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Copyright q 1996, American Society for Microbiology
A hallmark of herpes simplex virus type 1 (HSV-1) latent primary 8.3-kb LAT transcript been mapped. It has been pro-
infection is the propensity of the latent virus to reactivate at posed that LAT may function via some type of antisense reg-
various times and produce recurrent disease. Recurrent HSV-1 ulation of the important ICP0 and/or ICP34.5 viral genes, both
corneal infection, which can lead to blindness due to scarring of which the primary 8.3-kb LAT transcript completely over-
of the cornea, is the most common cause of infectious blind- laps in an antisense direction (47) (see Fig. 1). Presumably
ness in the developed world (21). During latency, LAT is the such an antisense interaction would block ICP0 or ICP34.5
only viral gene that is abundantly transcribed (28, 35). LAT is function, thereby leading to latency. Increased latency would
located in the long repeat region of the HSV-1 genome and is then result in increased reactivation rates. Although this in-
therefore present in two copies per genome. The primary LAT triguing hypothesis has received continued bolstering by the
transcript is 8.3 kb and gives rise to a family of LAT RNAs uniform inability to detect a LAT-encoded protein during la-
(LATs), including very stable ones of 2 and 1.5 kb (6, 9, 28, 32, tency in experimental animals, no direct supportive experimen-
34, 35, 40–44, 47). LAT is essential for efficient reactivation tal evidence has yet been presented.
from sensory neurons, since LAT transcription-negative mu- Determining the region(s) of LAT required for LAT’s func-
tants have been shown to reactivate poorly by explant or in- tion would be very helpful in determining if an antisense mech-
duced reactivation in the mouse (1, 11–13, 18, 19, 29, 33, 39), anism may be involved. Unfortunately, the same overlap with
by induced reactivation in the rabbit (2), and by spontaneous ICP0 and ICP34.5 that makes antisense a possible mechanism
reactivation in the rabbit (24). severely complicates attempts to make LAT mutants that would
Despite the large body of work indicating that LAT is re- address the likelihood of such an antisense mechanism. It is not
quired for efficient reactivation, the mechanism by which LAT possible to delete or mutate portions of LAT that are colinear
functions remains unknown. No LAT-encoded protein has been with ICP0 or ICP34.5 without also altering the colinear gene.
detected during latency, nor has the functional region of the Likewise, it is not possible to alter ICP0 or ICP34.5 without
altering LAT. If such mutants had an altered phenotype, it
would be very difficult to determine if the phenotype was due
* Corresponding author. Mailing address: Ophthalmology Research to the alteration in LAT or to the alteration in the overlapping
Laboratories, Cedars-Sinai Medical Center Research Institute, Davis gene.
Bldg., Room 5072, 8700 Beverly Blvd., Los Angeles, CA 90048. Phone: This concern would not apply to mapping that portion of the
(310) 855-6455. Fax: (310) 652-8411. LAT gene (approximately 1.8 kb) prior to the regions of ICP0
976
VOL. 70, 1996 LAT’S FUNCTION MAPS COMPLETELY WITHIN THE FIRST 1.5 kb 977
and ICP34.5 overlap. One approach might be to insert a tran- PacI site of the plasmid pV375Pac. The resulting plasmid was designated
scriptional stop site (i.e., a polyadenylation signal) prior to the pV375LAT3.2. This plasmid contains the HSV-1 McKrae LAT fragment de-
scribed above, bounded by UL37 and UL38. pV375LAT3.2 was then amplified in
region of ICP0 overlap. However, interpretation would be am- E. coli as described above.
biguous because it would not be possible to unequivocally LAT15a was generated by homologous recombination as we previously de-
prove that the stop site blocked transcription with 100% effi- scribed (23, 24, 26). Briefly, pV375LAT3.2 was cotransfected with infectious
ciency. Even a very small, undetectable level of readthrough dLAT2903 DNA (the LAT deletion mutant described above) by the calcium
transcription could result in maintenance of a biological func- phosphate method. Viruses from the cotransfection were plated, and isolated
plaques were picked and screened for insertion of the 3.2-kb LAT fragment
tion. We therefore took a novel approach. Starting with a LAT between UL37 and UL38 by restriction digestion and Southern analysis. Selected
mutant (dLAT2903) that we had previously made and which plaques were triple plaque purified and reanalyzed by restriction digestion and
lacks the LAT promoter and the first 1.6 kb of LAT (24), we Southern analysis to ensure that the 3.2-kb LAT fragment was present between
introduced the first 1.5 kb of the LAT gene along with 1.7 kb 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 59 end of the primary LAT
of upstream promoter sequences into a distant location in the transcript (see Fig. 6). A final plaque was purified and designated LAT15a
unique long region of the virus genome (see Fig. 1). This virus (LAT15 indicates 1.5 kb of the 59 end of LAT; a indicates addition).
(LAT15a) can transcribe only the first 1.5 kb of the 8.3-kb LAT Replication of virus in tissue culture. Cell monolayers at approximately 70 to
transcript from the inserted material. No LAT transcription 80% confluency were infected with virus at 0.01 PFU per cell, and all monolayers
were refed with exactly the same amount of minimal essential medium contain-
should occur from either original promoter-negative LAT ing 10% fetal calf serum. Virus was harvested for titration at various times by two
59-TCTCCCCCCCCCCTTCTTCACCCCCAGTAC-39, corresponding to LAT repeats (long terminal and internal repeats and short terminal
nt 550 to 580. and internal repeats). The long repeats are expanded in Fig.
Statistical analyses. Statistical analyses were performed by using Instat, a
personal-computer software program. For analyses with either the Student t test, 1A to show the relative locations and statuses of the LAT,
the Mann-Whitney rank sum test, the chi-square test, or the Fisher exact test, ICP0, and ICP34.5 genes. The primary 8.3-kb LAT transcript is
results were considered statistically significant when the P value was ,0.05. unstable and difficult to detect. It gives rise to the very stable
and easily detected 2-kb LAT, the location of which is shown in
RESULTS Fig. 1A; the location of the LAT promoter TATA box is also
indicated. Transcription of the 8.3-kb LAT (11) starts 28 nt
Structure of the LAT15a virus. The McKrae strain was used downstream of the TATA box (47).
as the original parental virus for all mutants because its high The LAT promoter mutant dLAT2903, which we have pre-
spontaneous-reactivation rate in rabbits allows changes in the viously described (24) is shown schematically in Fig. 1B.
spontaneous-reactivation rates of mutants to be detected and dLAT2903 contains a 1.8-kb deletion in both copies of the
analyzed statistically. The genomic structures of wild-type HSV-1 LAT gene (one in each long repeat). This deletion consists of
McKrae and dLAT2903R, a marker-rescued virus of the LAT 0.2 kb of the LAT promoter and the portion of the LAT gene
deletion mutant dLAT2903 (24), are shown schematically in encoding the first 1.6 kb of the 8.3-kb primary LAT transcript.
Fig. 1A. The HSV-1 genome contains a unique long region and As indicated in Fig. 1B, the deletion extends to LAT nt 1667.
a unique short region, both of which are flanked by inverted As we previously described (24), dLAT2903 was marker res-
VOL. 70, 1996 LAT’S FUNCTION MAPS COMPLETELY WITHIN THE FIRST 1.5 kb 979
LAT15a (18) 55/468 (11.8) 0.12 13/18 (72) 8/9 (89) 1.4
dLAT2903 (12) 0/312 (0) (P , 0.0001e) 0.0 (P 5 0.001 f) 0/12 (0) (P , 0.0001e) 0/6 (0) (P 5 0.001e) 0 (P 5 0.001 f)
dLAT2903R (wt) (24)g 70/624 (11.2) (P 5 0.85e) 0.11 (P 5 0.66 f) 18/24 (75) (P 5 1.0e) 10/12 (83) (P 5 1.0e) 1.6 (P 5 0.68 f)
a
Statistical analysis of the data presented in Fig. 5. Rabbits were bilaterally ocularly infected with dLAT2903 (virus with the LAT promoter and the first 1.6 kb of
LAT deleted), dLAT2903R (marker-rescued wild-type virus), or LAT15a (dLAT2903 containing the LAT promoter and the first 1.5 kb of LAT at a distant location)
as described in Materials and Methods.
b
Spontaneous reactivation was assessed beginning on day 30 postinfection by culturing tear films collected daily for 26 days as described in the text.
c
For each eye within each group, the fraction of days on which positive cultures were obtained was calculated (total HSV-1-positive cultures for eye/total cultures
for eye). The average is shown.
d
The total number of reactivations in each group, assuming that consecutive days of HSV-1-positive tear film cultures from a single eye are the result of a single
reactivation event or episode.
e
Versus LAT15a; determined by a two-sided Fisher exact test. The groups are considered significantly different if P is ,0.05.
f
Versus LAT15a; determined by a two-sided Mann-Whitney rank sum test. The groups are considered significantly different if P is ,0.05.
g
Wild-type (wt) virus. This marker-rescued virus is indistinguishable from the original parental McKrae virus.
BamHI cut is in the repeat and one is in the adjacent unique in this study was mapped to the first 1.5 kb of LAT must be
long region. The larger band contains LAT from the internal involved in spontaneous reactivation, not establishment of la-
repeat, while the smaller band is from the terminal repeat. tency. The similarity in intensity of hybridization to the LAT
Both of these bands are smaller in the original LAT deletion RT-PCR products from rabbits latently infected with wild-type
dLAT2903 (lane D) and LAT15a (lane 15a), indicative of the virus and LAT15a virus also suggests a wild-type level of la-
1.8-kb LAT deletion in each long repeat in these viruses. Lane tency with LAT15a, further suggesting a role for the first 1.5 kb
R1 (containing a spontaneously reactivated LAT15a virus) of LAT in reactivation.
contains bands identical to those seen in lanes D and 15a, The stable 2-kb LAT is not essential for wild-type levels of
confirming that the original LAT deletion was retained in both spontaneous reactivation. The 2-kb LAT is extremely stable
original copies of LAT in the LAT15a spontaneously reacti- compared with the remainder of the 8.3-kb LAT RNA (47).
vated virus. This stability makes the 2-kb LAT relatively easy to detect
These Southern analyses confirm the structure of the during latency compared with the rest of the LAT transcript.
LAT15a virus and demonstrate that the spontaneously reacti- Thus, for several years after the initial reports of LAT (28, 35),
vated LAT15a virus retained the 1.8-kb LAT deletion in both it was thought that the 2-kb LAT (and a 1.5-kb LAT derived
repeats as well as the 3.2-kb LAT insert in the unique long from the 2-kb LAT) constituted the entire LAT RNA. Because
region. This was found to be the case for all of the spontane- of its abundance during latency, it is still often assumed that
region makes it formally possible that it is this region rather 1994. Molecular analysis of herpes simplex virus type 1 during epinephrine-
than the region corresponding to the first 1.5 kb of the LAT induced reactivation of latently infected rabbits in vivo. J. Virol. 68:1283–
1292.
transcript to which the spontaneous reactivation function 3. Bohenzky, R. A., M. Lagunoff, B. Roizman, E. K. Wagner, and S. Silverstein.
maps. In fact, if the LAT promoter was bidirectional, transcrip- 1995. Two overlapping transcription units which extend across the L-S junc-
tion might occur in the upstream region during latency. A tion of herpes simplex virus type 1. J. Virol. 69:2889–2897.
small amount of such transcription in this region has been 4. Bolovan, C. A., N. M. Sawtell, and R. L. Thompson. 1994. ICP34.5 mutants
in herpes simplex virus type 1 strain 17 syn1 are attenuated for neuroviru-
reported to occur in latently infected mice (20); however, the lence in mice and for replication in confluent primary mouse embryo cells. J.
direction of transcription was not determined. Our computer Virol. 68:48–55.
analysis of the sequence in this region revealed three open 5. Chou, J., E. R. Kern, R. J. Whitley, and B. Roizman. 1990. Mapping of
reading frames, each beginning with an ATG and ending in a herpes simplex virus-neurovirulence to gamma 134.5, a gene nonessential for
growth in culture. Science 250:1262–1265.
typical termination codon. All three have a very high Fickett 6. Croen, K. D., J. M. Ostrove, L. J. Dragovic, J. E. Smialek, and S. E. Straus.
test code probability of .0.92, indicating that codon usage is 1987. Latent herpes simplex virus in human trigeminal ganglia: detection of
typical of a protein (IBI Pustell Sequence Analysis Program). an immediate early gene ‘‘antisense’’ transcript by in situ hybridization. N.
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7. Crowl, R., C. Seamens, P. Lomedico, and S. McAndrew. 1985. Versatile
additional study. expression vectors for high-level synthesis of cloned gene products in E. coli.
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