeat. Both of these bands are smaller in the original LAT deletion mutant, dLAT2903 (lane 1) and in LAT2.9A, LAT2.9AR, and LAT3.3A, (lanes 3 to 5), a finding indicative of the 1.8-kb LAT deletion in each long repeat in these viruses. This confirms that in these viruses the original LAT deletion was retained in both original copies of LAT and that these viruses are therefore incapable of transcribing any LAT RNA from either normal LAT gene. Combined, the Southern analyses shown in Fig. 2 confirm the structure of the LAT2.9A and LAT2.9AR viruses.
In vitro replication of LAT2.9A. Monolayers of CV-1 cells were infected with 0.01 PFU of LAT2.9A, wild-type McKrae, LAT3.3A, dLAT2903, or dLAT2903R per cell. The monolayers were harvested by freeze-thawing at the indicated times (Fig. 3A), and the virus yield was determined by standard plaque assays as described in Materials and Methods. As we previously showed (14, 17), replication of LAT3.3A, dLAT2903, and dLAT2093R, were all wild type in tissue culture. LAT2.9A also replicated with wild-type kinetics.
FIG. 3.
Replication of LAT2.9A (A) Semiconfluent monolayers of CV-1 cells were infected with 0.01 PFU of the indicated virus per cell. At various times, the infected cell monolayers were harvested by freeze-thawing and the amount of virus was determined by plaque assay on RS cells. Each time point is the average of two determinations. (B) Tear swabs were performed on the eyes of rabbits infected with the indicated virus at various times postinfection. The amount of virus present in individual tear swabs was determined by plaque assay on RS cells. Each point represents the mean titer from five eyes, each from a different rabbit. Error bars indicate the standard deviation.
In vivo replication of LAT2.9A.
Rabbits were infected with 2 × 105 PFU of LAT2.9A, dLAT2903, dLAT2903R, or LAT3.3A per eye. Tears were collected at the indicated times (Fig. 3B), and the virus yield was determined by plaque assay. As we have previously shown (14, 17), replication of dLAT2903 and LAT3.3A in rabbit eyes was similar to that of wild type (dLAT2903R) virus. Replication of LAT2.9A (Fig. 3B, open circles) appeared to be slightly lower than that of LAT3.3A and dLAT2903R (solid circles and squares) on days 3 and 5 but not on day 7. However, as indicated by the overlapping error bars, these minor differences were not significant.
Reduced survival of rabbits ocularly infected with LAT2.9A.
Eighteen rabbits per group were infected with 2 × 105 PFU of LAT2.9A, dLAT2903, dLAT2903R, or LAT3.3A per eye. Only 17% of the LAT2.9A-infected rabbits survived for 21 days (Table 1, experiment 1, column 3) compared to 33-39% survival for the other groups. Since dLAT2903, dLAT2903R, and LAT3.3A all have wild type parental McKrae virulence in rabbits (14, 17), it appeared that survival of LAT2.9A may have been reduced compared to wild type. However, the differences were not significant (Table 1, experiment 1, column 4). To determine if the tendency in experiment 1 for LAT2.9A to be more virulent than wild type was meaningful, additional experiments were done. In a second experiment, 22 rabbits per group were infected with LAT2.9A or dLAT2903R (Table 1, experiment 2). Again, the percentage of rabbits surviving infection with LAT2.9A appeared to be reduced compared to wild type (32 versus 55%). However, as in experiment 1, the differences was not statistically significant (Table 1, column 3).
TABLE 1.
Survival of rabbits infected with LAT2.9A
| Expt | Virusa | Survival (%)b | P vs LAT2.9Ac |
|---|---|---|---|
| 1 | LAT2.9A | 3/18 (17) | |
| dLAT2903 (LAT−) | 7/18 (39) | 0.26 | |
| LAT3.3A (wt) | 6/18 (33) | 0.44 | |
| dLAT2903R (wt) | 7/18 (39) | 0.26 | |
| 2 | LAT2.9A | 7/22 (32) | |
| dLAT2903R (wt) | 12/22 (55) | 0.22 | |
| 3 | LAT2.9A | 4/16 (25) | |
| LAT2.9AR | 7/16 (44) | 0.46 | |
| dLAT371 (wt) | 7/16 (44) | 0.46 | |
| dLAT371R (wt) | 11/16 (69) | 0.032 | |
| Totalsd | LAT2.9A | 14/56 (25) | |
| dLAT2903 | 34/68 (50) | 0.006 | |
| Wild-type viruses | 50/106 (47) | 0.007 |
Viruses designated “(wt)” have previously been shown to be have the same virulence rate based on rabbit survival as the parental McKrae wild-type virus.
Rabbits were ocularly infected as described in Materials and Methods. Survival was determined on day 21 postinfection as the number of surviving animals/total rabbits infected with each virus.
Fisher exact test. P < 0.05 indicates statistical significance.
Wild-type viruses are a compilation of all of the above wild-type viruses plus LAT2.9AR, which appeared to behave like wild type. dLAT2903 data was compiled from experiment 1 and the four most recent experiments in our laboratory that included dLAT2903 as a negative control.
In a third experiment, 16 rabbits were infected per group. This experiment included dLAT371 and its rescued virus, dLAT371R, as controls, since dLAT371 contains the same StyI-StyI deletion as LAT2.9A (Fig. 1). In addition, a marker-rescued version of LAT2.9A, designated LAT2.9AR, was included to determine if the apparent reduced virulence of LAT2.9A corresponded to the StyI-StyI deletion. Again, LAT2.9A appeared to be more virulent than each of the other groups (Table 1, experiment 3, column 3) although statistical significance was only reached between LAT2.9A and dLAT371R (Table 1, experiment 3).
Because the same pattern of a tendency toward increased virulence of LAT2.9A was seen in all experiments and between LAT2.9A and each of the wild-type or rescued groups within each experiment, it was statistically meaningful to compile and compare the totals (Table 1). Of 56 rabbits infected with LAT2.9A, only 25% survived. In contrast, 47% of 106 rabbits infected with wild-type virus, marker-rescued virus, or mutants with wild-type virulence survived. This difference was significant (P = 0.007). It was also of interest to compare LAT2.9A to dLAT2903, a virus that makes no LAT, since LAT2.9A is derived directly from dLAT2903. For this purpose, we compiled the results of experiment 1 with those of the four most recent experiments in our laboratory in which dLAT2903 had been included as a negative control for spontaneous reactivation. Like the wild-type viruses (P = 0.76; not shown), but in contrast to LAT2.9A (P = 0.006), 50% of the 68 dLAT2903-infected rabbits survived. These results strongly suggest that LAT2.9A was more virulent than either wild-type or LAT-negative viruses.
Reduced spontaneous reactivation of LAT2.9A.
Beginning at 31 days postinfection (at which time latency had already been established), all eyes from the surviving rabbits in experiments 1, 2, and 3 were swabbed once a day for 26 days to collect tear films for analysis of reactivated virus as described in Materials and Methods. In experiment 1, LAT2.9A was compared to dLAT2903, which has reduced spontaneous reactivation, and to LAT3.3A and dLAT2903R, both of which have spontaneous reactivation rates identical to wild-type McKrae. The cumulative number of virus-positive tear film cultures is shown in Fig. 4A. Because of the different numbers of surviving rabbits in the different groups, the data were standardized to represent cumulative positive cultures per eye. The cumulative spontaneous reactivation rate in rabbits latently infected with LAT2.9A (Fig. 4A, open circles; approximately 1.8/eye on day 26) appeared to be less than dLAT2903R and LAT3.3A (Fig. 4A, solid squares and circles; approximately 3 to 3.5/eye) and slightly greater than dLAT2903 (Fig. 4A, open squares; approximately 1/eye).
FIG. 4.
Spontaneous reactivation of LAT2.9A. Each panel shows an independent experiment, each of which was performed at a different time. Rabbits were infected with 2 × 105 PFU of the indicated virus per eye. Rabbits surviving past day 21 are considered to have a latent HSV-1 infection in both TG. Beginning 31 days postinfection (day 1 of the collection period), tear swabs were collected daily for 26 days from all eyes and plated on RS to look for the presence of spontaneously reactivated virus. The cumulative number of virus-positive cultures divided by the number of eyes in the group is shown. Panels A, B, and C indicate the results of experiment 1, 2, and 3, respectively. dLAT2903R, LAT3.3A, dLAT371R, all of which have previously been shown to have wild-type McKrae spontaneous reactivation rates, were used as positive controls in various experiments.
The cumulative data for positive (spontaneously reactivated) cultures versus total cultures (Table 2, experiment 1, column 3) indicated that 7.1% of the tear film cultures from rabbits latently infected with LAT2.9A virus contained spontaneously reactivated virus compared to 3.6% of the tears from eyes infected with dLAT2903. In contrast, 12.2 and 13.7% of tears from eyes infected with LAT3.3A and dLAT2903R, respectively, contained spontaneously reactivated virus. Thus, based on total cultures, without regard to the number of eyes, LAT2.9A appeared to reactivate less well than the wild-type viruses. However, in this experiment, the percentages of eyes that reactivated at least once were similar for LAT2.9A and the wild-type viruses (Table 1).
TABLE 2.
Spontaneous reactivation of LAT2.9Aa
| Expt | Virusb | No. positive/total eye cultures (%)c | P vs LAT2.9Ad | P (fraction of positive cultures/eye vs LAT2.9A)e | No. positive/total eyes (%) | P vs LAT2.9Ad |
|---|---|---|---|---|---|---|
| 1 | LAT2.9A | 11/156 (7.1) | 5/6 (83.3) | |||
| dLAT2903 (LAT−) | 13/364 (3.6) | 2/14 (14.3) | ||||
| LAT3.3A (wt) | 38/312 (12.2) | 10/12 (83.3) | ||||
| dLAT2903R (wt) | 50/364 (13.7) | 13/14 (92.9) | ||||
| 2 | LAT2.9A | 6/364 (1.6) | 4/14 (28.6) | |||
| dLAT2903R (wt) | 52/624 (8.3) | <0.0001 | 0.02 | 16/24 (66.7) | 0.04 | |
| 3 | LAT2.9A | 1/184 (0.5)f | 1/8 (12.5) | |||
| LAT2.9AR | 36/364 (9.9) | <0.0001 | 0.01 | 10/14 (71) | 0.02 | |
| dLAT371 (wt) | 36/300 (12)g | <0.0001 | 0.03 | 8/12 (67) | 0.7 | |
| dLAT371R (wt) | 63/527 (11) | <0.0001 | 0.01 | 15/22 (68.2) | 0.01 | |
| Totalsh | LAT2.9A | 18/704 (2.6) | 10/28 (36) | |||
| dLAT2903 | 45/1768 (2.5) | 0.98 | 0.32 | 12/68 (18) | 0.32 | |
| Wild-type viruses | 275/2584 (10.6) | <0.0001 | 0.01 | 72/100 (72) | 0.0007 |
Analyses of the experiment shown in Fig. 4.
Viruses designated “(wt)” have previously been shown to have spontaneous reactivation rates indistinguishable from that of the parental McKrae wild-type virus.
Virus-positive tear cultures per total number of cultures. The number of cultures is not always equal to the number of eyes times 26 days (the length of the study period) because of occasional deaths apparently unrelated to the latent infection.
Fisher exact test. P < 0.05 indicates statistical significance.
For each eye within each group the fraction of days on which positive cultures were obtained was calculated and the resulting fractions (one per eye) were then compared between groups by using the Mann-Whitney rank sum test. The groups were considered significantly different if P was <0.05.
One rabbit died prior to tear collection on day 15.
One rabbit died prior to tear collection on day 3. One rabbit died prior to tear collection on day 19.
See Table 1, footnote d.
The analysis and interpretation of the results of experiment 1 were greatly handicapped by the fact that only three of the LAT2.9A-infected rabbits survived. Based on our previous experience, this was too small a number for meaningful comparisons and statistical analyses are not shown. Additional data was obtained from experiments 2 and 3 (Fig. 3B and C; Table 1), both of which contained more surviving rabbits in most groups. In experiment 2, the cumulative spontaneous reactivation rate in rabbits latently infected with LAT2.9A appeared to be decreased compared to dLAT2903R (Fig. 3B, open circles compared to solid circles; approximately 0.4/eye versus 2.2/eye). The number of positive eye cultures versus total eye cultures (Table 2, experiment 2, column 3) indicated only 1.6% of the tear film cultures from rabbits latently infected with LAT2.9A virus contained spontaneously reactivated virus compared to 8.3% of the tears from eyes infected with dLAT2903R. This difference was highly significant (Table 2, experiment 2, column 4). Because the above analysis did not take into account the number of eyes in each of the groups, the data were also analyzed as follows. The fraction of virus-positive cultures for each eye in each group (i.e., the fraction of time each eye was virus positive) was calculated, and these fractions were analyzed by the Mann-Whitney rank sum test (Table 2, experiment 2, column 5). By this analysis, LAT2.9A also reactivated less well than dLAT2903R. The percentage of eyes that reactivated at least once in the LAT2.9A group was also significantly reduced compared to wild type (dLAT2903R) (Table 2, experiment 2, columns 6 and 7).
In experiment 3, the cumulative spontaneous reactivation rate in rabbits latently infected with LAT2.9A also appeared to be decreased compared to that of dLAT371 and dLAT371R (Fig. 4C). Both of these viruses have wild-type spontaneous reactivation rates (18). LAT2.9A was compared to dLAT371 in this experiment since, although dLAT371 transcribes the entire primary LAT while LAT2.9A only transcribes up to nucleotide 1,499, both viruses contain the same StyI-StyI deletion. The number of positive eye cultures versus total eye cultures (Table 2, experiment 3, column 3) indicated that only 0.5% of the LAT2.9A tear film cultures were positive, while 12 and 11% of the dLAT371 and dLAT371R cultures were positive. This reduction in LAT2.9A spontaneous reactivation was highly significant (Table 2, experiment 3, column 4). Analysis of the fraction of time each eye was positive for reactivated virus also indicated that the reduction in LAT2.9A spontaneous reactivation was significant compared to dLAT371 and dLAT371R (Table 2, experiment 3, column 5). The percentage of eyes that reactivated was also significantly reduced in LAT2.9A compared to dLAT371R (Table 2, experiment 3, columns 6 and 7). The lack of significance between LAT2.9A and dLAT371 was probably due to the small number of rabbits in the dLAT371 group.
To confirm that the apparent decreased spontaneous reactivation of LAT2.9A was not due to an undetected, unrelated, mutation in the virus, LAT2.9A was marker rescued by using a restriction fragment containing the intact first 1.5 kb of LAT as described in Materials and Methods. This rescuing fragment restored the StyI-StyI deletion in the 1.5-kb LAT insert in the unique long region of LAT2.9A. The deletion in both original copies of LAT was retained (see Fig. 2C). Assuming that there was no extraneous, undetected, spontaneous mutation elsewhere in the virus, LAT2.9AR should be structurally identical to LAT3.3A. The cumulative spontaneous reactivation rate in rabbits latently infected with LAT2.9AR appeared to be similar to that of dLAT371R and dLAT371 (Fig. 4C; 2.6 versus 2.9 and 3.1 positive cultures/eye on days 26). The number of positive eye cultures versus total eye cultures (Table 2, experiment 3, column 3) also indicated that LAT2.9AR was restored to wild-type levels (9.9% versus 11 and 12% for the dLAT wild-type viruses) and that spontaneous reactivation of LAT2.9AR was significantly greater than that of LAT2.9A (P < 0.001 and P = 0.01; Table 2, experiment 3, columns 4 and 5). Finally, the percentage of LAT2.9AR eyes that reactivated was similar to dLAT371 and dLAT371R (Table 2, experiment 3, columns 6 and 7; 71% compared to 67 to 68%) and greater than LAT2.9A (71% versus 12.5%; P = 0.02).
The totals of all three experiments were analyzed for spontaneous reactivation as they had been for virulence. In Table 2, as in Table 1, the wild-type viruses contain the results for all of the viruses designated “wt” combined with the results for the LAT2.9AR marker-rescued virus, while the dLAT2903 totals reflect the results of experiment 1 plus the combined totals of the last four experiments in our laboratory, in which dLAT2903 was used as a negative spontaneous reactivation control. The decreased spontaneous reactivation of LAT2.9A compared to wild type was highly significant for each of the parameters analyzed (Table 1, totals, columns 4, 5, and 7). In addition, the spontaneous reactivation rate of LAT2.9A was similar to that of the LAT null mutant dLAT2903 for all of the parameters. Thus, LAT2.9A was impaired for spontaneous reactivation and had a spontaneous reactivation phenotype indistinguishable from that of a LAT null mutant. Southern analyses of selected spontaneously reactivated virus detected in tears confirmed that the reactivated viruses were structurally unchanged from the infecting viruses (data not shown).
Transcription of LAT in tissue culture and the TG of rabbits latently infected with LAT2.9A.
To confirm that transcription of LAT in LAT2.9A was as expected, RT-PCR analyses were done. CV-1 cells were infected at a multiplicity of infection of 2, and RT-PCR was performed on total RNA. The primers used generate a 160-bp product specific for LAT nucleotides 471 to 631. The RT-PCR products were subjected to Southern analysis by using an internal 32P-labeled probe (LAT nucleotides 550 to 580) (Fig. 5A). As expected, a 160-bp RT-PCR product was detected in cells infected with dLAT371, LAT3.3A, or LAT2.9A (Fig. 5A). In addition, the apparent intensity of the RT-PCR bands in each lane was similar. Since we previously showed that the amount of LAT RNA in TG latently infected with dLAT371 or LAT3.3A is similar to that of wild-type McKrae (17, 18), these results suggest that in tissue culture the predicted LAT2.9A RNA was present in wild-type amounts. No RT-PCR product was produced from dLAT2903-infected cells, confirming that RNA corresponding to this region of LAT was not made by the dLAT2903 LAT null mutant.
FIG. 5.
Transcription of LAT in tissue culture cells and in rabbits latently infected with LAT2.9A. (A) CV-1 cell monolayers were infected with the indicated viruses at a multiplicity of infection of 2, and the total RNA was isolated, RT-PCR was performed with primers corresponding to LAT nucleotides within the predicted LAT2.9A RNA, and Southern analysis was performed with an internal 32P-labeled probe as described in Materials and Methods. (B) Total RNA was isolated from individual TG from rabbits latently infected with the indicated virus, and RT-PCR analyses were performed and analyzed by Southern analysis as above. Each lane shows the RT-PCR product from one TG. The lane labeled ddH2O is a negative control. The RT-PCR analyses shown in the last three lanes were done at a different time than the other analyses.
To confirm that LAT2.9A transcription was as predicted during latency, total RNA was prepared from the individual TG of latently infected rabbits. RT-PCR analyses were performed as described above. As we previously reported (17), a 160-bp RT-PCR product was detected in the TG from rabbits latently infected with LAT3.3A, while no RT-PCR product was detected in rabbits latently infected with dLAT2903 (Fig. 5B). The same RT-PCR product was also seen in the TG from rabbits latently infected with LAT2.9A (Fig. 5B). We previously showed that the amount of LAT RNA in individual TG from rabbits latently infected with the same virus can vary significantly when analyzed by either RT-PCR (17, 18) as done here or by in situ hybridization (20). Thus, the apparent different intensities of the RT-PCR bands in the two LAT2.9A lanes (Fig. 5B) was similar to the differences previously seen with other viruses. The similar intensity of the RT-PCR product in the rightmost LAT2.9A lane and the LAT3.3A lane (Fig. 5B), therefore, strongly suggests that the amount of LAT2.9A LAT present in latently infected TG was similar to that of LAT3.3A LAT. Thus, the unexpected LAT2.9A phenotype did not appear to be due to unexpected aberrations in LAT transcription.
DISCUSSION
In the experiments reported here, LAT2.9A had increased virulence (based on reduced rabbit survival) and decreased spontaneous reactivation. In contrast, its marker-rescued virus, LAT2.9AR, was wild type for both phenotypes. This very strongly suggests that the increased virulence and the decreased spontaneous reactivation of LAT2.9A were both due to the 371-nucleotide StyI-StyI deletion. Both of these phenotypes were unexpected.
In contrast to the StyI-StyI deletion in LAT2.9A, we previously showed that deletion of the StyI-StyI region in the context of the entire LAT gene (dLAT371) (18) did not effect spontaneous reactivation. In addition, we previously showed that insertion of the LAT promoter and the first 1.5 kb of LAT (LAT 3.3A) into an ectopic location in the LAT null mutant dLAT2903 restored wild-type spontaneous reactivation (17). LAT2.9A is identical to LAT3.3A except that it contains the StyI-StyI deletion. Thus, it was expected that LAT2.9A would have a wild-type rate of spontaneous reactivation.
Possible explanations for the reduced reactivation of LAT2.9A fall into at least three categories. (i) The first is the LAT copy number. It is possible that the StyI-StyI region is not essential for wild-type spontaneous reactivation when two copies of LAT are present in the genome. (ii) The second is the LAT location. It is possible that when LAT is located in its normal location in the long repeats, the StyI-StyI region is not required, but the StyI-StyI region is required for LAT function when LAT is not in its normal location. (iii) The third is the lack of LAT from 1.5 to 8.3 kb. The StyI-StyI region may not be required in the context of the otherwise-complete LAT gene, but it is needed when only the first 1.5 kb of LAT is present. This would suggest that there are one or more additional functional regions downstream of the first 1.5-kb region. Since both dLAT371 and LAT3.3A have wild-type rates of spontaneous reactivation, the region pre-1.5 kb and the region post-1.5 kb must each be able to produce wild-type levels of spontaneous reactivation, but the effects of this must not be additive. This would easily occur if each functional region resulted in the maximum spontaneous reactivation possible in the rabbit model. Although not easily detected in animal models, the presence of multiple independent functional regions within LAT may have beneficial effects in nature. It would also help explain why selective pressure has maintained the entire 8.3-kb LAT gene in all of the HSV-1 isolates examined, despite the fact that the first 1.5 kb of LAT alone is sufficient for maximum levels of spontaneous reactivation in the rabbit. Alternatively, functional regions after the first 1.5 kb of LAT may not be fully independently able to produce wild-type levels of spontaneous reactivation but may be able to compensate for the StyI-StyI deletion in the first 1.5 kb of LAT, perhaps via interactions with part of the remaining 1.5-kb region. It is also possible that a previously undetected splicing event occurs, either partially or completely within the StyI-StyI region, and that this splicing is required for LAT function in the context of the first 1.5 kb but that it can be compensated for by sequences downstream of the first 1.5 kb.
Although LAT may partially inhibit productive gene expression (2, 7, 11), there have been no previously reports of any LAT mutants with increased virulence. Over the course of numerous experiments in our laboratory, the virulence of the LAT null mutant, dLAT2903 (14), has always been similar to that of its wild-type parent. In addition, the virulence of LAT3.3A has been indistinguishable from that of both its direct parent, dLAT2903, and dLAT2903’s wild-type McKrae parent (17). In contrast, LAT2.9A, which is effectively a deletion of the StyI-StyI region from LAT3.3A, had significantly increased virulence. Moreover, the increased virulence of LAT2.9A was restored to the less-virulent wild-type level by rescue of the StyI-StyI deletion in the ectopic insert (LAT2.9AR). Thus, the increased virulence of LAT2.9A appeared to be due to the 371-nucleotide StyI-StyI deletion in its ectopic LAT insert.
Why does the deletion of LAT nucleotides 76 to 447 in LAT2.9A increase virulence when the deletion of LAT nucleotides −161 to +1667 (dLAT2903) does not? Asked another way, why does the insertion of LAT nucleotides −1800 to +76 plus 447 to 1667 between UL37 and UL38 in dLAT2903 increase virulence, whereas the insertion of LAT nucleotides −1800 to +1667 does not? or, from yet another perspective, why does preventing transcription of LAT nucleotides 76 to 447 along with LAT nucleotides 1667 to 8323 increase virulence, whereas preventing all LAT transcription does not alter virulence? This phenomenon, in which a small deletion has a larger effect than a larger deletion that encompasses the smaller deletion, is reminiscent of results often seen when mapping promoter activity. This is because promoters often contain numerous functional elements, some of which work in concert and some of which act antagonistically. A series of deletions that removes successively larger regions of the 5′ end of a promoter often produces a pattern of promoter activity that increases and decreases several times before all activity is gone. Since it is unlikely that LAT encodes a protein and since deletion of the StyI-StyI region does not appear to alter the expression of LAT itself, this parallel suggests that the LAT RNA may regulate the expression or function of one or more viral and/or cellular genes. This regulation could be direct or through the intervention of other gene products. In either situation, LAT could enhance spontaneous reactivation by being a key factor involved in intricate, controlled regulation of viral and/or cellular genes involved in the latency-reactivation cycle.
We previously reported that d34.5, a McKrae-based mutant deleted for both copies of γ34.5, the gene for ICP34.5 (one in each long repeat), had dramatically decreased virulence and poor spontaneous reactivation after infection of rabbits with 2 × 105 PFU/eye (19). However, after an extremely high-dose ocular infection with over 108 PFU/eye, d34.5 was still avirulent but its spontaneous reactivation rate was wild type (16). A second mutant, derived from d34.5 and designated d34.5A, contains one copy of γ34.5 inserted into the same ectopic location used for LAT2.9A. d34.5A, has wild-type spontaneous reactivation while remaining much less virulent than wild type (13). These previous studies showed that decreased virulence does not necessarily result in decreased spontaneous reactivation. The results reported here extend and complement the above findings by showing that increased virulence does not necessarily result in increased spontaneous reactivation. In 2.9A, increased virulence did not result in increased spontaneous reactivation and, in fact, was coincident with decreased spontaneous reactivation. These results confirm that the phenotypes for spontaneous reactivation and virulence are separable.
Nonetheless, since LAT2.9A shows that LAT affects virulence as well as reactivation, is of interest to consider the possibility that in this mutant the increased virulence and the decreased spontaneous reactivation were related. It is possible that the increased virulence resulted in the elimination of neurons that would otherwise have become latently infected. This might result in fewer surviving latently infected neurons, resulting in a smaller pool of latently infected neurons capable of reactivation. This, in turn, would be expected to result in reduced spontaneous reactivation. Thus, one possible hypothesis that could be derived from these studies is that the normal function of LAT is to protect acutely infected neurons from death, thereby producing a larger pool of latently infected neurons. This larger pool of latently infected neurons would subsequently allow for increased spontaneous reactivation. Consistent with this, it has been proposed that the bovine herpes virus LR-RNA (latency-related RNA, comparable to LAT) encodes products that promote neuronal survival (8, 22). Unfortunately, in the rabbit model it has not yet been possible to detect decreased amounts of latent viral DNA or decreased numbers of latent viral DNA-positive neurons in the TG (13), and so this hypothesis remains to be tested.
ACKNOWLEDGMENTS
This work was supported by Public Health Service grants EY07566 and EY10243, the Discovery Fund for Eye Research, and the Skirball Program in Molecular Ophthalmology.
We thank Anita Avery for her expert technical support.
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