Vol. 65, No. 10

A Herpes Simplex Virus Type 1 Mutant Lacking the ICPO IntronsReactivates with Normal Efficiency

RAJESWARI NATARAJAN,'t SATISH DESHMANE,' TIBOR VALYI-NAGY,' ROGER EVERETT,2AND NIGEL W. FRASER'*

The Wistar Institute, 36th and Spruce Streets, Philadelphia, Pennsylvania 19104,1 and Institute of Virology,MRC Virology Unit, University of Glasgow, Glasgow, GIl 5JR Scotland2

Received 29 April 1991/Accepted 2 July 1991

Previous evidence suggests that the latency-associated transcript (LAT) gene of herpes simplex virus type 1appears to have a role during reactivation of latent virus because viruses which are null mutants in this gene

reactivate slowly or less efficiently than wild-type viruses. Mapping studies have shown that the LAT gene

covers a region of about 8.5 kb that overlaps the ICPO gene in the repeat long region of the herpes simplex virusgenome. Previously, we had constructed a mutant with a deletion in the region of the LAT gene encoding a

stable 2-kb RNA species (that accumulates to high levels in latently infected cells) and had shown that itreactivates normally (T. M. Block, J. G. Spivack, I. Steiner, S. Deshmane, M. T. McIntosh, R. P. Lirette, andN. W. Fraser, J. Virol. 64:3417-3426, 1990). We now show that a mutant which has two deletions downstreamof this region (deleted in both ICPO introns) reactivates normally in explant cocultivation assays. Thus, the slowor inefficient reactivation phenotype of herpes simplex virus type 1 LAT null mutants is not assignable to thisregion of the LAT gene.

Following the primary infection of humans and laboratoryanimals, herpes simplex virus establishes latency in neurons

of sensory ganglia innervating the peripheral site of infection(7, 8, 20). Latent virus can reactivate spontaneously or inlaboratory animals can be induced to reactivate by severalmethods, including explantation of latently infected ganglia(21). During latency, viral replication and protein synthesisare not detectable. Furthermore, viral gene expression islimited to the latency-associated transcripts (LATs). ByNorthern (RNA) blot analysis, three transcripts, 2.0, 1.5,and 1.45 kb in size, have been detected in latently infectedganglia. These map to the BamHI B and E fragments of theviral genome partially overlapping sequences encoding theviral immediate-early gene ICPO; however, they are tran-scribed in the opposite direction (17, 18, 22). By using themore sensitive technique of in situ (RNA) hybridization,transcription has been mapped to a considerably largerregion (approximately 8.5 kb; Fig. 1) that covers all of theICPO gene (2, 12). The location of the LAT promoter hasbeen established by deletion mutant analysis (4, 19). Thefunction of the LATs is presently unclear, but mutationalanalysis suggests that LAT transcription may be importantfor efficient reactivation.

Interestingly, herpes simplex virus type 1 (HSV-1) mu-

tants with deletions in their LAT promoter region have beenreported to reactivate inefficiently (10, 19), whereas mutantshaving deletions within the 2-kb LAT RNA have beenreported to reactivate with kinetics similar to those ofparental wild-type virus strains (1, 9). Since it has beendemonstrated in tissue culture that the 2.0-kb LAT RNAbehaves like an intron spliced out of a large primary tran-

* Corresponding author.t Present address: Department of Endocrinology, Mt. Sinai Hos-

pital, New York, NY 10029.

script (3, 6), interruptions within the 2.0-kb RNA may notinterfere with normal function of the LAT gene. Thus, it isimportant to know whether interruptions in the putativelarge primary transcript of the LAT have any effect on theprocess of reactivation.Downstream and overlapping the 3' end of the 2-kb LAT

RNA is the ICPO gene (Fig. 1). It has previously beenreported that, in certain circumstances, mutants in the ICP0gene replicate inefficiently (16) and are inefficient in estab-lishing latent infections (11). Thus, any LAT gene interrup-tions within the overlapping ICP0 gene must be carefullyconstructed so that they do not interfere with the normalfunctioning of ICPO. The ICPO gene has two introns in itscoding region. Mutants having deletions within these intronswould be particularly useful in interrupting the LAT genewithout compromising ICP0 function. The construction andproperties of virus 11OC1 (an intronless mutant of ICPO)have been described earlier (5). In brief, it is a mutant withdeletions of both introns of ICPO which expresses ICP0 in a

fashion similar to that of parental virus in tissue cultureexperiments. In this study, mutant 11OC1 was tested for itsability to establish and reactivate latent infection in themouse HSV-1 latency model.Subsequent to corneal scarification, 4- to 6-week-old fe-

male BALB/c mice (Jackson Laboratory, Bar Harbor,Maine) were infected with approximately 106 PFU of HSV-111OC1, 11OC1R, or 17+ per eye. Virus l1OClR is a rescuantof 11OC1, which has had the intron-deleted ICPO genereplaced by the normal gene. At a minimum of 4 weekspostinfection, latently infected mice were sacrificed by cer-

vical dislocation and the trigeminal ganglia were explantedand incubated with monolayers of CV-1 cells. The cells wereinspected daily for signs of cytopathic effect. Ganglia were

transferred to new cells every 5 to 6 days and observed untilreactivation occurred or for a maximum of 27 days. Whenvirus reactivation occurred, the virus-containing media were

removed and saved for DNA analysis.

5569

JOURNAL OF VIROLOGY, Oct. 1991, p. 5569-55730022-538X/91/105569-05$02.00/0Copyright ©D 1991, American Society for Microbiology

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FIG. 1. (A) The genome of HSV-1 showing the unique long and short regions and the repeat elements (boxed). The long internal andterminal repeats encode ICPO and the LATs. (B) The internal repeat region and the joint between the long and short regions of the genomeare shown along with their transcripts. The regions deleted in mutant 11OC1 are stippled. (C) The DNA fragments used as probes are shown.Numbers are nucleotide numbers from the sequence of the HSV-1 (17+) genome (13).

In mice infected with strain 17+ wild-type virus, thereactivation process was detected on day 7 and by the 13thday postexplant, 94% of the ganglia (15 of 16) were positive(Fig. 2). A similar situation was evident with 11OC1 mutantvirus-infected mice. The first signs of reactivation were seenon the 9th day postexplant, and by 13th day postexplant, themajority of ganglia (92.8% [26 of 28]) were positive. Thepatterns of reactivation of mice infected with marker-res-cued revertant (liOC1R) were comparable to those of 17+-infected mice. At the end of the experiment (27 days

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FIG. 2. Explant cocultivation reactivation assay. Trigeminalganglia from mice latently infected with wild-type HSV-1 17+,mutant 110C1, and marker-rescued revertant 110ClR were explantcocultivated on CV-1 cells. The cultures were observed daily forvirus-induced cytopathic effects. The ganglia were transferred ontonew cell monolayers every week and observed for 27 days. Thegraph represents accumulated data from three independent experi-ments.

postexplant), 100% of 17+-infected ganglia (16 of 16), 100%of 11OC1 mutant-infected ganglia (28 of 28), and 93.3% ofrevertant 1101CR-infected ganglia (28 of 30) were positive byexplant cocultivation assay.To determine the effect of the deletion in the LAT gene of

11OC1 on LAT transcription, in situ hybridization was con-ducted by using a nick-translated BstEII (0.9 kb) probe anda BamHI SP probe, which detected transcription upstreamand downstream of the deletions from the LAT gene, respec-tively (Fig. 1). Interestingly, the patterns of hybridization ofthe two probes to tissues latently infected with 11OC1 did notdiffer from the patterns of hybridization to 17+- or 11OClR-infected tissues (Fig. 3). Both probes hybridized to someneurons (and a few satellite cells) of trigeminal ganglia ofanimals latently infected (28 days) by HSV-1 strains 17+,11OC1R, and 110CL. The hybridization was stronger andmore cells were detected with the BstEII (0.9 kb) probe,which is specific for the stable 2-kb LAT RNA. The produc-tion of 2-kb LAT RNA was confirmed by Northern blotting(Fig. 4). Thus, the accumulation of LAT does not appear tobe affected by the deletion of the ICPO introns.To confirm that the genome of the reactivated mutant

11OC1 virus did not change during latent infection or reacti-vation, a Southern blot DNA analysis was undertaken.Virion DNA was prepared, digested with BamHI, resolvedby agarose gel electrophoresis, Southern blotted, and probedwith a nick-translated viral probe (Fig. 5). Since the deletionwithin one of the introns results in the loss of a BamHI site,digestion of virion DNA followed by Southern blotting andhybridization with a radioactive BamHI SP probe wasexpected to yield a characteristic pattern for the parental orrevertant and mutant viruses. BamHI digestion of parental17+ virus followed by hybridization with BamHI SP proberesults in the detection of three fragments, namely, BamHI Swhich is derived from long terminal repeat, BamHI P de-

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FIG. 3. In situ hybridization for the detection of HSV-1 transcripts in trigeminal ganglia of mice latently infected with 17+ (A, D), 11OC1(B, E), or 11OClR (C, F). Panels A, B, and C, BstEII-BstEIl probe; panels D, E, and F, BamHI SP probe. Trigeminal ganglia of micesacrificed 28 days postinfection with strain 17+, 11OC1, or 11OClR were removed and processed for in situ hybridization as previouslydescribed (2). DNA fragments for 35S-labeled nick-translated BstEII or BamHI SP probes (Fig. 1) were obtained from cloned fragments ofHSV-1 F from B. Roizman (15). DNA inserts were gel purified and nick translated as described previously (2). The specific activities of thenick-translated 35S-labeled probes were 1 x 108 to 2 x 108 cpm/,ug.

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FIG. 4. Detection of LAT RNA by Northern blot analysis. Eachlane contains 10 p.g of RNA from trigeminal ganglia of uninfectedmice (lane 1) or mice infected with HSV-1 11OC1 (lane 2), HSV-111OClR (lane 3), and HSV-1 17+ (lane 4). The blot was hybridizedwith a 32P-labeled 0.9-kb BstEII-BstEIl probe (Fig. 1). The autora-diograph was made with XAR-5 film for 36 h.

rived from short terminal repeat, and BamHI SP derivedfrom the junction region. However, with the loss of theBamHI site in the mutant virus long terminal repeat, BamHIS and SP are absent and four new larger fragments are

detected in one large band (BamHI B-SP, B-S, E-SP, andE-S). Figure 5 shows the differences in the patterns of DNAfragments. The patterns of inoculum and reactivated 11OC1virus were identical, and thus the fast reactivation was notdue to repair of the lesions in 110CL.Thus, viruses having deletions within the introns of ICPO,

in the minor LAT region, can efficiently establish andreactivate from a latent infection. Mutant 11OC1 also showsno significant difference from wild-type virus in the timecourse of growth or efficiency of expression of ICPO in tissueculture studies (5). It was indistinguishable from the parentvirus in terms of growth, particle-to-PFU ratio, and viralpolypeptide expression in variety of cell types. Therefore,the function of the introns in ICPO remains unknown.

In the present communication, we have shown that 11OC1establishes latency in mice after corneal scarification as

efficiently as wild-type virus, expresses 2-kb LAT RNAduring latency, and reactivates from explant culture with an

efficiency similar to that of wild-type virus. Thus, mutationsaffecting LAT gene sequences encoded in the ICPO gene

introns do not affect the ability of the virus to establish,maintain, or reactivate from HSV-1 latency. The search forthe LAT sequences which cause the slow or inefficientreactivation phenotype shown by the LAT null mutants is

FIG. 5. DNA analysis of reactivated virus isolates. Reactivatedvirus isolates of mutant (11OC1) and marker-rescued revertant(ilOClR) along with their original inoculum virus strains were usedto infected confluent monlayers of CV-1 cells. Infected cells were

harvested at 24 h postinfection, and virion DNA was extracted fromthe cytoplasmic fraction as described by Pignatti et al. (14). BamHI-restricted 110CI DNA from inoculum virus (lane 1) and its fivereactivated isolates (lanes 2, 3, 4, 5, and 6) were resolved on 0.8%agarose gels, transferred onto nitrocellulose, and hybridized with32P-labeled nick-translated BamHl SP DNA probe. With strain11OC1R, the probe detects three bands: BamHI SP derived from theLS junction, BamHl S derived from the L terminus, and BamHI Pderived from the S terminus (lane 7, inoculum virus; lanes 8 through12, reactivated isolates). Since the deletion in 11OC1 results in theloss of a BamHI site in each of the long repeat sequences, BamHl Sand SP remain joined to the adjacent BamHI fragments (BamHI Band E) and migrate as larger DNA fragments. These serve as a

diagnostic marker to differentiate the mutant from the wild-type or

revertant virus. The location of the BamHI fragment on the viralgenome is shown in Fig. 1.

continuing with the construction and examination of mutantsin other regions of the 8.5-kb LAT gene.

We thank Allan Dillner and David Sole for technical assistance.Jordan Spivack is thanked for reading the manuscript and for helpfulsuggestions, and Cheryl McFadden is thanked for help in preparingthe manuscript.

This work was supported by Public Health Service grant A123968from the National Institutes of Health.

REFERENCES1. Block, T. M., J. G. Spivack, I. Steiner, S. Deshmane, M. T.

McIntosh, R. P. Lirette, and N. W. Fraser. 1990. A herpessimplex virus type 1 latency-associated transcript mutant reac-

tivates with normal kinetics from latent infection. J. Virol.64:3417-3426.

2. Deatly, A. M., J. G. Spivack, E. Lavi, and N. W. Fraser. 1987.RNA from an immediate early region of the HSV-1 genome ispresent in the trigeminal ganglia of latently infected mice. Proc.Natl. Acad. Sci. USA 84:3204-3208.

3. Devi-Rao, G. B., S. A. Goodart, L. M. Hecht, R. Rochford,M. K. Rice, and E. K. Wagner. 1991. Relationship betweenpolyadenylated and nonpolyadenylated herpes simplex virustype 1 latency-associated transcripts. J. Virol. 65:2179-2190.

4. Dobson, A. T., F. Sederati, G. Devi-Rao, W. M. Flanagan, M. J.Farrell, J. G. Stevens, E. K. Wagner, and L. T. Feldman. 1989.Identification of the latency-associated transcript promoter by

v

SP -

9.49-7-46-

2-37-

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NOTES 5573

expression of rabbit beta-globin mRNA in mouse sensory nerveganglia latently infected with a recombinant herpes simplexvirus. J. Virol. 63:3844-3851.

5. Everett, R. D. 1991. Construction and characterization of herpessimplex type 1 viruses without introns in immediate-early gene1. J. Gen. Virol. 72:651-659.

6. Farrell, M. J., A. T. Dobson, and L. T. Feldman. 1991. Herpessimplex virus latency-associated transcript is a stable intron.Proc. Natl. Acad. Sci. USA 88:790-794.

7. Fraser, N. W., J. G. Spivack, Z. Wroblewska, T. Block, S. L.Deshmane, T. Valyi-Nagy, R. Natarajan, and R. Gesser. 1991. Areview of the mechanism of HSV latency. Curr. Eye Res.1O(Suppl.):1-14.

8. Hill, T. J. 1985. Herpes simplex virus latency, p. 175-240. In B.Roizman (ed.), The herpesviruses, vol. 3. Plenum PublishingCorp., New York.

9. Ho, D. Y., and E. S. Mocarski. 1989. Herpes simplex virus latentRNA (LAT) is not required for latent infection in the mouse.Proc. Natl. Acad. Sci. USA 86:7596-7600.

10. Leib, D. A., C. L. Bogard, M. Kosz-Vnenchak, K. A. Hicks,D. M. Coen, D. M. Knipe, and P. A. Schaffer. 1989. A deletionmutant of the latency-associated transcript of herpes simplexvirus type 1 reactivates from the latent state with reducedfrequency. J. Virol. 63:2893-2900.

11. Leib, D. A., D. M. Coen, C. L. Bogard, K. A. Hicks, D. R.Yager, D. M. Knipe, K. L. Tyler, and P. A. Schaffer. 1989.Immediate-early regulatory gene mutants define different stagesin the establishment and reactivation of herpes simplex viruslatency. J. Virol. 63:759-768.

12. Mitchell, W. J., R. P. Lirette, and N. W. Fraser. 1990. Mappingof low abundance latency associated RNA in the trigeminalganglia of mice latently infected with herpes simplex virus type1. J. Gen. Virol. 71:125-132.

13. Perry, L. J., and D. J. McGeoch. 1988. The DNA sequences of

the long repeat region and adjoining parts of the long uniqueregion in the genome of herpes simplex virus type 1. J. Gen.Virol. 69:2831-2846.

14. Pignatti, P. F., G. Meneguzzi, N. Chenciner, and G. Milanesi.1979. Herpes simplex virus DNA isolated from infected cellswith a novel procedure. Virology 93:260-264.

15. Post, L. E., A. J. Conley, E. S. Mocarski, and B. Roizman. 1980.Cloning of reiterated and nonreiterated herpes simplex virus 1sequences as BamHI fragments. Proc. Natl. Acad. Sci. USA77:4201-4205.

16. Sacks, W. R., and P. A. Schaffer. 1987. Deletion mutants in thegene encoding the herpes simplex virus type 1 immediate-earlyprotein ICPO exhibit impaired growth in cell culture. J. Virol.61:829-839.

17. Spivack, J. G., and N. W. Fraser. 1987. Detection of herpessimplex virus type 1 transcripts during latent infection in mice.J. Virol. 61:3841-3847.

18. Spivack, J. G., and N. W. Fraser. 1988. Expression of herpessimplex virus type 1 latency-associated transcripts in the trigem-inal ganglia of mice during acute infection and reactivation oflatent infection. J. Virol. 62:1479-1485.

19. Steiner, I., J. G. Spivack, R. P. Lirette, S. M. Brown, A. R.MacLean, J. Subak-Sharpe, and N. W. Fraser. 1989. Herpessimplex virus type 1 latency-associated transcripts are evidentlynot essential for latent infection. EMBO J. 8:505-511.

20. Stevens, J. G. 1989. Human herpesviruses: a consideration ofthe latent state. Microbiol. Rev. 53:318-332.

21. Stevens, J. G., and M. L. Cook. 1973. Latent herpes simplexvirus in spinal ganglia of mice. Science 173:843-845.

22. Stevens, J. G., E. K. Wagner, G. B. Devi-Rao, M. L. Cook, andL. T. Feldman. 1987. RNA complementary to a herpes virusgene mRNA is prominent in latently infected neurons. Science235:1056-1059.

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