Vol. 62, No. 12

Detection and Preliminary Characterization of Herpes Simplex VirusType 1 Transcripts in Latently Infected Human Trigeminal Ganglia

PHILIP R. KRAUSE, KENNETH D. CROEN, STEPHEN E. STRAUS, AND JEFFREY M. OSTROVE*

Medical Virology Section, Laboratory of Clinical Investigation, National Institute of Allergy and Infectious Diseases,Bethesda, Maryland 20892

Received 6 July 1988/Accepted 29 August 1988

RNA extracted from human trigeminal ganglia was examined for the presence of herpes simplex virus type1 (HSV-1) transcripts by Northern hybridization (RNA blot) analysis. By using cloned DNA and single-stranded RNA probes, two abundant colinear HSV-1 transcripts (1.85 and 1.35 kilobases) were detected inganglia from 9 of 17 individuals. These RNAs overlap the 3' end of the transcript for immediate-early gene

ICPO but are transcribed from the opposite strand; thus, they are antisense relative to the ICPO mRNA. Wealso report evidence by in situ hybridization that these latently infected ganglia contain HSV-1 RNAhomologous to the BamHI SP region of the genome which is transcribed in the same direction as the otherlatency transcripts. In Vero cells productively infected with laboratory strain HSV-1 KOS and in culturesinfected with different low-passage clinical isolates, only the 1.85-kilobase transcript was detected, and therewas variation in the size of this larger latency transcript. These novel transcripts may play a role in maintainingHSV-1 latency in human ganglia.

Herpes simplex viruses (HSV) establish lifelong latentinfections of sensory neurons which innervate the site ofinitial infection. In humans, the virus may reactivate repeat-edly to produce either silent or symptomatic infections of themucocutaneous tissues served by these sensory nerves (3).Most of the information regarding HSV latency has beengathered from experimentally infected mice, in which HSVdoes not spontaneously reactivate.There is increasing evidence that limited regions of the

HSV genome are transcribed in latently infected mouseganglia (15, 19). By in situ and Northern hybridization (RNAblot) analysis, RNAs homologous to sequences within thelong repeats of the HSV genome are detected. Specifically,they overlap the gene which encodes the immediate-earlyprotein ICPO (5, 18) (the sequence and splicing pattern ofwhich has been reported [14]) but are transcribed from theopposite DNA strand and thus in an antisense directionrelative to that of the ICP0 transcript (18). RNA homologousto a portion of the BamHI SP restriction fragment (whichstraddles the junction between long and short repeats; Fig. 1)has also been detected by in situ hybridization (5, 21).During latency in mice, three HSV-1 transcripts of sizes 2.0,1.5, and 1.45 kilobases (kb) have been mapped by Northernhybridization (17). RNA homologous to the same region ofthe HSV-1 genome has also been detected in rabbits latentlyinfected with HSV-1 (16).HSV-1 latency in humans has been studied in less detail.

HSV-1 can be recovered in cultures from a high proportionof human trigeminal ganglia (1) and has been detected bySouthern hybridization (6). Even in individuals from whomexplanted trigeminal ganglia failed to yield virus, evidencefor the presence of HSV-1 DNA sequences has been foundby coculture with mutant HSV-1 strains (2). Other data fromin situ hybridizations have also suggested limited transcrip-tion of the HSV-1 genome in ganglia during human latency(20), although one study mapped the RNAs to the left-hand30% of the long unique segment (7).Our laboratory has presented in situ hybridization data

* Corresponding author.

demonstrating HSV-1 RNA in human trigeminal ganglia (4).The RNA we detected in human ganglia was found to bederived from the same region of the HSV-1 genome as thatfound in mice. As in mice, the HSV-1 RNA was shown tooverlap the gene encoding ICP0 and to be transcribed in an

antisense direction relative to that of the ICP0 message.Similar data have recently been reported by another labora-tory (9). We now report that these RNAs include twocolinear HSV-1 transcripts and that RNA homologous to theHSV-1 BamHI SP region is also present in latently infectedhuman ganglia. We also describe variation in the size of thelarger latency transcript among different clinical isolates ininfected cell cultures and in human latent infection.A total of 33 human trigeminal ganglia, removed at au-

topsy from 17 individuals 12 to 24 h after death, were frozenimmediately and stored at -700C. None of the individualshad evidence of an active herpetic infection at the time ofdeath or was known to have immune-impairing illnesses.Total cellular RNA was extracted from the 33 ganglia,infected (harvested at 16 to 20 h postinfection) or uninfectedVero cells, and human brain by homogenization (mincing,polytron treatment, and Dounce homogenization) in guani-dinium thiocyanate, centrifugation through cesium chloride,and precipitation in ethanol as described elsewhere (13).RNA yields were assessed spectrophotometrically andranged between 20 and 50 i,g per ganglion. RNA extractedfrom the paired trigeminal ganglia of a single individual werepooled. Approximately 5 jig of RNA was loaded per well in6% formaldehyde-1.5% agarose gels, subjected to electro-phoresis, and transferred to nitrocellulose paper. Hybridiza-tions with DNA probes were carried out in 60% formamideat 56°C, and those with RNA probes were carried out in 60%formamide at 68°C. Wash conditions were comparably strin-gent. Blots were exposed to Kodak XAR-5 film in cassetteswith intensifying screens at -700C for 1 to 7 days. RNAswere sized by using 18S and 28S internal markers, as well as

high-molecular-weight size markers (Bethesda ResearchLaboratories, Gaithersburg, Md.). In situ hybridization was

performed as described elsewhere (4, 8, 11).HSV-1 DNA probes were subcloned from a BamHI E

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FIG. 1. Map of the areas of the HSV-1 genome from which RNA transcription is detected during latent infection in humans. Northernhybridization analysis corresponding to each gel-pure [32P]dCTP-radiolabeled DNA probe is shown above the corresponding region of themap. RNAs shown are from Vero cells infected with laboratory strain HSV-1 KOS and uninfected Vero cells (UNINF) and from humantrigeminal ganglia 1 through 9. Sizes (in kilobases) were determined from 18S and 28S internal markers and high-molecular-weight RNAmarkers run in separate lanes of the gels. On the basis of these data, the locations of the latency transcripts and their approximate terminiare shown by the dashed lines. The directionality of the transcripts and the approximate 5' end of the 1,350-base (b) transcript is based onthe data in Fig. 2.

genomic clone (Fig. 1). SphI cleavage fragments of about2,600, 786, and 2,866 base pairs (bp) in length (the latter twowere determined precisely from sequence information kindlyprovided by D. McGeoch) were cloned into plasmidpGEM3Z (Promega Biotec, Inc., Madison, Wis.). This vec-tor contains a multiple cloning site flanked by T7 and SP6RNA polymerase promoters and allows synthesis of radio-labeled single-stranded RNA probes (12). To detect byNorthern hybridization RNA transcribed in the same (sense)or opposite (antisense) directions relative to that of authenticICPO mRNA, single-stranded RNA probes were synthesizedin the presence of [32P]UTP by using the 786-bp SphIfragment as a template. Similar RNA probes for in situhybridizations were prepared with [35S]UTP. DNA probeswere prepared by cleavage with the appropriate restrictionendonucleases, electrophoresis in agarose gels, electroelu-tion, and labeling by nick translation with [32P]dCTP. The

2,600- and the 786-bp SphI fragments were gel purified freeof their plasmid vectors prior to nick translation. The 830-bpSphI-SalI subfragment probe was obtained by cleaving the2,866-bp SphI insert with Sall, and the 1,256-bp SalI-HincIIand 780-bp HincII-SphI probes were obtained by appropri-ate double digestions of the 2,866-bp SphI insert (Fig. 1).Northern hybridization analysis of Vero cells infected

with HSV-1 KOS by using the DNA probes spanning thelong repeat (Fig. 1, lanes KOS) revealed the 2.6-kb ICPOtranscript (830-bp SphI-SalI, 1,256-bp Sall-HincII, and 780-bp HincII-SphI DNA subfragment probes). This transcriptwas not detected with the 786-bp SphI probe, which does notoverlap ICPO. A transcript of 1.85 kb was also detected inKOS-infected Vero cells with probes spanning the 786-bpSphI, 830-bp SphI-SalI, and 1,256-bp SalI-HincII DNAsubfragments (Fig. 1). This 1.85-kb transcript overlaps theICPO gene but is not transcribed as an immediate-early gene,

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FIG. 2. Northern hybridization analysis using the 1,256-bp Sall-HincIl DNA subfragment probe of RNA from Vero cells infectedwith laboratory strain HSV-1 KOS, uninfected Vero cells(UNINF.), and Vero cells infected with three different HSV-1clinical isolates (A, B, and C). Sizes are shown at right in base.

since its transcription is blocked in the presence of cyclo-heximide (P. Krause and J. Ostrove, unpublished observa-tion). Neither the ICPO mRNA nor the 1.85-kb transcriptwas seen in uninfected cells (Fig. 1, lanes UNINF). HSV-1-infected Vero cells were also found to contain a family oftranscripts homologous to the 780-bp HincII-SphI probe(Fig. 1). Since no cellular RNAs were detected by this probein uninfected Vero cells, these transcripts may representalternative splicing products of the ICPO message.

Hybridization with the 1,256-bp Sall-HincII probe dem-onstrated variability in the size of the 1.85-kb transcript inVero cells infected with laboratory strain KOS or withdifferent low-passage clinical isolates of HSV-1. In some

cases, two or three transcripts averaging 1.85 kb in lengthwere detected (Fig. 1, 1,256-bp probe, and Fig. 2). This mayimply either that a family of different RNAs can be tran-scribed from this region of the genome of clinical isolates orthat different-sized RNAs are synthesized from diversesubpopulations of virus within the uncloned clinical isolatepools. Alternatively, this variation may result from differ-ences in the extent (if any) of polyadenylation of the tran-scripts, alternative processing in different ganglia, or multi-ple RNA start sites.To determine if human trigeminal ganglia contain tran-

scripts comparable with those reported in murine modelsand homologous to those mapped to this region by in situhybridization, Northern hybridizations with DNA probes ofRNAs from human trigeminal ganglia were performed andare shown in Fig. 1. Lanes 1, 2, and 6 to 9 show HSVtranscripts in ganglia during latency, while the ganglia inlanes 3 to 5 were negative. RNAs extracted from thetrigeminal ganglia of 17 individuals were probed with the786-bp SphI DNA fragment. A 1.85-kb transcript (whichvaried slightly in size; Fig. 1, lanes 8 and 9) was detected in8 of the 17 ganglia RNAs. This 1.85-kb transcript was alsodetected by using the 830-bp SphI-SalI DNA probe and the1,256-bp SaII-HincII DNA probe. In a sample from oneadditional individual (lane 7), a weak signal at 1.85 kb wasdetected with the 830-bp SphI-SalI subfragment DNA probeand with the 1,256-bp SaII-HincII subfragment DNA probebut not with the other DNA probes. The 1.85-kb transcriptsdetected in latently infected trigeminal ganglia and in KOS-infected Vero cells were detected in exactly the same regionof the genome. Hybridizations of ganglia RNA with the830-bp SphI-SalI DNA probe and the 1,256-bp Sall-HincllDNA probe also detected a 1.35-kb transcript which was notseen with the 786-bp SphI or the 780-bp HincII-SphI probes.

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FIG. 3. Northern hybridization analysis using single-stranded[32P]UTP-radiolabeled RNA probes prepared from the HSV-1 SphI786-bp subfragment, which detect transcription from the samestrand ('sense') and from the opposite strand ('antisense') relative toICPO RNA. RNAs are from Vero cells infected with laboratorystrain HSV-1 KOS, uninfected Vero cells (UNINF.), human brain,and human trigeminal ganglia numbers 10 and 11. Sizes are shown atright in base.

This smaller transcript was found exclusively in those gan-glia which were positive for the 1.85-kb transcript and wasnot detected in infected or uninfected Vero cells. Therelative amounts of the 1.85- and 1.35-kb transcripts variedin the different ganglia (Fig. 1, lanes 8 and 9). The 780-bpHincII-SphI probe detected no transcripts in either gangliontested, including one which was strongly positive with theother probes. Northern hybridization analysis using probesspanning the 2,600-bp SphI fragment and the region to theright of these probes, including the entire HSV-1 BamHI SPfragment, also detected no latency-associated transcripts inganglia or in Vero cells infected with HSV-1 (data notshown).

In hybridizations using the single-stranded RNA probes ofeither polarity prepared in vitro from the 786-bp SphItemplate, the 1.85- and 1.35-kb ganglion RNAs were shownto be transcribed in the ICPO antisense direction (Fig. 3). Aweak signal at 1.85 kb was detected in a sample from oneindividual by using this RNA probe (Fig. 3, lane 10) but notby using the corresponding DNA probe (data not shown).The single-stranded RNA probes were of specific activitiesapproximately equal to those of the corresponding DNAprobes, but the RNA-RNA hybridizations were carried outunder less stringent conditions. Thus, the 1.35-kb transcriptswere readily detected with the RNA probe but not with thecorresponding DNA probe, suggesting that the body of thesetranscripts may originate only a short distance to the left ofthe SphI recognition site. The 1.85- and 1.35-kb transcriptsdrawn as dashed lines in Fig. 1 are based on the hybridiza-tion data using all of the probes described.

Detection of HSV-1 transcripts by Northern blot analysisof ganglion RNAs corresponded to the results of parallel insitu hybridizations performed on fragments of the sametissues. Detection of RNA in trigeminal ganglia by in situhybridization has been shown by us to correspond to sero-positivity of the individual (4). Of the samples from 10individuals tested by both methods, 5 showed evidence ofHSV-1 RNA transcription by both Northern and in situhybridization analyses, while 3 were negative by both meth-ods. One ganglion was weakly positive by in situ hybridiza-tion but negative by Northern blot analysis, and another wasnegative by in situ hybridization but weakly positive by

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a :r *t h. } s M-FIG. 4. In situ hybridization of human trigeminal ganglia by using [35S]UTP-radiolabeled RNA probes from the HSV-1 BamHI E and SP

regions. Probes were synthesized to detect transcription in the direction opposite to that of the ICPO message. Panel A is typical of thebackground signal detected by using the 2,866-bp SphI RNA probe on a ganglion in which no positive neurons were seen. Panels B and Cshow positive signals obtained by using the BamHI SP and 2,866-bp SphI clones, respectively. Hybridization conditions and probe specificactivities were equivalent for all three hybridizations. Slides were exposed for 4 to 5 days at 4°C and photographed at x400 magnification.Probes transcribed in the opposite directions from these regions gave negative signals equivalent to that in panel A.

Northern blot analysis, possibly reflecting a low abundanceof the transcript and its unequal distribution throughoutthese ganglia.

In situ hybridizations of human trigeminal ganglia usingprobes homologous to the BamHI SP region detected HSV-1RNA transcribed from the same strand as the 1.85- and1.35-kb latency transcripts. Only ganglia that were positiveby in situ hybridization for the other latency transcriptsyielded signals homologous to the BamHI SP probe. Thesesignals were present in fewer cells (8 to 50% as many) andwere considerably weaker than those obtained in the sameganglia with probes complementary to the latency tran-scripts mapping in the BamHI E region (Fig. 4). In adjacentsections of each ganglion, no correlation was observed in theintensity of the signals between the BamHI E and BamHI SPprobes. Our failure to detect transcripts from the BamHI SPregion by Northern hybridization may be a reflection of thesizes or of the low abundance of these RNAs.The data presented in this report regarding HSV-1 tran-

scription in latently infected human trigeminal ganglia areconsistent with those previously reported for mice. The 1.85-and 1.35-kb latency transcripts described in human trigemi-nal ganglia are slightly smaller than the 2.0-, 1.5-, and1.45-kb transcripts detected in mice. Nevertheless, the ori-entations and map locations of these latency transcripts areidentical in the murine and human systems. The 1.35-kbtranscript may represent a spliced or otherwise processedversion of the 1.85-kb transcript. The evidence for transcrip-tion homologous to the HSV-1 BamHI SP fragment, whichwe now report for humans, is also consistent with thatreported for mice. Thus, we consider the latent infections ofHSV-1 in humans to be essentially identical to that in theexperimental mouse model. The slight differences in the datamay be attributable either to a true distinction in the patternsof HSV-1 transcription in mice and humans or to differences

in sizing the transcripts and our inability to resolve a doubletat 1.35 kb.

In summary, we detected two HSV-1 latency transcriptsin naturally infected human trigeminal ganglia. We alsofound evidence by in situ hybridization in latently infectedhuman trigeminal ganglia of RNA transcription homologousto the BamHI SP region of HSV-1. The 1.85-kb RNA is alsotranscribed in HSV-1-infected Vero cells and may vary insize depending on the virus strain. It is noteworthy that the1.35-kb transcript has only been detected in trigeminalganglia and not in cells acutely infected with HSV-1.Whether this represents a spliced product of the largertranscript or is a unique RNA transcribed solely during thelatent state remains to be determined. These are the onlyHSV-1 transcripts now known to be expressed during la-tency. The exact role of these transcripts is not known, buttheir abundance and unique localization relative to the ICPOgene suggest that they are biologically important to theestablishment or maintenance of viral latency in humansensory nerve ganglia. Latency may be facilitated by theiractivity as veritable antisense transcripts, a mechanismshown to be regulatory in other naturally occurring systems(10), or they may encode proteins which are inhibitory tovirus reactivation.

We thank Alison Freifeld for helpful suggestions and Holly A.Smith and William C. Reinhold for technical assistance. Also specialthanks to John Smialek and Mario Golle of the Baltimore MedicalExaminer's office for assistance in obtaining the tissues and toDuncan McGeoch of the Institute of Virology, University of Glas-gow, for unpublished HSV-1 sequence information.

LITERATURE CITED1. Bastian, F. O., A. S. Rabson, C. L. Yee, and T. S. Tralka. 1972.

Herpesvirus hominis: isolation from human trigeminal ganglion.Science 178:306-307.

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2. Brown, S. M., J. H. Subak-Sharpe, K. G. Warren, Z. Wro-blewska, and H. Koprowski. 1979. Detection by complementa-tion of defective or uninducible (herpes simplex type 1) virusgenomes latent in human ganglia. Proc. Natl. Acad. Sci. USA76:2364-2368.

3. Corey, L., and P. G. Spear. 1986. Infections with herpes simplexviruses. N. Engl. J. Med. 314:686-691, 749-757.

4. Croen, K. D., J. M. Ostrove, L. J. Dragovic, J. E. Smialek, andS. E. Straus. 1987. Latent herpes simplex virus in humantrigeminal ganglia. N. Engl. J. Med. 317:1427-1432.

5. Deatly, A. M., J. G. Spivack, E. Lavi, and N. W. Fraser. 1987.RNA from an immediate early region of the type 1 herpessimplex virus genome is present in the trigeminal ganglia oflatently infected mice. Proc. Nati. Acad. Sci. USA 84:3204-3208.

6. Efstathiou, S., A. C. Minson, H. J. Field, J. R. Anderson, and P.Wildy. 1986. Detection of herpes simplex virus-specific DNAsequences in latently infected mice and humans. J. Virol. 57:446-455.

7. Galloway, D. A., C. M. Fenoglio, and J. K. McDougall. 1982.Limited transcription of the herpes simplex virus genome whenlatent in human sensory ganglia. J. Virol. 41:686-691.

8. Gendelman, H. E., S. Koenig, A. Aksamit, and S. Venkatesan.1986. In situ hybridization for detection of viral nucleic acid incell cultures and tissues, p. 203-223. In G. Uhl (ed.), In situhybridization in brain. Plenum Publishing Corp., New York.

9. Gordon, Y. J., B. Johnson, E. Romanowski, and T. Araullo-Cruz. 1988. RNA complementary to herpes simplex virus type 1ICPO gene demonstrated in neurons of human trigeminal gan-glia. J. Virol. 62:1832-1835.

10. Green, P. J., 0. Pines, and M. Inouye. 1986. The role ofantisense RNA in gene regulation. Annu. Rev. Biochem. 55:569-597.

11. Haase, A., M. Brahic, and L. Stowring. 1984. Detection of viralnucleic acids by in situ hybridization. Methods Virol. 7:189-226.

12. Melton, D. A., P. A. Krieg, M. R. Rebagliati, T. Maniatis, K.Zinn, and M. R. Green. 1984. Efficient in vitro synthesis of

biologically active RNA and RNA hybridization probes fromplasmids containing a bacteriophage SP6 promoter. NucleicAcids Res. 12:7035-7056.

13. Ostrove, J. M., W. Reinhold, C. Fan, S. Zorn, J. Hay, and S. E.Straus. 1985. Transcription mapping of the varicella-zoster virusgenome. J. Virol. 56:600-606.

14. Perry, L. F., F. J. Rixon, R. D. Everett, M. G. Frame, and D. J.McGeoch. 1987. Characterization of the IE110 gene of herpessimplex virus type 1. J. Gen. Virol. 67:2365-2380.

15. Puga, A., and A. L. Notkins. 1987. Continued expression of apoly(A)+ transcript of herpes simplex virus type 1 in trigeminalganglia of latently infected mice. J. Virol. 61:1700-1703.

16. Rock, D. L., A. B. Nesburn, H. Ghiasi, J. Ong, T. L. Lewis,J. R. Lokensgard, and S. L. Wechsler. 1987. Detection oflatency-related viral RNAs in trigeminal ganglia of rabbitslatently infected with herpes simplex virus type 1. J. Virol. 61:3820-3826.

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. Stevens, J. G., E. K. Wagner, G. B. Devi-Rao, M. L. Cook, andL. T. Feldman. 1987. RNA complementary to a herpesvirusalpha gene mRNA is prominent in latently infected neurons.Science 235:1056-1059.

19. Stroop, W. G., D. L. Rock, and N. W. Fraser. 1984. Localizationof herpes simplex virus in the trigeminal and olfactory systemsof the mouse central nervous system during acute and latentinfections by in situ hybridization. Lab. Invest. 51:27-38.

20. Tenser, R. B., M. Dawson, S. J. Ressel, and M. E. Dunstan.1982. Detection of herpes simplex virus mRNA in latentlyinfected trigeminal ganglion neurons by in situ hybridization.Ann. Neurol. 11:285-291.

21. Wagner, E. K., G. Devi-Rao, L. T. Feldman, A. T. Dobson, Y.Zhang, W. M. Flanagan, and J. G. Stevens. 1988. Physicalcharacterization of the herpes simplex virus latency-associatedtranscript in neurons. J. Virol. 62:1194-1202.

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