• No results found

Human herpesvirus 6 infects cervical epithelial cells and transactivates human papillomavirus gene expression.


Academic year: 2019

Share "Human herpesvirus 6 infects cervical epithelial cells and transactivates human papillomavirus gene expression."


Loading.... (view fulltext now)

Full text



Copyright © 1994,AmericanSocietyforMicrobiology



Herpesvirus 6 Infects Cervical Epithelial Cells and

Transactivates Human Papillomavirus Gene Expression










Laboratory ofBiology,1 Laboratory of Tumor Cell Biology, 2 and Laboratory of Cellular and Molecular Biology,3 National Cancer

Institute, Bethesda,



Received 14 October 1992/Accepted 25 October 1993

To examine whether human herpesvirus 6 (HHV-6) is capable of infecting human cervical epithelial cells and altering expressionofhuman papillomavirus (HPV) genes, HPV-immortalized or -transformed carcinoma cell lines were infected with HHV-6 variant A. No cytopathiceffectwas observed in infected cervical cells. However, immunofluorescence indicated that infected cells expressed early-late proteins of HHV-6 by day 3 postinfection. HHV-6 DNA was also detected by Southern blot hybridization after infection and persisted through continued subculture in an episomal state as proven by Gardella gel electrophoresis and fluorescence in situ hybridization. HHV-6 infection enhanced expression of HPV RNAs encoding the viral oncoproteins E6 and E7. Transient transfection assays showed that two HHV-6 molecular clones, pZVB-70 and pZVH-14, upregulated transcription 9- to 15-fold from a reporter plasmid containing the HPV type 18 regulatory sequences which control transcription in vivo.Cervical carcinoma cells infected with HHV-6 induced more rapid development of tumors in mice than did noninfected cells. These results are the first evidence that human cervical epithelial cells can be infected with HHV-6 and that HHV-6 contains transactivators which stimulate the HPV-transforming genes.

Experimental and epidemiologic evidence indicates that human papillomaviruses (HPVs) are important etiologic agents for cervical cancer (5, 39, 46). Infection with HPVsis necessary but not sufficient for development of this disease. For example, the long latency between HPV infection and development of cervical cancer and the low prevalence of cancerin the infectedpopulation emphasize the importance of other etiologic and host factors for the progression to malig-nancy. Current experimental evidence indicates that HPV infection results in a cell population with an increased expo-sure to other cancer-causing factors, such as smoking and humanherpesvirus2.Epidemiologicdataindicate correlations between cervical cancer and smoking (7, 44) and, more re-cently, between cervicalcancerandherpes simplex virustype2 (16).

Recent reports suggest that human immunodeficiency virus (HIV)-positivewomenwith cervicalcancerhavehigher recur-rence and death rates than HIV-negative controls (33). Both squamouscellneoplasia ofthecervixand HIV type 1 (HIV-1) infection are sexually transmitted diseases; HIV-1 has been isolated fromcervical biopsy material of HIV-positivewomen with cervicitis (12, 36). Human herpesvirus 6 (HHV-6), first isolated from patients with lymphoproliferative disorders and AIDSpatients in 1986 (25, 37), isaT-lymphotropic virus (32) widely distributed in the general population. Preliminary evi-dence correlates active HHV-6 infection with rapid progres-sion of HIV infection (la, 19, 28). HHV-6proteins

transacti-*Corresponding author. Mailing address: Laboratory ofBiology,

National Cancer Institute, 9000 Rockville Pike, Building 37, Room 2A19, Bethesda, MD 20892. Phone: (301) 496-6441. Fax: (301) 496-3238.

tPresentaddress: UniversityofAntwerp(VIA), Antwerp,Belgium.

vatethe long terminal repeat ofHIV(10, 17, 18, 31). HHV-6 caninfect cellsnotbelongingtotheT-lymphocyte lineage,such asBlymphocytes(2, 22, 24)andepithelialcellsof thebronchus andsalivary glands (13, 27). However, noinformation regard-ing interaction betweenHHV-6and theHPVsassociatedwith cervicalcancerexists.To studypotential interactionsbetween HPV and HHV-6, cervical carcinoma cell lines and HPV-immortalized genital epithelial cell lines were exposed to HHV-6 (strain GS, variant A) to determinewhether human cervical epithelial cells could be infected with HHV-6 and whether specific HHV-6 sequences influenced HPV gene expression in infected cells.

Several cervical epithelial cell lines or primary cultures of humanforeskinepithelial (HKc) cellswereused.Immortalized cervical cell lines CX16-2S and CX16-5S were originally ob-tainedby transfection ofprimaryexocervical cells with recom-binant HPV type 16 (HPV-16) (45). Tumor lines C4-1 and QG-HarespontaneouscervicalcarcinomasharboringHPV-18 and HPV-16, respectively (3, 41). As a positive control, an immature human T-cellline (HSB-2),which is permissivefor HHV-6,wasalsoinfectedwith thesamevirusstockbymethods previously described (2). All cultures were propagated in F12-Dulbecco's medium, supplemented with5% fetal bovine serum.

No generalized morphologic change was observed in any cervical cell line or HKc examined on days 3, 7, 10, and 14 postinfection with HHV-6 (1,00050% tissue culture infective doses of filtered virus per ml). A limited number of cells in HHV-6-infected cultures became detached; some became roundandenlarged. When cells fixedonslideswereexamined by indirect immunofluorescent assay (IFA) with monoclonal antibodies (MAbs) to early-late viral proteins 9A5D12


(4) andC-5 (p41/38)(21), anaverageof10to 15% ofthe cell 1173

on November 9, 2019 by guest



1174 NOTES

FIG. 1. Immunofluorescencestainingof cervicalepithelialcells infected with HHV-6(GS),aswellasofpositiveandnegativecontrols.(A, D, G,andJ)HSB-2 T-cell lineinfected with HHV-6(positive control); (B, E, H,andK)mock-infected CX16-2S cervicalepithelialcells(negative control); (C,F,I, and L) CX16-2S cellsinfected with HHV-6. Cellswerecollected either 3days (AthroughI) or10days(J throughL)after infection ormock infection. Antibodies used werefluorescein-conjugated goatanti-mouse immunoglobulin Gantibodyalone (A throughC), 9A5D12(p41) (D through F),C-5 (p41/38) (Gthrough I),and 2E2(gpllO)(Jthrough L).

population (CX16-2S, CX16-5S, QG-H, and C4-1 cell lines) wereHHV-6 antigen positive.The results for HSB-2(positive

control) andCX16-2SareshowninFig. 1.Immunofluorescent

staining in some cells was nuclear and in other cases was diffuse, with the entire cell stained.ForCX16-2S,


of the cellsexpressed early-late proteins(p41 and/or p41/38) (Fig.1F and I), whereas <5% of the cells werefound toexpress late protein asdetected byMAb 2E2(gp110) (21) (Fig. IL).Cells expressinglateproteinweredetected by day7 andthereafter,

whereas early-late protein-expressing cells were observed by

day 3 (<15%). Mock-infected HPV-16-immortalized cells

(HCX16-2S) (Fig. 1B,E,H, and K), aswellasmock-infected normal HKc and QG-H and C4-1 tumor cells, did not react with HHV-6 MAbs (data not shown). When infected HCX16-2S cellsweresubculturedandexamined for expression

of HHV-6antigens by IFA,<1% of the cellswerepositivefor p41 andp41/38,but nostainingwasobserved forgpllO.After subsequent subculturing, no HHV-6 antigen-expressing cell was observed by any MAb, suggesting that the virus either became latent or was eliminated from the cell. In contrast, infection ofthe HSB-2 T-cell line resulted in lysis of infected cells. The staining pattern was similar to that observed with HHV-6-infected cervical cells. However, by day 3,

approxi-mately35% ofHHV-6GS-infected HSB-2 cellswere antigen

positive as determined by IFAwith all the MAbs; by day 7, >70% of the cells exhibitedHHV-6antigens(Fig. ID, G,and J).Byday 14, >90% oftheinfected HSB-2 cellswereHHV-6 antigen positive and themajority of the cells showed typical HHV-6-inducedcytopathicmorphologyasreportedpreviously






on November 9, 2019 by guest



1 2 3 4 5 6 7 8 9 10

8.7kb-FIG. 2. Characterization ofHHV-6 DNA ininfected cervical cells. Southern blotanalysisofHHV-6(GS)-infected andnoninfected HPV immortal, cervical carcinoma cell linesand HSB-2was done 10days postinfection. DNA was digested with Hindlll and hybridized with

32P-labeled pZVH-14HHV-6 DNA insert. Lanes 1 through 4and 9, noninfectedC4-1, QG-H, CX16-2S,CX16-5S,and HSB-2cells, respec-tively; lanes 5 through 8 and 10, infected C4-1, QG-H, CX16-2S, CX16-5S,and HSB-2 cells,respectively.





and loss of HHV-6 DNA, Southern





with infected cervical cell lines after subcultivation. DNAsextracted from HHV-6-infected and mock-infected cell lineswere




sequences under

stringent hybridization








probe pZVH-14,

an 8.7-kb restriction

fragment coding

for the

putative large



(23), hybridized strongly

with all infected cell linestested,






2,lanes 5


8 and


noninfectedcell lineswere



2, lanes 1


4 and

9). Interestingly,

HHV-6 sequences



detected in infected carcinoma cells even

after in vivo passage and tumor formation in nude mice,


the lack of HHV-6

antigen expression by





This indicated that HHV-6


in a latent state in HPV-transformed



To examine the


state of the


HHV-6 DNA, molecular


and fluorescence in situ



on infected cells after 16








HHV-6. A South-ern blot


based onthe method






for detection of


closed circles of DNA in bacteria and modified


Gardella et al. for DNA molecules in mammalian cells


wasused. This




in that

gel loading





at40Cand that cell





inthe wellof

a horizontal

gel (0.8%




hybridization probe

was a 22.3-kb HHV-6 sequence


the GGGITA tandemrepeatand





HHV-6 hasa lineargenome; less than 10% of HHV-6 DNA is


tobe circularized

(29, 34).

HHV-6 DNA molecules were detectable as both


closed circles and linear forms in infected C4-1 cells

(Fig. 3B3,

lanes 4 and


butnotin mock-infected C4-1 cells


3B, lane


The lower band contains linear HHV-6 DNA because its



com-A B

- ccc


1 2 3 4 5

FIG. 3. Presence of episomal HHV-6 DNA in HHV-6-infected C4-1 cells as demonstrated by Southern blot hybridization of a Gardellagel with pZVB-70. (A) Lanes1 and 2, positive control HSB-2 cellsinfected with HHV-6. Both have covalent closed circle (CCC) and linear HHV-6. (B) Lane 3, non-HHV-6-infected C4-1; lanes4and 5, HHV-6-infected C4-1. Lanes4and 5 show both covalent closedcircle and linear HHV-6.

parabletothatobtained with HHV-6-infected HSB-2 cells that containpredominantly linearHHV-6 (Fig. 3B, lanes1 and2). The broad aspect observed in the lower linearDNAband of the infected HSB-2 cells may reflect the presence of variable lengthsof theterminal directrepeatsof theHHV-6genome as describedby Lindquester and Pellett(29). WhenthepZVB-70 probe was biotinylated and applied to HHV-6-infected C4-1 cells,multiple randomly distributed fluorescentsignals associ-ated with chromosomes were observed on 38% of the 100 metaphase spreadsand52% of the 1,000 nuclei examined. The pattern wasidenticaltothat obtainedwithaBurkittlymphoma (Raji) (20) cell line usedas apositive control. Uninfected C4-1 cellsdidnotshow anyhybridization signal. Thus, fluorescence in situhybridization analysis confirmed thepresence of episo-mal HHV-6in50% of the overallpopulation. These results,as wellasotherswithsalivary glands from normal individuals and bronchialepithelia fromHIV-positive patients (13, 27), clearly demonstrate that HHV-6 DNA is capable of persisting in a latent state in humanepithelial cells.

DisregulationandoverexpressionofHPVoncogenes E6and E7have beenproposedascentral mechanisms intheinduction of cervical carcinoma (46). Because HHV-6 sequences influ-encetranscription ofother virusesincluding HIV(10, 17, 18,

22), studies were undertaken to examine whether HHV-6 altered HPV gene expression. The effect of HHV-6 on the expression of HPV-16 and HPV-18 RNA was examined in C4-1 andCX16-5S cell lines (Fig. 4)which containedHPV-18 and HPV-16 DNA sequences, respectively. Northern (RNA)

blot analysis showed that both cell lines expressed 1.7- and 4.2-kb transcripts representing spliced and unspliced RNAs encoding E6 and E7. Both transcripts are found in a wide variety of HPV-immortalized cell lines (8, 26, 35, 45). C4-1 cellsalsoexpressedhigh-molecular-weight HPV RNAswhich may originate or terminate within host sequences. HHV-6 infection of both C4-1 and CX16-5S cell lines increased steady-state levels of HPV-18 and HPV-16 RNA relative to those ofmock-infected controls (Fig. 4, lanes 2). Films were

monitored with a scanning densitometer to quantitatively measuredifferencesin RNAexpression.Althoughthe patterns of HPVtranscriptsin HHV-6-infectedand mock-infected cell lines were similar, the level of HPV RNA expression was

on November 9, 2019 by guest


[image:3.612.] [image:3.612.373.502.74.222.2]

1176 NOTES

A. HPV18

1 2





B. HPV16

1 2



4.2 Kb

1.7 Kb



FIG. 4. Upregulation of HPV geneexpression byHHV-6 in C4-1

tumor(A)andCX16-5S immortal(B)cell lines. Northern blotanalysis

shows enhancement of HPV-16 and HPV-18 RNA expressionafter

HHV-6(GS)infection.rRNA markersare ontheright,andkilobase

markers are on the left. Lanes 1,mock-infected cells; lanes2, cells

infected with HHV-6. Hybridization was done with 32P-labeled

HPV-18 (A) orHPV-16(B) and GAPDH DNAprobes.The 1.7-kb

transcript (slightlybelow the18S position) isasplicedRNAencoding

E6 and E7. The 4.2-kb transcript located slightly below the 28S

positionisanunspliced, complete early region transcript.

upregulated byHHV-6 two- to threefold inC4-1 tumor cells and to a lesser extent in CX16-5S immortal cells. Because approximately half of the infected cells demonstrated the HHV-6 genome by the fluorescence in situ hybridization technique, the observed two- to threefold increase in expres-sion of HPV RNA actually represents a four- to sixfold increase in itsexpressioninthe subset of the cells infected with HHV-6.

Transient transfection analyses were undertaken to deter-mine whether specific HHV-6 sequences mediated

transcrip-tional activation of the HPV-18 promoter. Inthese studies, a

DNAfragmentfrom thelongcontrolregionofHPV-18,which is responsible for regulating virus gene transcription in vivo,

was cloned in front of a chloramphenicol acetyltransferase

(CAT) reporter plasmid (43). These constructswere cotrans-fected into cervicalcells in combination with eitheroneoftwo HHV-6genomicclonespZVB-70andpZVH-14,whichencode proteinsknown to transactivate the HIVlong terminalrepeat

1 2 3 4 5 6

FIG. 5. Transient transfection analysis showing transactivation of HPV promoterby HHV-6 molecular clones. HeLa cellswere

trans-fected withpHl8CATalone(lane 1), pHl8CAT andpZVB-70(lane

2), pHl8 CAT and pZVH-14 (lane 3), pHl8 CAT and no vector

pZVB-9 (lane 4), pHl8CAT andvector IBI-31(lane 5),orpBluescript

vectoralone(lane6).Cellswereharvested for CATanalysis48 h after transfection.CATassayswereperformedfor 30minat37Cby using 100 jig of total cellularprotein; productswereseparated byascending thin-layerchromatography.

(18). Cotransfection was performed with lipofection reagent

according to the manufacturer's procedure (GIBCO,

Be-thesda, Md.). Cellswere lysed bysonication and assayed for CATactivityasdescribedbyGorman et al.(15),with volumes ofextractscorrespondingto 100 ,ugofprotein.Percent acety-lationwasdeterminedby quantitationofcountsin acetylated

and unacetylated spots. The independent HHV-6 molecular

clones,pZVB-70andpZVH-14,transactivated thepH18-CAT

promoter in HeLa cells (Fig. 5). Although each experiment

was done in triplicate, results of a single experiment are presentedbecause of datasimilarity.Little CATactivity (1%) wasdetected in unstimulated HeLa cells transfected withpH18 CAT alone(Fig. 5).Increased CATexpression,corresponding to 15.2- and 8.8-fold CAT conversion, was induced upon cotransfection withpZVB-70andpZVH-14, respectively.This transactivation by pZVB-70 and pZVH-14 was specific be-cause cotransfection withpZVB-9, an 11-kb molecular clone that contains the major DNA-binding protein (6) ofHHV-6, failed to transactivate as did plasmid containing vector-only

sequences(pIBI-31forpZVB-70andpZVB-9andpBluescript for pZVH-14). These data suggest that specific molecular clones of the HHV-6 genome transactivate HPV-18, a clini-cally important pathogenicagentincervicalcancer. Thus,the data support the hypothesis that HHV-6 functions in the multistage carcinogenesis process leadingtocervicalcancerby

upregulating expressionof HPV E6 and E7 oncogenes. Todetermine whether theupregulationof HPVbyHHV-6 influenced the tumorigenicity of the infected cervical carci-nomacell lines, infected and uninfected cultureswere inocu-latedsubcutaneouslyinto nudemice. Cervical carcinoma cells C4-1 andQG-Hinfected with HHV-6producedtumorswitha shorterlatencyperiod (3weeksversus6weeks)than that of the parent line. All injected animals developed tumors, and ani-mals were killed when the tumorswere 2.5 cm in diameter.

Explantedtumorscontained detectableHHV-6-specificDNA

(data not shown). Histopathological examination revealed increased numbers of multinucleated cells and foci of kerati-nizationcomparedwith those in theparentalC4-1 andQG-H

cells.However,noanimalinjectedwith theHPV-immortalized cell lines (CX16-2S and CX16-5S), whether they contained HHV-6 ornot, developedatumorduringa6-month observa-tion period. Thus, HHV-6 enhancedtumorigenicity of estab-lished carcinoma lines but did not convert immortal lines to malignancy.

Similartootherherpesviruses, HHV-6is likelytopersistin a latent form after primary infection. Its reactivation may contributetodiseasemanifestation. TherateofHHV-6 reac-tivation is high in asymptomatic HIV-1-positive individuals


and in AIDS patients


(1). Furthermore, HIV-1 and HHV-6 are both infectious and cytopathic for

CD4+ cells in vitro (31), and HHV-6 is capable ofinducing

CD4 in CD8+ T cells, rendering such cells susceptible to HIV-1 (30). Therefore, such interaction in vivo could leadto severeimmunesuppression.Ageneral depletionofT

lympho-cytes,with CD4helpercellsmoredepletedthan CD8 suppres-sorcells, has also been observed inpatientswith HPV infec-tionsandcervicalintraepithelial neoplasia(42).On the basis of such observations, one can hypothesize that reactivation of HHV-6may contribute to immunosuppression and transacti-vate papillomavirus oncogenes in cervical carcinoma. The increased expression of HPV-18 RNA post-HHV-6 infection inC4-1 carcinoma cells each withasinglecopyofHPV-18(40)

supportssuch aconcept. Additional studiesto determine the interaction among HPVs, HHV-6, and HIV in the develop-mentof cervical carcinomasarewarranted.




on November 9, 2019 by guest



Experiments utilizinginsituhybridizationonfixedtissueare in progress to determine how frequently HHV-6 is found in cervical cancer. Preliminaryresults indicate that a small pro-portionofHPV-positivecervicalcancershaveHHV-6 DNA in epithelial cell nuclei, supporting a possible relationship be-tween HPVand HHV-6 invivo. Thus, HHV-6may influence HPV


and inturnbe involved in the progressionof cervicalcancer.

FrancoiseThierryprovidedthepH18 CAT,anHPV-18 BamHIlong controlregion fragment-linkedCAT.

REFERENCES 1. Ablashi,D. V.Unpublisheddata.

la.Ablashi, D. V., S. F. Josephs, A. Buchbinder, K. Hellman, S. Nakamura, T. Liana, P. Lusso, M. Kaplan, J. Dahlberg, S. Memon,F.Imam,K. L.Ablashi,P.D.Markham,B.Kramarsky, G.R. F. Krueger,P. Biberfeld,F.Wong-Staal,S.Z.Salahuddin,

and R. C. Gallo. 1988. Human B lymphotropic virus (human

herpesvirus-6).J.Virol. Methods 21:65-88.

2. Ablashi, D. V., P. Lusso, C. L. Hung, S. Z. Salahuddin, S. F. Josephs,T.Llana,P.Biberfeld,P. D.Markham,and R.C. Gallo. 1988. Utilization of humanhematopoieticcell linesfor the prop-agationandcharacterization of HBLV(human herpesvirus-6).Int. J. Cancer 42:787-791.

3. Auersperg, N.,and A. F. Hawryluk. 1962. Chromosome observa-tionsonthreeepithelialcell culturesderived from carcinomas of the human cervix. J. Natl. Cancer Inst. 28:605-627.

4. Balachandran,N., R. E.Amelse,W. W. Zhu, and C. K.Chang. 1989. Identification of proteins specific for human herpesvirus 6-infected humanTcells.J. Virol. 63:2835-2840.

5. Bauer,H.M.,Y.Ting,C. E.Greer,J.C.Chambers,C.J.Tashiro, J.Chimera,A.Reingold,and M.M.Manos.1991.Genital human

papillomavirus infection in female university students as deter-minedbyaPCR-based method.JAMA265:472-477.

6. Berneman,Z. N.,and D. V. Ablashi. 1992. Molecularbiologyof human herpesvirus 6 (HHV-6), p. 81-96. In D. V. Ablashi, G. R. F. Krueger, and S. Z. Salahuddin (ed.), Human herpesvi-rus-6. ElsevierScience Publishers B.V.,NewYork.

7. Brinton,L.A.,C.Schairer,W.Haenszel,P.Stolley,H.F.Lehman, R.Levine,and D. A.Savitz. 1986.Cigarettesmokingandinvasive cervicalcancer. JAMA255:3265-3269.

8. Durst, M., R. T. Dzarlieva-Petrusevska, P. Boukamp, N. E. Fusenig, and L. Gissmann. 1987. Molecular and cytogenetic

analysis of immortalized human keratinocytes obtained after transfection with human papillomavirustype16 DNA.Oncogene 1:251-256.

9. Eckhardt, T. 1978. A rapid method for the identification of

plasmid desoxyribonucleicacid in bacteria. Plasmid 1:584-588. 10. Ensoli,B.,P.Lusso,F.Schachter,S.F.Josephs,J.Rappaport,F.

Negro, R. C. Gallo, and F. Wong-Staal. 1989. Human

herpesvi-rus-6increases HIV-1 expressioninco-infectedTcells via nuclear factorsbindingto theHIV-1 enchancer. EMBO J. 8:3019-3027. 11. Feinberg,A.P.,and B.Vogelstein.1983.Atechnique forlabeling

DNArestriction endonuclease fragmentstohigh specific activity.

Anal. Biochem. 132:6-13.

12. Feingold,A. R., S. H.Vermund,R. D. Burk, K. F.Kelley, L. K. Schrager, K. Schreiber, G. Munk, G. H. Friedland, and R. S. Klein. 1990. Cervical cytologicabnormalities andpapillomavirus

in women infected with human immunodeficiencyvirus. J.


ImmuneDefic. Syndr.3:896-903.

13. Fox,J.D.,M.Briggs,P. A.Ward,and R.S. Tedder.1990.Human


6in salivary glands.Lancet336:590-593.

14. Gardella, T., P. Medveczky, T. Sairenji, and C. Mulder. 1984. Detection of circular and linear herpesvirus DNA molecules in mammalian cellsby gel electrophoresis. J. Virol. 50:248-254. 15. Gorman,C.M.,L. F.Moffat,and B. H. Howard. 1982.

Recombi-nantgenomeswhichexpresschloramphenicolacetyltransferasein mammalian cells. Mol. Cell. Biol. 2:1044-1051.

16. Hildesheim,A.,V.Mann,L.Brinton,M.Szklo,W.Reeves,and W. Rawls. 1991. Herpes simplexvirus type 2: apossible interaction with human papillomavirus types 16/18 in the development of

invasive cervicalcancer. Int. J.Cancer 49:335-340.

17. Horvat, R.T., C. Wood,andN.Balachandran. 1989.Transactiv a-tion of human immunodeficiency virus promoter by human her-pesvirus 6. J. Virol. 63:970-973.

18. Horvat, R. T., C. Wood, S.F.Josephs,and N.Balachandran.1991. Transactivation of the human immunodeficiency virus promoter by human herpesvirus 6 (HHV-6) strains GS and Z-29 inprimary humanTlymphocytes and identification of transactivating HHV-6(GS) gene fragments. J. Virol.65:2895-2902.

19. Huemer, P. H., C. Larcher, H.Wachter, and M. P.Dierich. 1989. Prevalence of antibodiestohumanherpesvirus-6 in human immu-nodeficiency virus 1-seropositive and -negative intravenousdrug addicts. J. Infect. Dis. 160:549-550.

20. Hurley,E.A.,S.Agger, J.A.McNeil,J. B. Lawrence, A. Calendar, G. Lenoir, and D. A. Thorley-Lawson. 1991. WhenEpstein-Barr virus persistently infects B-cell lines, it frequently integrates. J. Virol. 65:1245-1254.

21. Iyengar,S., P. H. Levine, D. V. Ablashi, J. Neequaye, and G. R. Pearson. 1991. Sero-epidemiological investigations on human herpesvirus-6 (HHV-6) infections using a newly developedearly antigen assay. Int. J. Cancer 49:551-557.

22. Jarrett,R. F., S. Gledhill, F. Qureshi, S. H. Crae, R. Madhok, I. Brown, I. Evans, A. Krajewski, C. J. O'Brien, R. A. Cartwright, P. Venables, and D. E. Onions. 1988. Identification of human her-pesvirus 6-specific DNA sequences in two patients with non-Hodgkin's lymphoma. Leukemia 2:496-502.

23. Josephs, S. F.,D. V.Ablashi,S. Z.Salahuddin,L. L.Jagodzinski, F.Wong-Staal,andR. C. Gallo.1991.Identification of the human herpesvirus6glycoproteinH andputative large tegument protein genes.J. Virol. 65:5597-5604.

24. Josephs,S.F.,A.Buchbinder,H. Z.Streicher, D. V. Ablashi, S. Z. Salahuddin,H.-G.Guo,F.Wong-Staal,J. Cossman, M. Raffeld,J. Sundeen, P. Levine, R. Biggar, G. R. F. Krueger, R. I. Fox, and R. C. Gallo. 1988. Detection of human B-lymphotropic virus (human herpesvirus 6) sequences in B cell lymphoma tissues of threepatients. Leukemia 2:132-135.

25. Josephs,S.F., S. Z.Salahuddin, D.V. Ablashi,F. Schachter,F. Wong-Staal,and R. C. Gallo. 1986. Genomicanalysis of human B-lymphotropicvirus (HBLV). Science 234:601-603.

26. Kaur, P.,andJ.K.McDougall. 1989.HPV-18immortalization of humankeratinocytes. Virology173:302-310.

27. Krueger,G. R.F.,K.Wassermann,L. S. DeClerck,W.J.Stevens, N.Bourgeois,D. V.Ablashi, S. F. Josephs, and N. Balachandran. 1990. Latent humanherpesvirus-6insalivary and bronchial glands. Lancet336:1255-1256.

28. Levy, J. A., D. Greenspan, F. Ferro, and E. T. Lennette. 1990. Frequentisolation of HHV-6 from saliva andhigh seroprevalence of the virus in thepopulation.Lancet 335:1047-1050.

29. Lindquester,G.J.,and P. E. Pellett.1991.Propertiesofthe human herpesvirus 6 strain Z29 genome: G + C content, length, and presenceofvariable-length directly repeated terminal sequence elements.Virology 182:102-110.

30. Lusso, P.,A. DeMaria,M.Malnati, F.Lori,S. E. DeRocco,M. Baseler,and R.C.Gallo. 1991.Induction of CD4 and susceptibil-itytoHIV-1 infection in human CD8+ Tlymphocytes byhuman herpesvirus6. Nature(London)349:533-535.

31. Lusso, P., B. Ensoli, P. D. Markham, D. V. Ablashi, S. Z. Salahuddin,E.Tschachler,F.Wong-Staal,and R. C. Gallo. 1989. Productive dual infection of human CD4+ T lymphocytes by HIV-1 and HHV-6. Nature(London)337:368-370.

32. Lusso, P., P. D. Markham, E. Tschachler, F. Veronese, S. Z. Salahuddin,D.V.Ablashi,S.Pahwa,K.Krohn,and R. C.Gallo. 1988. In vitro cellular tropism of human B-lymphotropic virus (human herpesvirus-6).J.Exp. Med.167:1659-1670.

33. Maiman, R., R. Fruchter, E. Serur, J. Remy, G. Feuer, and J. Boyce. 1990. Human immunodeficiencyvirus infection and cervi-calneoplasia. Gynecol.Oncol. 38:377-382.

34. Martin,M. E.D., B.J. Thomson,R. W. Honess,M. A.Craxton, U. A.Gompels, M.-Y. Liu, E. Littler, J.R. Arrand, I.Teo,and M. D.Jones.1991.The genome of humanherpesvirus6: mapsof unit-lengthandconcatemeric genomes for nine restriction endo-nucleases. J. Gen.Virol. 72:157-168.

35. Pecoraro, G.,D.Morgan,and V. Defendi. 1989.Differential effects

on November 9, 2019 by guest



1178 NOTES

of humanpapillomavirus type 6, 16, and 18 DNAson

immortal-ization andtransformation of human cervical epithelial cells. Proc. Natl. Acad. Sci. USA86:563-567.

36. Pomerantz, R. J., S. M. de laMonte,S. P. Donnegan, T. R. Rota, M. W. Vogt, D. E. Craven, and M. S. Hirsch. 1988. Human immunodeficiency virus infection of the uterine cervix. Ann. Intern.Med. 108:321-327.

37. Salahuddin, S. Z., D. V. Ablashi, P. D. Markham, S. F. Josephs, S. Sturzenegger, M. Kaplan, G. Halligan, P. Biberfeld, F. Wong-Staal, B. Kramarsky, andR. C. Gallo. 1986. Isolation ofa new

virus, HBLV, in patients withlymphoproliferative disorders.


38. Sambrook, J., E. F. Fritsch, and T. Maniatis. 1989. Molecular cloning: a laboratory manual, 2nd ed., p. 916-923. Cold Spring

HarborLaboratory Press, Cold Spring Harbor, N.Y.

39. Schiffman, M.1992. Recentprogressindefiningtheepidemiology of humanpapillomavirus infection and cervical neoplasia. J. Natl. Cancer Inst.84:394-398.

40. Schwarz, E.,U. K. Freeze, K. Gissmann, W. Mayer, B. Roggen-buck, A. Stemlau, and H. zur Hausen. 1985. Structure and transcription of human papillomavirussequencesincervical

car-cinomacells. Nature (London) 314:111-114.

41. Shirasawa, H., Y. Tomita, S. Sekiya, H. Takamizawa, and B. Simizu. 1987. Integrationand transcription of human papilloma-virustype16 and 18 sequencesin cell linesderived from cervical carcinomas.J. Gen.Virol. 68:583-591.

42. Tay,S. K.,D.Jenkins, andP. Maddox.1987. Lymphocyte

pheno-typesincervicalintraepithelial neoplasia and human

papillomavi-rusinfection. Br.J. Obstet. Gynaecol. 94:16-21.

43. Thierry, F., and M. Yaniv. 1987. The BPV1 E2 trans-acting protein

canbeeitheranactivatororarepressorof theHPV18regulatory region. EMBO J. 6:3391-3397.

44. Winkelstein,W. 1977. Smoking andcancerof the uterinecervix: hypothesis. Am. J.Epidemiol. 106:257-259.

45. Woodworth, C. D., P. E. Bowden, J. Doniger, L. Pirisi, W. Barnes, W. D. Lancaster, and J.A. DiPaolo. 1988. Characterization of normal humanexocervical epithelial cells immortalizedinvitroby papillomavirustypes 16and 18 DNA. Cancer Res. 48:4620-4628. 46. zurHausen, H. 1989. Papillomaviruses in anogenitalcancer as a

modeltounderstand the role of viruses in humancancers.Cancer Res.49:4677-4682.


on November 9, 2019 by guest



FIG.1.control);9A5D12G,infection and Immunofluorescence staining of cervical epithelial cells infected with HHV-6 (GS), as well as of positive and negative controls
FIG. 2.Southernpostinfection.32noninfectedtively;CX16-5S,immortal,P-labeled Characterization of HHV-6 DNA in infected cervical cells
FIG. 4.tumorshowsHHV-6HPV-18markersE6positiontranscriptinfected Upregulation of HPV gene expression by HHV-6 in C4-1 (A) and CX16-5S immortal (B) cell lines


Related documents

According to Gacuru (2015) who did research on factors affecting efficiency in logistics performance of trading and distribution firms based in Jomo Kenyatta International airport

Because we felt that the situations at our two institutions were not that different, and we could imagine process evolution at either place that would take it in the direction of

sical MD method, which assumes an appropriate interatomic interaction, the so-called empirical potential. The former can be used to perform highly accurate calculations, but

These sequence data show that no specific viral and corresponding to cellular flanking sequences at the integra- cellular sequences were found at the junctions in these tion site,

were treated with LV-IFN for 24 h prior to their addition to infected macrophages, and again the cultured cells were observed for fusion. fibroblasts and

The major herpes simplex virus DNA-binding protein, designated ICP8, binds tightly to single-stranded DNA and is required for replication of viral DNA.. The sensitivity of

[35S]methionine-labeled virions of reovirus T3 (Dearing) purified from infected mouse L cells were added to Laemmli sample buffer and boiled for 2 min before electrophoresis in a 5

The PDF reflects the full information of the stochastic signal in terms of its randomness. To control the shape of the PDF, the relationship between the PDF and the control signal