JOURNAL OF VIROLOGY, Oct. 1991, p. 5597-5604

Vol. 65, No. 10

0022-538X/91/105597-08$02.00/0 Copyright C 1991, American Society for Microbiology

Identification of the Human Herpesvirus 6 Glycoprotein H and Putative Large Tegument Protein Genes STEVEN F. JOSEPHS,'t DHARAM V. ABLASHI,2 S. ZAKI SALAHUDDIN,lt LINDA L. JAGODZINSKI,3 FLOSSIE WONG-STAAL,'§ AND ROBERT C. GALLO'* Laboratory of Tumor Cell Biology,' and Laboratory of Molecular and Cellular Biology,2 Division of Cancer Etiology, National Cancer Institute, Bethesda, Maryland 20892, and Cambridge Biotech, Inc., Rockville, Maryland 208503

Received 8 March 1991/Accepted 16 July 1991

Determination of the nucleotide sequences of two molecular clones of human herpesvirus 6 (HHV-6) (strain GS) and comparison with those of human cytomegalovirus (HCMV) has allowed the identification of the genes for the glycoprotein H (gH) and the putative large tegument protein of HHV-6. Two molecular clones of fragments of HHV-6, the BamHI-G fragment (7,981 bp) of the clone termed pZVB43 and a HindlIl fragment (8,717 bp) of the clone termed pZVH14, represent approximately 10% of the HHV-6 genome (16,689). An open reading frame within the BamHI-G fragment was designated the gH gene of HHV-6 because of the extensive sequence similarity of its predicted product (79,549 Da) to the HCMV gH gene product. The predicted product (239,589 Da) of an open reading frame within clone pZVH14 showed homology to the predicted product of the proposed gene of HCMV representing the large tegument protein. Computer analyses indicated a closer relationship of the predicted peptides of these HHV-6 genes to those of HCMV than to those of the other human herpesviruses Epstein-Barr virus, herpes simplex virus type 1, and varicella-zoster virus. The gH gene was more conserved among the human herpesvirus group, while significant sequence similarity of the tegument gene could be found only with that of HCMV. The data reported here with one conserved gene (gH) and a more divergent gene (tegument) support previous reports that HHV-6 and HCMV are more closely related to each other than to the other well-characterized human herpesviruses. Human herpesvirus 6 (HHV-6) (initially named human B-lymphotropic virus) (3) was originally isolated from a number of patients with AIDS and other lymphoproliferative disorders (43). It was distinguished from other human and

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reported worldwide (2, 24, 28, 42, 49, 50). HHV-6 infection commonly occurs early in life, and the virus is the etiological agent for roseola infantum (exanthem subitum) (48, 50). HHV-6 is also etiologically linked with approximately 12%

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animal herpesviruses by immunologic and nucleic acid homology criteria and by morphology (5, 19, 43). Since its isolation in 1986, numerous instances of virus isolation from patients with various diseases and normal donors have been

*

of heterophile-negative and Epstein-Barr virus (EBV)- and human cytomegalovirus (HCMV)-negative infectious mononucleosis (4, 37, 47). In a variety of fresh lymphoma and leukemia tissues tested, HHV-6 sequences could be detected in three B-cell lymphomas and in tissues obtained from a patient with T-cell leukemia (16, 18) but not in a large number of tissues from other similar cases. Since HHV-6 can infect CD4+ cells (31), which are also the target of human immunodeficiency virus (HIV) infection, it has been proposed that HHV-6 may play a cofactor role in the progression to AIDS (30). Recent studies have shown that HIV type 1 (HIV-1) expression is elevated in cells dually

Corresponding author.

t Present address: Applied Sciences, Baxter Healthcare Corp.,

Round Lake, IL 60073. t Present address: Department of Internal Medicine, University

of Southern California, Los Angeles, CA 90033. § Present address: fornia at San

Diego,

Department

of Medicine,

University of Cali-

La Jolla, CA 92093.

5597

5598

J. VIROL.

NOTES .

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8

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. TTAAAAT TCTGGGAGCACAGTTAT TGTACATAAAACTGACCGAAGAAT CCT TGCGGATGGCA-418 TGCC CAACACTAACATATACCGCAGATCCGCCATGCGTAGAGTTACTACCAT ACAAACGGTTGTGATACATGTATATGCGCT CTAGCGGCTACGCATCTCAATGATGGTAAAT T TTAAGACCCTCGTGTCAATAAACTGTAT TT TGACTGCCCT TCT TAGGAACGCCCTACCGT 42 F F N Y T S F V Y F L L Y N T T S C V P " S K F S M S N L T R S L T S F T S E ATGAGCAAAT T T TCCAACTCGAATCTGACACGGTCAT TGACT TCGTTCACGTCAGAAATCT TCT TTAAT TACACCACCT TTGTTTACTTCTTGCTCIATAACACAACAYCATGCGTCCCT ~4m0 T ACTCCGTTTAACCGT TGAGCCTTAGCTGCTGCCCAGT AACTCA AGCAACGTGCCAGT CT TT AGAAGAAAT TAATGCTGCCTCCAAACAAATGCAAGAACGAGAT ATTGCTGCTTGTAGTACGCAGGCA I

3378

GGAT CCGCCACAATAAAAAAAGACT T TCAAT TGCAACCAAGCCAGACCGTAAATAAT GTTGCAT CGTGTATGCAAGAT TGGAACAT TAT T TCCCAAT TT TGGCCCTCAT TAGC TGATAT GTCC_ 12Z0 CCTACGCGGT GT TATT T TTT TCT GAAAGT TAACGTTGCCT TCGGTCT GGCAT TTATTACA,ACTAGCACATACT TC TAACCT TG TAATAAAGCCTTAAAACCGGAGTAAT CGACTATACAGG R W L L F L S E A V L G S R L Y H 0 D H F P V N N G L K P R N L Q Y T W

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AATACACCAAAACATCAT TCT TAATGGCCCCCGTGTAAACGAGCT TCAACATCAAAATT TTGCACTAATT *TTCTCACCGGCAATT TT T TGCAT TACCCCT*C TACCTAACAACAT CT TTATAA_ 240 T TAT GTGGT TT T GTAGTAAGAAT TACCCGGGGCACA TT TGCTCGAAGT TGTAGTTT TAAAACTGAT TAAAAGAGTGGCCCCTTAAAAAACGTAATGGGGAGATGCGATTCGTTCGTAGAAATAT T Y V L V D N K K E G P A G H L R A E V D F N Q S K 0 M V G R G L L N K L GCACATGATTCCCGAAT GT GTT TT C TGCATAAT GCT TGCCYTTTTC TCAT GGGAACAAAAAAT T TCATACAAGAAGGATCAT TAAA TCCAAAA TAGGT GCCAGAAGGAGAAACAT TT T TCA 3.60 CGT GTACTAAGGGCTTACACAAAAGACGTAT TACGAACGGAAAAGAGTACCCTTGT TTT*T*TAAAGTAT GTT*CT TCCTAGTA,AT TTAGGY TT*TATCCACGGT C TTCCTCTT* GTAAAAAGT

2298

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A TGCT GTAAT T TCACT*GCCGAGTAA TCT*AAATATCACAGGCAAGAGGGT TAT AACAT CTAC TAG.GT T GTACGT TCGTCGATCTGCTGAAT TCTCATMAG TGGAATAAAAAGCTG*CCAGGA_ 480 TACGACATYTAAAGTGACGGCTCAT TAGAT T TATAGTGCT CCGT TCT CCCAATATTGCT AGATGAT CCCAACATGCCAAGCAGCTAGACAC *TTAAGAGT ATT*CACCT*TAT TT TT*CGACGGT CCT 2258 A T E S G L L R F V P L L T V D V L T T R E D 0 S N E Y T s Y F A A L

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ACAACAAC CAACCAAAGCAT CAAAAAALAT AGTTAAACGT TCAGA TT T TCCT CGACCCATGCTACACCCCAACCGACAT TCCAACTC T TACAA TCCCGT CTTAACGCCCACTAT TAAAACC_5 160 TGTTGCCCTTTGTTGCCCTTTCGTAGT TT T TTTATCAATT TGCAACTCYAAAACCACCTGGGTACATGTCGGTTGGCTGTAAUT TGAGAATGCT TAGGCAGATTGCGGTGATAATTTTGG N N

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R P K T F E S A T V T T N A T O K 202 E 0 N T E N P K S P P K P T N F E N T T CAAACCCAACAAAT CCACCAAAACCAACAAATT TTGCAAAAT ACCACOAAT CAGAAT TCCCAAAACCTTTGCACGACCCCTACACGT AACA ACAAACCCAACCCAAAAGAT C_5 280 CTTGTTTTGTGGCTTT TGGGTTTT TCAGGTGGT TTTGGTTGTTTAAAO,CTTT TATGGTGT TAGTCT TAAGGGT TTTCCAAACTCTCGCCCATGT CATTGTGTCTTGTCCCTTGGGTTTTCTAG

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GAAAGCACCACCT TCACAACAAT AGGAAT CAAGGAAA T TAACGGCAATACC TA T TCT TCAC CAAAAAACTCTAT TTATCT TA AGAGCAAATCACAGCAGAGTACAACAAAAT TCACCGAC_5400 CT TTCGTGGTGGAAGTGT TGTTAT CCT TAGT TCCT TTAAT TGCCCCTTATGGATA,AGCAGTGCCTT TT T TGAGATAAATAGAAT TCT CGTTTAGTGCTCGTCTCATGCT TGCTTTTAAGTGGCTGC F

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GCCGCAACACACCACTCCGAT T TTAAAGT TTACCACTTGCCCAAAACACGGCACGCACAT ACATGCAGTCACAACACAGCAGCTACAAAACATGACCGACAGAT TCCAGAGGACAACCTTGA,AA_5520 CGGCTTGCTGTGGTGAGGCT AAAAT TT CAAATGGCTGCAACCGTT T TGTGCCCCTGCCCTGTATG7ACT CAGTGCTTGCTGTCTTCATGCTTTTGTACT CCCTGT C TAAGGTCTCCTGCTTGGAACTT T T N M A L P S S N 32Z S S N E L P T O T L S V T P K 0 K L P S M V T A K T E V H T CCT CAAACGAGCTACCTACCA T TCAGACGT TGCTCTGCTCAC T CCAAAACAAAAACT ACCGT CGAATGCTAAC T CCCAAAACTGCAAGTACACATAACTAACAACGCT TTACCAT CTAGTAA T_940 AGGAGTTTGCTCGATGGATGGTAAGTCTGCAACAGACAGTGAGGT TT TGTTTT TGATGGCAGCTTACATTGACGGTTTTGACTTCATGTGTAT TGATTGT TGCGAAATCCTAGATCAT TA

840

N N T E A O T P L N A H 36Z T E V T K E V K H T R N S A S T H E E S S Y S TCATCAT ACTCAATCACTGCAAGT CACTAA,AGACCGTAAAGCATAC TAGAATGTCAGCGCTCCACTCACGAACAGATAAACCACACAGAAATAC.CACAAATAACACCAAT TCT TAACGCT CAC_S5760 AGTAGTATGAGTTAGTGACTTrCAGTCAT TTCTCCATTTCGTATGATCTTACAGTCGCAGGTGAGTGCT TCTCTAT TTCCTGTGTCTTTATCGTGT TTAT TGTGUTTAAGAATTGCCCAGTG

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K V T P N T P T P T P E P P P V T K N S T K L H T D T P S T N L T S P H 12Z CTCACATCACCACATATCCCTAGTACCAACATACCAACCCCAGAACCTCCCCCCCGTAACAAAAA.ACTCTACAAAACTGCATACAGACACCATAAAAGTTACACCGAACACACCCACr-ATA_5040 TTCAATGTGGCTTGTGTGGGTGGTAT GAGTGTAGTGCCTGTATAGGGATCATGGTTGTATGGTTGGGGTCTTGGAGGGGGCAT TGCTTTTTTCAGATGTTT TGACGTATGTCTGTGGTATT G G G T G L L N G V G F F V F C V S V N F T V G F V G V N V D G

CGACGT TTACCTCCCCATCATTCGTAAACAGCAAAGCATCCTCTATCGCT TTCAAATGAGAAT TCAAAACGTCACT GTCTTT TTGCAT CTT T TTTTGCAGTGCGTCTAAT TCGGTTTT TA- 960 GCT*GCAAATGGAGGGGTAGT AAGCAT TT GTCGT T TCGTAGGAGATAGCGAAAGT TTAC TC TTAkAGT TT*TGCAGT GACAGAAAAACGT AGAAAAAAACG TCAC GCAGATT*AAGCCAAAAAT 252 98

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T ACACAAGGCTGC T TCCAGGT CTGACAT TTCACT TCCGAATATAGGATACACCGT CGCTTGCC TCCAACGCATTCACGGTAACT GC TAGT AAC TCTT TT CAAATAGT CAGAGAAT CCACAT- 600 ATGCTGCT TCCGACGAAGGT CCAGACT GTAAAGTGAAGGCT TATATCC TAT GT GGCAGCGAACGAGGT TGCGTAAC TGCCAT TGACGA TCAT TGAGAAAACT TT*ATCAGT CTC TTAGGTGTA C L A A E L D S M E S G F P Y V T A Q E L A N V T V A L L E K S T L S D V N

*TTTCTCCTCTAAAATCGTCTGTAAAT CT TATAAAGGGGTCGAGTGGGGT TATAGCGACCTGAT TGAATT *TTTCCAGCTT*CT CAGGAGATT*TCGGTATCAGT TGCGCATCAGCT *TCGT TT AAAGAGGAGAT T TTAGCAGACAT TTAGAATAT TTCCCCAGC TCACCCCAA TATCGCTGGAC TAACT TAAAAAGGT CGAAGAGT CC TCTAAAGCCATAGT*CA.ACGCG TAGT CGAAAGCAAA E G R F D D T F R A V 0 N F K E L K E P S K P F P D L P T L 0 A D A K T E

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T CAAATGCAT CAATYAT T TTAAACAGT CGCCAAAACC TATAA ATGT TACTACT TCCTT TGGACGA,ACCATAGTAA ACTT TGATTCGATACTAAC TACGACACCATCATCGACGTCAGCGT CT .4920 AGTTTACTAGTTATAAAATTTG GCTGUCCTTT TGGATATTTACAATCATGAAGGAAACCTGCTTGGTATCAYT TGAAACTAAGCTATGAT TGATGCCTGTGGAGTAGCTGCAGTCGCAGA

103

T AGT AAGT TT TAAAGCCCT TCT TAAACCGGGCTCA T TGT AAAAAACCC TCTTGCAGCT TAT AGAACCAAGACCCCAACGTAAAATT*CCACAT CTGGGCAT TAT TGAAAAAT GTA T TGAT CA- 720 ATCAT TCAAAAT TTCGGGA,AGAAT TTGGCCCGAGTAACATT*TTT TGGGAGAACTCGA,ATATCT TGGT TCTGCCCT TGCAT TTTAAGGTGTAGACCCGTAATAACTTfTTTACATAACTAGT 178 T L K L A R R L G P E N Y F V R K L K Y F W S G F T F N W N 0 A N W F F T N M

138

A

L

GGCGCAT TCT TAGCAACTT*CTCGCGATGCCCCTTGATCTAT TTTCTCCCGAAGTCTTAGTGAGGACTGACT TTGGAGCTTTTCGACAT CT*T TTTCTGTACAAATCAGTAT GTI1 TCTGACAT 1080 CCGCGTAAGAAT*CGT*T*GAACAGCGCTACGGGAACTAGATAAAAGAGGGCTT*CAGAATCAC TCCT CACTGAAACC TCGAAAAGCT*GTAGAAAAAGACATG *TTTAGT CATACAAAGAC TGT A I L D T HK 0C 237 A N K A V 0R S A RS R E G S T K T L V SK P A K R C R K 58 R R L L K DR H G 0D IK E R L L S S 0S 0 L K E V D E TC L N RVH

L L S T T P K E P 102 L T W S S E K S T T P Q R S F T A E T F L T T S S K P A ACAT CGGAAAAATCAACTACACCTCAACr.GTCCT TCACCGCTGAAACGT TCT TAACGACATCT TCAAAGCCCCCCCATCCTAACCTGGTCAAAT TTAT TAT CAACAACGCCCTAAGGAACCA_5880 TGTAGCCT T TTTAGTTGCATGTGGAGTTGCCAGGAAGTGCGCACTT TGCAAGAATTGCTGTAGAAGT TTCGGGCGGTAGCATTGGACCAGTT TAAATAATAGTTGTTGCGGATTCCTtTGG

GCCCT T CTAAAAT TGG TGTAAT TGGG TAT TGATAAAAGAGA T TGCACT*GCGCCAT CCTGCAGACC TGGCCCAACAAATCACG TATAAACGAAAATGCACAACCGC T TGGT CT TCCCCGAAAAC_1200 CGGGAAGATT T TAACCACAT TAACCCAT ............. TT G AACTAT TT T CTCTAACGTGACGCGGTAGGACT CT*GGACCGCTT *GT T TAG *GCATAT T TGCT T TTACT GT TGGCGAACCAGAAGGGGCTT --------------------------------------197 A I I I 11 I I . I -- ---------------It A I I . It I I I I . 11 A IIISIRICIKG.l

T T0 Y 442 L T T S M R T Q S A K L T K A N S S Q T T 8 T S L R W T D 5 L 8 T T TAACGAAT ACAAG TCTAACCGTGGACAGAT CA TA TCACAACACAC.CTAACGACTAGCAATAGAAC TCAATCAGCCCAAACTAACAAAAGCCTAACA TCT CGTCACAAACGACT AACAT CT AC_00 T TGCTGATTGTAGATG AAT TGCT TATGTTCAGAT TCCACCTGTCTAGTATAGTGT TGTGT CCATTGCTGATCGT TATCTTGAGTTACT CGGTT TGAT

P

TGCTTYTTCGATTGTAGAGCAGTGT

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CCCCAAACAATCACGGGACGATCTACAGAGGT TTAAAAAAAAAGT TCCACAAAAAGTCGGAGGCTAACGCAACTCTACCTGGAAACAACGAT TACCGCATCACCGATAAAAACTCACAC_612Z0 GGGGT T TGTTAGTGCCCTGCCTAGATGTCTCCAAAT TT TTT TTTCAAGGTGT TTTT CAGCCCTCCGATTGCCCTTGAGATUGACCT TGTTTGCTAATGCGCTAGTGGCTAT TTTTGAGTGTG

TTTTTAAAAAAATCTAACTTGAGTCCCCACTGCCTGAAAAAACAGATTACGGGAACAACGTYCCACGAAACCTCGTCTGCGATTAAATATTGATCCAAGAACGAAATAGACTGTTGCAAC-1320

AAAAATTTTTTTAGATTGAACTCAGGGGTGACGGACTTTTTTGTCTAATGCCCTTGTTGCAAGGTGCTTTGGAGCAGACGCTAATTTATAACTAGGTTCTTGCTTTATCTGACAACGTTG 157K K F F 0 L K L G W 0 R F F C V P V V N W S V E D A L Y 0 D L S S 0 0 L

TGTCTCAAACCTTGCAAATATTGCGCCTGGTGAATGCAGTTGTGATGCACAGACTGCATCGTCGACTTCCCCAAAGTCGTCTTAAACTCGAAAAAATAACAATGAAAAGCAAACTCATCT_1440

ACAGAGTTTGGAACGTTTATAACGCGGACCACTTACGTCAACACTACGTGTCTGACGTAGCAGCTGAAGGGGTTTCAGCAGAATTTGAGCTTTTTTA7TGTTACTTTTCGTTTGAGTAGA 1170 R L G 0 L Y 0 A Q N C N N N V S 0 M T S K G L T T K F E F F Y C H f A F E D

TTGACATCTACACTCTTTAGCAAGACGATACAATCCGGAATGCGCGGCCCTAGACGCACCTCGAAAAACACCGAGACAGATGCCGATCGCAAATTAGCCGGAAACACGTCTCCTAAAAAA 1560 AACTGTAGATGTGAGAAATCGTTCTGCTATGTTAGGCCTTACr.CGCCGGGATCTGCGTGGAGCTTTTTGTGGCTCTGTCYACGGCTAGCGTTTAATCGGCCTTTGTGCAGAGGATTTTTT 77K V D V S K L L V C D P R P G L R V E F F V S V S A S R L N A P F V D G L F CAAT GSAACT T TAT CAAAAGCCAGATCGAAGAAAACGAAT T CAAGACGATCTCATAGGTATGCGAATGGCAtCTCTGAGAA TAAAAGTCCAC T T CCGTAGCGGCCCCCAAAAACAAG_6600 GT TAT T T T T TTCTAAATAGT T T TCGGT CTAGCT T CTT TY G CTTAAGT TCTGCTAGAGTAT CCATACGCT TACCGTGAGACCTCT TAT TTTCAGGTGAAGGCATCGCCGCGCCT T T T TGT T C

TAATTTAGTCGCGCAAAATTTCCTTGAAAAGATAAGATCTGTTTATATATCCTCAAGTGGTGAAACTGTCCAATTTTTTTCCGGATATCAGGCAAACCTGTCAGAGACATGTTATTTCAC-1680

ATTAAATCAGCGCGTTTTAAAGGAACTTTTCTATTCYAGACAAATATATAGGAGYTCACCACTTTGACAGGTTAAAAAAAGGCCTATAGTCCGTTTGGACAGICTCTGTACAATAAAG7G 37Y N L R A F N G 0 F S L 0 K Y R L N N F 0 G K K R D P L G T L S N

AATGGAAAAGAGCCAGAGACTGACATATAACGCT TGCCT TGCCCCT T T TCTTACAACTCATCTTAAGCGT TAT T TTCAACTAGT CT TCGCGTCGACGTGATTMACACAGACAGCTCTCTG_6720 TTACC T T T TCTCGGTCT CTGACTGTATAT TCGCAACGGAACGCGAAAAGAATGTTGAGTAGAATT CGCAATAAAAGTTGATCAGAAGCGCAGCTCCACTAAT TGTCGTC TGTCGAGAGAC

kAC0TTCAAAGAATA6CA6AACCG 1800

GGGGAASAAAAACAC GGAGAA T TT GAA CCCCTTTTTTTCTGTGCCTCTTAATCTT

TTGAAAGTTTCTTATCGTCTTGCC

N

N F E N P V R P G F T F N F Y T K N D T R Y 0 V P K C L L G S D T Y H L 71 AAACTTTGAAAATCCAATCGT TCGCCCCGGCT TTATAACAT TTAACT T TTATACAAAAAACGACACTCCCATATATCAMGTCCCTAAAT GCT TACTCGGC TCCGATATCACATACCACCT_2040 TT TGAAACT T TTAGGT TAGCAACMCCCCCCGCAAATAT TGTAAATTGAAAATATGTTT TT TGCTGTGAGCCTATAT AGTTCAGGGATTTACGAATGAGCCCCAGGCTATAGTGTATGGTGGA D

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GT TTGATGCCATCAACACGCAAGAT CGTTAACAAAT TATGAAAAACGCGT TACACGTT T TTATGAGCCGCCAATGAMCGATAT T TTAAGACTT TCT CCT GTACCT TCGGT CAAACAATT_2160 CAAACTACCCGTAGTTGCTGCCTGT CTTAGCCMTTGCTTTAATACT TT TTGCCCCAATGTGCAAAAATACT CGCGGCTTACT TGCC TATAAAA T TCTGCAAAGAGGACATGCCAAGCCACGT TTGTTAA N L D R S Q P a v v Y S L N N Y P S 0 G Y Y V R V V E V R Q N Q Y D N V S C 151 TA'ACT TAGATCGCCTCTATCCA,ACCGCAGGTTGTGTAT TCC TTAAACATGCTACCCT TCACAGGGAAT TTAT TACGTCAGGGTCCGTAGAUAGTTCUCACAGTGCCAATACGACAACGTT TCCTG_22W0 AT T GAATCTAGCGAGATAGGTTGGCGCTCCAACACAT AAGGAA T TTGTACATGCCCAAGTGT CCCT TAAATAATGCCAGTCCCAGCAT C TTCAAGCTGTCTACGTTATGCTGT TGCAAAGGAC T R Y V G E D F P V O V R C A K Y T H F F T P D F M L 191 S L K E L T AAGCT GCCTAATT C TCTCAAGGAACTAATAT TTCCAGT CCAAGTCAGATGCCCCTAAAAT TACGCCCCTATGTGCCCCUAACACAT CTATACCCAT T TCT TTAC TCCGCAC T TTATGATACT_2400 AT TCGACGGATTAAGAGAGT TCCTTGCAT TATAAAGCTCAGGT TCAGTCTACGCCCAT T TTAATGCGCGCATACACCCGCTT CTGTAGAT ATGCCCTAAAGAAATGCAGGCCTGCAAATACTATGA

L

K

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N F K A P Y K K S S F Y 0 k P A G D L T N N Y G N T T S F K 0 T L T D D L 231 GTACAT CCAGAATCCCGCCCCCAGAT CTGCACTATGCA TGTACGGAAACACCACCAGCAT AAACT TTAAAGCCCCT TATAAAAAGT T CAT TCATAT TCAAACAGACAT TGACAGACGATT T_2520 CAT GTAGGTCTTAGGGCGCCCCTCTAGACT CATACTACATGCCCTT TGTGGTGCCTCGTAT TTGCAAAT TT CGGGGAA T ATTC TT T TCAACGTAAGTA TAAGT T TGCTCTGCTAACTGCTC TGCCTAAA

L L K F N 271 0 AC TATTGCATAGT CGAAAAAGACGTAATACATGTACAATACCGT TTCATATCAGATGCCCACAT TCGTAGACGAAACGT TGAATGACGTAGATGCAAGTAGAAGCTCTAC TAC TCAAATT TAA_2640 TGATAACTATCAGCT TTTTCTGCATTATCTACATGT TATGGCAAAGTATAGTCTACGCTGTAAGCATCTGCTT TGCAACT TACTGCATCTACT TCATCTTCGAGATGCATGAGTTTAAAT T

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TAACCTAGGAAT CCAAACCCTAT TAAGAGGAGACTGTAAAAAACCCAACTATGCCGGCCATACCGCAGATrCATGT TT CT TTACGGTATCGTACAT TYTCT CATATAGCACAAAAAACACAGG_2760 ATTGGATCCT TACCT TTGGGATAAT TCTCCTCTGACATT T TTTGGGCTTGATACGGCCGCTATGCGCT CTACTACAA AGAAATGCCCATAGCATGTAAAGAGTATAT CGTGTT TT TTGCTGTCC P

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F 351

K E

ACCAATGCCCGCTGTTAAGAGTGCTTAAAGACACACGAA AATCT CCTGCTCCAT CGACTCAT TTGCTCAACCGATGCTGTGAACGT ATCGGCMCCTACGT TACAATACCCAAAAATGAAGCCAAT T_2880 TGGTTACGGGCACAAT TCTCACAAT T TCTGTGTGCT T TTACAGGACAGGT AGCTGAGTAAACAGTTGCCCTACACACT TGCATAGCCCT TCCATGCAA TGCT TATGCCCT T TTTAC TTCCT TAA L K Y E P S 0 Y s Y T K N K S S V S T L L T Y L A T A Y E S N V T S K Y K 391 T TT AAAATACGAGCCCCTCGGACTATAGCTACATAACCAAAAACAAAT CCAT TTCCGTAT CT ACGCTGCTCACGTACT TAGCCCACAGCGT ACGAATCCAATCGTAACGAT T TCCAAGTACAA_3000 AAAT T TTATGCCTCGGGAGCCTGATAT CGATGCTATTGGT TT TTGCT TTAGGT AAAGGCATAGATGCCCACGAGT GCATGCAAT CGCT G TCGCAT GC TTAGGT TACAT Tr.CTAAAGGT TCAT GT T

D

W T

N

A

T

L

N

a

E

Y

K

N

H

F

F

N

T

T

L

S D

F

E

R

T

N

F

L

E

A

L

N

A

P 431

GTGCACTGACATTGCCCAACACT CT ACAAAACAT C TATGAAAAACACATGTT T TTTACTAATCTGCACATT T TCCGATAGGCUAAC T CTAT TCAT GC TAGCCGAAAT AGCGAA TATCAT CCC_3120 CACCTGACTGTAACGCT TGTGAGAUTGT TTT GTAGATACTT TT TGTGTACAAAAAAT GAT TAGACTGTAAAAGGCTAT CCCT TTGAGATAAGTACGAT CGGCT TTATCCGC TTATAGTAGGG D

T

E

N

R

Q

H N

R

0

G N

L

L

L

N

C

P

E

V

S W A

V

N

R

T

L

A

R

D

A

N

P

471

N

E

L

T ACCGATGAACGCATGCCAGAGGCACATGCAAC TACTAAT TGGAAACCTGTGCTAACCCCGTAGAAATAGT T TCATGGGCCCCCCATGCT TACAGCTGCACAGGGCACCGAAT CTAGAAAATAT_3240 ATGCGCTACTTGCCGTACGTCTCCGTGTACGT TGATGAT TAACCTT TGGACACAT TGGGGCAT CT TTATCAAAGTACCCGGGCGTACGAA TGTCGAC TGTCCCGTGGCT Ir.AUTCT T TTATA Y

S

P

C

A

S

P

E

R

R

V

R

D

N

T

V

F

S

L

K

T

V

T

L

S

A

Y

L

D

R

Y

R

S

M

D

N

N

E

L

S

Y 511

V

T TAT TCGCCTTGCTGCCCTCCCCCCGTACGCAGACAT CTAACAAAT TCT T TTCTAAAAACAGT TCT CACGTACGCTYTCCCT TGACCGT TA TCGAT CAGACATGATGGUCATGCTATCCGTATA_3360 AATAAGCCCCAACACGGACGGGCCCATGCCCTCTC TACATTGCT TTAAGAAAAGAT TT T TGTCAAGAGTGCCATGCCCAAGGGAAC TGGCAA TAGCCTAGT CTGCTACTACCT CTACGATAGGCATAT R

P

N

P

N

V A A

0

C

S

L

P

E

S

P

A

S

A

L

N

P

L

T

T

V

F

S

V

S

P

Y

G 551

K

V

CAGACCGCCAAATATCUCAr.GATAGCGGCTAT TCAGT GTC TCT CCCCAAGTGAACCAGCAGCT TCT CTCACTC TGCCGAATGCTGCACATT TGCTAAT TT C TCCCT CTTACGTGATTAAAGG_3480 GTCTGGCGGCT TTATACCTCTCTCATCGCCCCATAAGT CACAGAGAGGGT TCACTTGGT CGTCGAAGAGACTGCAGACGCCT TACAC TGTAAACAT TAAAGAGGGAGUATGCAC TAA TTTCC V S L T T T T T A V A T S P L b S T C V S T N Y K Y A G 0 D L L V L 591 AGTGCAGCCTAACAATT GAACACAAT TGTGGCT ACGAGTATAATMAT CACAGCCAT ACC TC TCAAT TCCACCTGCCCT TTCAACCAACTATAAATATGCCAGGACAAGATC TGCT TGTGCT_3600 T CACTCGGAT TGCTTAATGCTTGCCTGCTTAACACCGATGCCT CATAT TAT TAGTGCTCGGTATGCCAGACGTTAAGGTGGACGCAAAGTTGGCT TGATAT TTAT ACCGTCCTGTTCTAGAUCGAACACGA

N

R

S

T

0

S

C

E

V V M

C 0 S

F

E

Y

0 G

D

D

P

L

0

Y

Y

K

D

H

E

L

T

K

5 E

S A

Y

S E

K

T

T

O R

L

O T

S S R Y

E

F

F

V

F

R N

V

N

D

E 36

TCACCTGGCAAA^AATGTAUCACGCAATTTCAGAAAAGATCTCCGCAAACGAT TACCCTACAGAGACAAACGACCTCGACATAXCAT TGAATTTTTCGTCT TTCGAAATGTGGACATCAATGAA_6540

M L L R L W V F V L L T P C Y G W R P L N S N S S H C R N CG31 T TTCACACT TCATCGATGAAGACAACTATGC TCCTCCGACTCTGGGTC T T TGTCCTGT TGACT CCCTGT TACGGT TGGAGACCGT TGAACAT AT CGAAC T CGAGCCAT TGTAGAAATGCC_ 1920 AAAGTGTGAAGTAGCTACTTCT TGTTTCATACUGCAGGAGCTGAGACCCAGAACAGCACAACTGAGGGACAATGCCAACCTCT GGCAACT T GTATAGCT TUAGCTCGGTAACAT CTTTACC

AGTGGACCTT TTTACATGCTCGCCCTTAA,AGTCTT TTCTAGAGGCTT TGCTAATGGGATGCTCTCTGTT TGCTCCAGCTCTATGTAACT TAAAAAGCAGAAAGCT T TACACCTGTAGTTACT T

P L 76 S E D K T H D V W P A V N K K 5 f K K f L E N E L T S Y O R P L W T T D CT T TGGAC TACAGACA TAT C TGAAGACAOAAACGCACGATGCTGCTGCCCCCGG CTG7AUTAAGAAAT CATTTAAAAATT T CTAGAAAACGAACTAACC TCT TA TCAACGCCCCAA TCCCGCTT _6960 TAGGGCGAA GAA,ACCTGATGTCTGTATAGACT T CT GT T T T GCGTGCTACACACCGCCGACCATT TATTCT T TAGT AAAT T T T T TAAAGATCT TT TGCTTGAT TGG^AGIATAGT T

GCCCCCT

P a N G T V S K T C K K E K 0 R E T D C V N Y E R K H G N P V T F Y P R H 116 L G C TAGGTAT T CCGCAAAATGGAACAGTGTCGAAGACCTGCAAAMACACAGAACAAAGAGAAACCGAT TGTGT TAACTATGAAAGGAAACACGGCAACCCAGT AACT T T CTACCCAAGGCAC GATCCATAAGCGCT TT TACCT TGTCACAGCTTCTGGACGT T TTTTCTCT TCGTTTCTCTT TGGCTAACACAATTGATACT TT CCT TTGTGCCGTTGGGTCAT

TGAAAGATGGGTTCCGTG

7D0M

T L V 156 S E E P S L V S H H R N S K N D V F M D T N K D T C TGGACGT T T T TAT GGACACCAACAAAATCACACT CGTA 7200 Cr.CGCAAAAAGAAATGCAAATACGG ACACT TGTAT T T CGGAAGAACCAT CTAT T T TAGTAAGCCAT T GCGCGT TT TTC T TTACGT TTATGCCCTGCTGAACA TAAAGCCT TCT TGCCTAGATAAAAT CAT TCGGTAGCTCTCCT TATCATT

R A

K R N A N

W

E

T

CACAGGAATAGT^AAAA

TTACCTGCAAAASATACCTGCTGGTTGT TTAGTGTGCAGCAT

R

W V P

L

H D 0 V R

V K

L

S L

D

P

Y

D G F F

G

T

V

G M

S N

D V F C

AATAGAGAACT CATATGGGTGCCCCCACGATCAAGT T CGA ATCGTAAAACTAGATAT TTCCCTGTACATCCCAGATGGTT T CT T CGGAGT TAT TAC TGGCCACAGCAACC.ACGTAT TCTGCC T TAT CT CT TGAGTAT ACCCACGCGCT CCTAGT TCAAGCT TAGCATT TTGCAT CTATAAAGGGACATGCTAGGGTCTACCAAAGAAGCCT CA ATAATGACCGGTGTCGT TGCTGCATAAGACG S V F L N N L S E N S L N GAATGCCCTCACTGAAATAATAACCGACGAAACCGCAUT T T CAGT T T T T CTAATGAACT TATCCGAGCACT CT C TTACGCAGTGCACT TTAT TATTGCCCTGCCTTTGCCCTGCTAAAGT CAAAAAGAT TACT TGAAT E C V T

T

E

C Y

P E

D A N

Y

D E

P W E N

T

D

N

L

L

L

P G

E

F

A

I

E

D V E

N F F S TCT CAAT CAACT

196

3Z0

L P 236

CTAATGCCT TCTCCCAGGAGATGTGCCAAT TTCTCCCT_7"0 AGGCTCGTGCAGAGATTACCAAGAGGGTCCT CTACACC TTAAGAGT TAGT TGAAAGAGGGA

S P P E

F A

F

H L K A

S R

K

P N

S Y

T

O Y

W N

K D

T

P S

316

N

R 356

F

276

TGCTACAT CCCGGAACATGGGCAGACATCAT ATCTGCTCTCCCCCCCGAAT TTGCCAATT T TCCAT CTAAAAGCATCUCAGAAAT T ATAGCTAAACCGAACAGCTATACGATACUATACTT C_7560 ACGATGCTACCCCCT TGGTACCCTCT7ACTAGT TAACAGCAGGGGGGCT TAAACGT TAAAACCGTAGATT T TCGTYAGGTCTCT TAAATAT CGATTTYGCCT TGTCGATAT CCTAT GT TATGkAG V C A D

E

L

K A

L N

A

P S K E

K

L

G L

L

F

T C G

T

Y

GACGCCUT GT ACGT T TGCGCCGAT GAACTGAAGGCT CTUTCATGAT CCCT CAAACAGAAT T GCCUAACTCGGCT TAT TGATCGAAACCTACATCTGGCCAAAGAACAUAT TCCCTCCUTA_7680 CTGCCGGTACATGCAAACGCGGC TACT TGACT TCCGAGAGTACTAAGGGAGT TT TCT TTAACGGTTTGAGCCGCAATAACTAGCCTTTGCCATGCTAGACCT TGCT TTCTGTGCTTAAGGGAGGTAT K

F

T

I

I

T

P

T G

C

A

R

H F

C L S L N P E R A

T T T TACT TGCGGCCAUT T T T TGCCT UCAT T TAATCCCTGAACCTGCUATAAACCCC 7S00 AAGATCT TUA~CTCCACACGCAAACTATCT ACAT CCCTACAGCAUTCT GCAT CGCCAGGATAATTAAAAAT G TACGGACT TGCACGTTAT GAACGCCTGTAAAAACGGAGT T TCTAGAAGT TGAGGTGTGCCCT T T TGAT AGAT T AGGCGATGTCCGTAGACGTAGCGGTCCTAT

CUAAT

TTGCCC

A 376 V L D A k S S F L F H Y A T L CTACAGGTACTAGACGCCAAT TCCTCT TT CT TGT T TCATTATGCCACCT CCAATAACGCT TAACTATACACT CT CACCT TCAGACTCAACGCGTAT CGATT TAACCT 7 T T CACTTGGAGC_7920 GATOG CCUT GAT CT GCGGT T AAGGAGAAAGAACAAAGT AA TACGG T GGAGGT TAT TGCGAAT TGATAT GTGAGAGT GGAAGT CT GAGT T GCGCATAGCT AAAY TGGAAAAGT GAACCT CG

a

S N

N

TTCCGTCACAACATTTGGCAGCGCTTGATATAATCCCGTCTCCTCATGGAGAATCGGATCC_7981

AAGGCAGTGTTGTAAACCGTCGCGAACTATATTAGGGCAGAGGAGTACCTCTTAGCCTAGG

FIG 2. N ucleotide sequence of the H HV-6 clone pZV B43 BtitmHI-G fragment and translation of all reading frames of greater than 300 bp or 100 amino acids with an initiator methionine residue. The HHV-6 putative gH peptide begins at nucleotide position 1829. The hydrophobic leader and transmembrane regions of this peptide are overlined, and the potential glycosylation sites are underlined.

T 631

L

ACGAAACAT CT CAT CT CAAACATGCGAGT TCTGCTCAGACCCTAGTCATGCCAATATGATGATAT CGACGGTCCC TTACAATACAT TTACAT AAAAAACAT AGACGAACT AAA,AACAT TGAC_3 720 TGCT TTGTAGAGTAGAGTTTGTACGCTCAAGACAGTCTCGCATCAGTACCT TATACTACTATAGCTGCCAGGGAATGTTATGTAAAT GTAT T TTT TGTATCTGCT TCATT T TTGTAACTG P N N N L L V P N T R T H Y L L L A K k G S V F E N S E V G D D Q V S I1671 AGAT CCCAACAACAAT TTAC TTGCT TCCCAACACCAGGACGCACTAT CT TT TGCTTAGCCCAAAAACCCCTCTCGT TT TT ACATGCTCTGCAAGTCGGAATCGA TATAGACCAAGTGCT CT A TCAT_384 0 TC TAGGGT TGCTTGCTTAAATGA,ACAAGGGT TGTGGTCCTGCCCTGATAGAAAACAATCGGTT TTTGCCCCAGACAAAAAC TCTACAGACT TCAGCCCT TAGC TATAT CTGGCT TCACAGATAGTA

D

L V Y L A A L F G L Y R L R L C 694 AT TGGTTATCAT TTATATTCTGATCGCAATAAT TGCT TTAT TCGGATTATATAGACTTATCAGATTGTGT TCAATAAACGT TT TAT TTATCT TAGGTAT TAGATGAAT TTGTCCGTATAC_3960 TAACCAATAGTAAATATAAGACTAGCGTTAT TAACGAAATAAGCCCT AATATAT CTGAATAGT C TAACACAAC TTAT TTGCAAAATAAATAGAA TCCAT AAT CTACT TAAACAGGCATATG

AGC TAAACGCCGACAACGTGCCTCGTATAAATAT CGCCAT TAAATCCGAT TATCCCCCCGGTAAAT TAC TAGT AA TAACAT TAAAATAGCCAGGGCAT TCGTAAT CGCGGT CCA ACACAGCA_4080 TCGATTTGCCCCCTGTTGCACGAGCATATT TATAGCGGTAATT TAGGCTAATAGCGGGCCAT TTAATGATCATTAT TGTAAT T TTATCGGTCCCGTAAGCAT TAGCGCCAGGT TGTGTCGT M H L V V 0 S Y K K S K Y Y F S H T F Y L Y K F V V N S P D N L H S 0 L 40 TGCCAT TTGGAGGTCATCGTCCAAT CCTATAAAAAAAGCAAATACTAT T TTAGTCACACAT TCTACT TGTACAAAT TCAT TGTGGTCAACTCACCCGACATGCT TCACATCTCGCAACTCG_4200 ACGT AAACCTCCAG TAGCACGT TAGGUTAT T TTT T TCGT TTATGCAT AAAATCAGT GTGT AAGATGAACATGCT TTAAGTAACACCAGT TGACGTGGGCTGTACGAAGTGCTAGACCCGTTGCAGCC

E

G

L

F

L

G

L

F

A

V

M

H

S

A

N

L

K

Y

T

S

D

P

L

E

A

F

K

I

V

N

R

H

N

W

S

D

E Q

R

S0

GCCCT TTTT CTGCGGCCTTTTCGCCCAT AGTCATGCCAC TCCGCCTAATC TAATAAA,ATACACGT CTGCATCCCT TAGAAGCT T TCAAAACCCGTCAACCGCCACAATTGCCACCCACGAACAAAGAG_4320 CGGAAAAAUCCCGGAAAAGCCCCTATCAGTACGTGCAGCCCAT TAGA TTAT T TTATGTGCCAGAC TAGGGAATC TT CGAAAGTT TTGCCCAGT TGGCCCCCTGCTTAACC TCGC TGCC TTGCTTTCT C E

H

F

Y

D

L

R

N

L

K

S

A

V

N

L

S

P

L

L T N V F S W D R D S Q C 120 Y T S F C 0 T N L S L D C F T 0 AACATTTTTACGACCTCCGAAACT TGTATACAAGT TTT TGTCAGACAAATCTATCCCTCGACTGCTTCACTCAAAT T TTAACTAACGT TTTCTCT TGGGUCAT TCUAGAT TCACAATGCA_4440 T TGTAAAAATGCCTGGUGGC TT TGAACATATGCTTCAAAAACAGTC TGT TTAGATAGGGUGCTGCACGATGACGT TTAAAAT TGAT TGCAAAAGAGAACCCTGCTAAGCT CTAAGTGCTTACGT

0

N

L

P

R

T

E

T

K

V

L

S

S

T

A

A

N

K

S

A

S

S

F

S

L

F

Y

L

160

AGTC TGCCCCTTAACTTGCTCT CCAT TACAGAATT TACCGCCCCACAGAAACCAAAATAG7CTCTAT CGTCGACGGCT GCAAACAAAT C TATCATCGCTAGCAGTT T TTCT CTAT T TTATCT T C_456C TCAGACGCCAATTGUACACAGAGTAATGTCTTAAAT GGCGCCTGCTCT TTGGCT T TTAT CACGAT AGCAGCTGCCCCACGT TTGT TTAGAT AGTAGCCCATCGTCAAAAAGUCATAAAATAGAAG

infected with HIV-1 and HHV-6 in vitro (30) and that HHV-6 infection can upmodulate HIV-1 long terminal repeat-directed gene expression in vitro (12, 15), as has been reported for other human DNA viruses (EBV, HCMV, and herpes simplex virus [HSV]) and human T-cell lymphotropic virus type I (10, 13, 20, 35, 36, 39, 40, 45). Furthermore, HHV-6 infection was recently shown to upmodulate expression of CD4, a receptor for HIV-1, and thus could expand the range of cells susceptible to HIV-1 infection (29). However, other investigators reported that HHV-6 suppressed HIV-1 replication in vitro (6, 25, 38). A lack of correlation between HHV-6 infection and the course of HIV-1 infection in vivo,

NOTES

VOL. 65, 1991

5599

ID

CD

EBV

HCMV 650-

658-

I/

ID

t

I I

5.-

50

se

59

831

see

HSV- 1

VZV

FIG. 3. Homology of the HHV-6 putative gH to the gH peptides of other human herpesviruses. Shown are the LFASTA comparisons with HCMV, EBV, HSV-1, and VZV.

in which HHV-6 antibody titers were not different in patients who progressed to AIDS and those who had a delayed onset of AIDS, was also reported (46). It must be pointed out that both Carrigan et al. (6) and

4

SW

am

be

=

Lusso et al. (30), using the GS isolate of HHV-6, did observe enhanced cell lysis in HHV-6- and HIV-1-coinfected cells. HHV-6 contains a linear double-stranded DNA genome of approximately 170,000 bp, arranged as a long unique region

=

4

Sewm pcs ts

4W

4W

4W

4W

=

=

W

=

=

=

n

FIG. 4. Hydrophobicity profile of the HHV-6 putative gH peptide. The hydrophobic leader (L) region and the hydrophobic transmembrane domain (TM) are marked by thick lines.

J. VIROL.

NOTES

5600

MS ER Y L L L AlK N G|R KN G TV L E V T DIV H Y L3L IA L H Y L L L ITF N G T V L |N G T VII HLLAF L|I T K A|| |F_P|N G T Vl V T IL LSIG

IH

HHV-6 HOWV EBV HSV-1

L

vzv

FIG. 5. Alignment of the potential glycosylation site within the gH genes of the human herpesviruses. The regions conserved among more than one virus are boxed.

flanked by variable-length directly repeated sequences (17, 26, 32). To further characterize this genome, we have determined the nucleotide sequences of two molecular clones of HHV-6 (isolate GS). One of these clones (8 kb) contained an open reading frame which was assigned as the glycoprotein H (gH) gene because of the extensive similarity of its predicted peptide to the gH (UL75) gene product of HCMV. HHV-6 gH was more distantly related to the gH genes of the other human herpesviruses. Another open reading frame contained in the second clone (8.7 kb) was similar to the UL48 gene of HCMV and is most likely the large tegument protein gene. The findings presented here are consistent with those previously published that showed a closer genetic relationship of HHV-6 to HCMV than to the other human herpesviruses (11, 23). Virus was concentrated by continuous-flow centrifugation from supernatants of HHV-6 (isolate GS)-infected HSB2 cells (1). The viral DNA was isolated by cesium chloride gradient centrifugation as previously described (17). A BamHI library was constructed by using plasmid pIBI31 (International Biotechnologies, Inc.) as previously described (17). Clones were identified by their hybridization to HHV6-infected HSB2 cell DNA but not to uninfected HSB2 cell DNA. Clone pZVB43 contained two inserts, the BamHI-G and BamHI-L fragments. Clone pZVH14, which contains an 8.7-kb HindIll fragment of HHV-6, was previously described (19). DNA sequencing was performed by the Maxam-Gilbert method (33) or by using Sequenase protocols (U.S. Biochemicals) on M13 single-stranded DNA (44). Both strands of all subclones were sequenced. Open reading frame, translation, and hydrophobicity analyses were performed with the SAP sequence analysis package developed by Universal Biotechnology, Inc. DNA and peptide similarity searches were performed with the PFASTA and LFASTA programs included in the Intelligenetics program package (version 6.0; Intelligenetics, University of Wisconsin Biotechnology Center) by utilizing the GenBank and PIR data bases (14, 27). A BamHI plasmid library was constructed from purified

OFI

I

3

l

|

l-[-

F

_

RF2

|| |

11111 10Liiilglll5

i 1lilNlilI 1ll 1zl . ..

I

.

..

.I .

E

l

E

411 |||||||| |F| | |||||| ||||| |||| |||LI| ~~~~~~11 ~1~IIII3 111 1111111

I l~ I.

1l 1lRF5

R.F3

I I I . I r I I T Hill A I E 1 I I 110 l11 E lI1111 11ll lll i iiillit Il Il || 11 |||"|ll

|

2

HHV-6 DNA. One plasmid clone contained two fragments which hybridized specifically to HHV-6-infected cell DNA. These were the BamHI-G and BamHI-L fragments, as determined by their correspondence in size when run in parallel with BamHI-restricted HHV-6 DNA (17). The BamHI-G fragment (clone pZVB43) was 7,981 bp in length and had a G+C content of 40.3%. A termination codon scan of the sequences showed eight possible open reading frame regions of 300 bp or more, as depicted in Fig. 1. Five of these, RF1 to RF5, were found in the right reading frame, and three, LF1 to LF3, were found in the opposite strand, or the left reading frame. All of these regions potentially encoded peptides of 100 or more amino acids in length, with all except RF1 having an initial methionine residue, as shown in the translated sequence (Fig. 2). A search of the GenBank data base with the DNA sequences of this fragment was performed but yielded no statistically significant matches. In contrast, a search of the PIR data base with the predicted translation product from one open reading frame region, RF2 (bp 1817 to 3911) (Fig. 2), showed significant sequence identity to the HCMV gH (UL75) gene product (7). This HHV-6 peptide was 694 residues long and had a calculated molecular mass of 79,549 Da and an estimated pl of 6.528. An identity of 23.9% was found in a 685-residue overlap of HHV-6 and HCMV gH peptides with an optimized score of 860. As shown in Fig. 3, LFASTA analysis demonstrated that sequence similarity with HCMV gH extended throughout the entire region. For EBV gH (BXLF2), the 24.2% identity in a 314-amino-acid overlap (optimized score, 267) was lower than that for the HCMV gH and the HHV-6 gH peptides. The identity was lowest with gH peptides of varicella-zoster virus (VZV) and HSV type 1 (HSV-1) at 21.8% for a 124-amino-acid overlap (optimized score, 64) and 17.4% for a 310-amino-acid overlap (optimized score, 69), respectively. The carboxyl-terminal portions of the human herpesvirus gH peptides were more conserved, as found in previous analyses (9, 34). This is shown by the diagonal toward the right for HSV and VZV compared with HHV-6 in Fig. 3. The hydrophilicity profile of the HHV-6 peptide was similar to those of the peptides of the other four human herpesviruses (HCMV, HSV-1, EBV, and VZV) (34) in that a hydrophobic leader sequence was found from residues 1 to 23 and a predicted hydrophobic transmembrane region extended from residues 668 to 694 (Fig. 4). Fourteen potential N-glycosylation sites of the consensus sequence N-X-S/T were noted, as underlined in Fig. 2. This is in contrast to 6, 6, 4, and 10 potential N-glycosylation sites found in the gH proteins of HCMV, HSV-1, EBV, and VZV, respectively. One of the potential N-glycosylation sites of HHV-6 gH

..

1000

.

I

.-

.

..

I

. . .

2 000

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3000

I -18lIIlll0ls .

I

.

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'468

.. .

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FIG. 6. Termination codon scan of the DNA sequence of the HHV-6 clone pZVH14 HindllI

...

1 1l& .I

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.

Iii\5 11iiflll]l I.

. ..

I

..

.

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-1800 00ee fragment. (See legend to Fig. 1.)

5

VOL. 65, 1991

NOTES

5601

AAATGGGATAGATAGTCTTTTTACAAGCATCTAAAGATATTGTCAGTCTTTGGGAGCCTGTCTCGAAGGCTTTAGGATCGGCGTTAGCTTAGGTAGGATCGCCTAGGGTCGTTTTTGTTC T V V N C F

8

TY

TTMMTGDATYC

888NL

YP S

0 L

FE

KT1LD 8Q

TF

TN

FD TS

LD KF

3D

GTTAATTTCCTCTCAATGCACTAT08CGTCGAAGC 1TG 1CACAACTTAG 0CCCGTAA 1TTTAACTC_86

AACTTAC1GAGAAAATGATATGGAGAGTTAGGGAGTTACTG 8A1GTTTCGCACCGTAGTTGA 0TG 801AGTATT 8A1AC 118AG G810P18

A LYI

FSSC1KIPFS1K58L11A0 DI191L8Y IY

T

18L 8EKT 1DL

1578

ACTTATAA11 AGTT9 GTTAAATATTTAAA1 ACTCCACCCGGAAAGGATAATACTTC1CT8TTAAACGCCTTC

4 198

L

102

88TTTT 8GGT

111GCCT ETCCAA 1TAAGTAGTGTGATTTGCTGAAGT

8

ILF 8 LIDFLGTE 8 GLIYNIL 1 01 0 6QKT 0 1 1 8 1E0 I1 P80 ERE8DLN

278

S 0 L E

K

E

G K N E

L

S T

E N K Q T A A

T

T

K 0

L

G D 0 E

755

T

P

D F

Y

L

N

L

N

S A Y

N

L P N

T

T

T

K N

V

L

L

N

F

N

T

795

E Q R P D

L

A N

TTAACCCCCATTTACGATTTCCTGCACCACCTCTCTGCTTATAACCTACCTAATACTACAACCGTAAAAAATTTACACCTCCATTYCATCTTAGAACAACGACCAGATATCGCTATGACT 48W AATTGGGGGTAA,ATGCTAAAGGACGTGGTGGAGAGACGAATATTGGATGGATTATGATGTTGGCATTTTTTAAATGTGGAGGTAAAGTAGAATCTTGTTGCTGGTCTATAGCGATACTGA R G L V V V T

L

F

F

C t W K N K S C R G S

K

A

V

L N D K 0 S L D Y V 0 D N L N D T V P E 0 T F S T V L F L V D L F P N S 835 CTACACGATAAAATACAATCCATACTTGACATATATGTAGACGACATGCTCAATGACATAACGGTCCCTGA,ACAGACGT T T TCAACCGT TCTAT TTCT TGTAGAT CTCT TCCCAAACAGC_.M0 GATGTGCTAT TT TAT GTTAGGTATGAACTGTATATACATCTGCTGTACGAGTTACYGYAT TGCCAGGGACT TGTCTGrJkAMGT T-OCUGA TAAAGAACATCTAGAGAAGGGT T TGTCG C D N S S N Y T S S N S L S N V T G S C V N E V T R N R T S R K G F L

76 R C S L

E K T A L

T

0818GCGGTC1GT111TAGTTTTCAATC 1ATTTATA

E N

ACAGTCGTAMTATAGMATGATCAAATCGCAACTTGAAGAACTGGGAAAAATGGAAATCTCCACCACCGAAAACAAACAGACAGCTGCAATCACAAAACAAATCCTCGGGGACCAAGAG 4w TGTCAGCATTTATATCTTTACTAGTTTAGCGTTGAACTTCTTGACCCTTTTTACCTTTAGAGGTGGTGGCTTTTGTTTGTCTGTCGACGTTAGTGTTTTGTTTAGGAGCCCCTGGTTCTC

E

F

S V

L

7

L

0 L A K K C A k L

R

K T L D E

F

D 0 L A 0

7

F

T

S 875

N

ACCGAAAAAACAGCACTGTTCGA,ATCCGTTCTCACGCTACGGCAATTAGCAAAGAAATGCGCTAACTTAAAAACTCYAGATGAATTCGACGATCTAGCCCAATTCATCACGACGAATAGC_5040

T GGCTT TT T TGTCGTGACAAGCTTAGGCM"GTGCGATGCCGT TAATCGTTTCT T TACGCGATTGAAT T T T TGOkGATCTACTTAAGCTGCTAGATCGGGT TAAGTAGTGCTGCTTATCG 36 V S F V A S N S 0 T R V S 0 C N A F F N A L K F V R S S N S S R A W N N

E

Q

L

Q

9 R Q N

N

N

F

G K K

P

T

L

G N

N

K

F

L

Y

K N A

L

S 0 K

T

0 915

E E K N W

T

GAACAACTTCAAAACATGATGAGAIA-CATTYTGGAAAAAAAATACCAACGCTGATGGGACATATCAAATTTCTCYACTCTCAAAAAATAATCACCACTGAAGAGAAAAACTGGATACAG_5160 CTTGTTGAAGTTTTGTACTA,CTCTGTCGTAAAACCTTTTTTTTATGGTTGCGACTACCCTGTATAGTTTAAAGAGATGAGAGTTTTTTATTAGTGGTGACTTCTCTTTTTGACCTATGTC R A K T V V

T

S P E

E

L

T

A

F

A

L

T A P

T

C K P D L D K A L a 4 N 955

T

0

GGTCGTCATCACATCTCCCGAAGAACTGACAGCCTTCTTAGCCACTGCACCCACTAAACACGCCTTACAAACTTGCAAACCTGACCTCGATAAAGCACTACAACGACAT_5280

AGA

TCTCGTTTTTGCCAGCAGTAGTGTAGAGGGCTTCTTGACTGTCGGAAGAATCGGTGACGTGGGTGATTTGTGCGGAATGTTTGAACGTTTGGACTGGAGCTATTTCGTGATGTTGCTGTA E

N

E 0 0 K Q T

E

A

N

0 K K

R

T

L

N

T

S

E

L

R

K

D

N

L

L

L

L

D G Q

K

S 995

F

ATGGAAGAGCAAATGAAACAA,ACTGCCGAGAACGACAAAAAACACATCCT CACAATCAGAAGCACCCTCGAAAAAAGACT GAACGACATCT TACT TAT TCTAAAGGATGGTCAAT T T T CG_5400

TACCTTCTCGTTTACTTTGTTTGACGGCTCTTGCTGTTTTTTGTGTAGGAGTGTTAGTCTTCGTGGGAGCTTTTTTCTGACTTGCTGTAGAATGAATAAGATTTCCTACCAGTTAAAAGC S

E

T

F L K Q L Q D N N V L L E N F T N A L L P V L K D E T T 1035 TCTCTGGAAACAATGCATTTGA,ATCTTT TAGAAACCT T TCTGAAACAACTGCAAGATAACAATGTCATCATTCACTTTACCCATGCACTACTGCCAGTGT TAAAAGACATAGAAACGACC_55ZO L

N

L

N

T

N

A"GACCTTTGTTACGTAAACTTAGAAAATCTTTGGAAAGACTTTGTTGACGTTCTATTGTTACAGTAGTAAGTGAAATGGGTACGTGATGACGGTCACAATTTTCTGTATCTTTGCTGG 808E 1 L10019115lC1A8TcmP1v8T 0 PE

yI

1290

S K

S D V

E

K

L

X

T

P

L

E 0 N S K E E 0 K Y T

P

N

ATCAGCAAGATCATCTCCGACGTTATTGAAA,AGATATTAATCAAAACTCCTCTAAACCCCGRACAGATGTC

P

L

S

L

F

S K

L

iO75

F

ATACCCCTTTACTCAGTTTCTTATCCAAGTTC_5"0

TAGTCGTTCTAGTAGAGGCTGCAATAACTTTTCTATAATTAGTTTTGAGGAGATTTGGGGCTTGTCTACAGGTTCCTTCTTGTTTTTATATGGGGAAATGAGTCAAAGAATAGGTTCAAG

K K T T F C T E D V K T E D 0 N 0 K S T F L K X A T S A N K N T R L S N S M5 AAAAAAACAACAT T CTGCACAGAAGATGTAAAAACAGAAATCGACCAAAT GCAAAAGAGTATCACGT TCCTCAAGAAAAT 7 GCTACCTCCGCCAATA"CATACTAGACTCAGCCACTcc_5760

TTTTTTTGTTGTAAGACGTGTCTTCTACATTTTTGTCTTTAGCTGGTTTACGTTTTCTCATAGTGCAAGGAGTTC7TTTAACGATGGAGGCGGTTATTTGTATGATCTGAGTCGGTGAGG G Q E

Y N I D C L F N V

1L11 E SI

CACTC9CGTTCTCACGCTTCCTCG 8TCTT

G

51AAA STTCC8

1

8

8

0

YK

7 GN 11FE6

GGACGEAAG LTAAA SCA8TCSAACA

CCATT

01AATGGG3AC20

L8 ICK8EVo YI

SD 8

L

N

E

Y

E

R

E

T

R

L

K E

T

K N K

N

E

S K E

L

9

Y A L

E

A

K K

L

1155

L

S S Q 0 A K T 0 K Y L V L N T N T L K E K N T Q F I E T A F A K A L T V E v 1195 TCGTCACAAGAT GCAAAAACTGACAAAAT TYATCTAGT TCTA"TACGCACACGCTGAAAGAAATAAAAAACACTCAAT TCA"GAAACAGCAT TCGCTAAAGCT CTCACGGT GGOLAGTC_6000

AGCAGTGTTCTACGTTTTTGACTGTTTTAA,ATAGATCAAGATTTATGCGTGTGCGACTTYCITTATTTTTTGTGAGTTAAGTCTCTTTGTCGTAAGCGAYTTCGAGAGTGCCACCTTCAG

GTTGTAAAAGTCGGGGGCTGAAATTTAAGTTCTCTGTCATTTTAAAAGTTGAAGTTTCCAT STS 8 O N PI TTL DE oLR VDAFVA TYVNSTK PNI

L

ATCTACGGACAAGAATTAAACCTCTACGAAGAACGCATTACAGAGCTGAGAAAAGAGACG"CAAAATGAMGOkACAGCTCTCCA,AAGA,ATACGCATTAGCTGAAAAAAAAATTCTTCTC_5M TAGATGCCTGTTCTTAATTTGGAGATGCTTCTTGCGTAATGTCTCGACTCTTTTCTCTGCTTGTTTTACTTTCTTGTCGAGAGGTTTCTTATGCGTAATCGACTTTTTTTTTAAGU"G

13A631 GTTTTCCTCCTCCGACACCACATTCGAACTTAAACTGGTAGAACGAAAGCACGGGTCCGAGA10 P

N

N

E

K

0

N

L

Q

E

L

L

N

F

N

A

N

L

N

K A K N 0 0 N

7

N

K

S

L

D T K W T A F V i235

F

AACAATAAAGAAAATCAACTCCAGGAATTGTTAAACCACTTTAACGCACACCTAAAGGCGAAAATGGATCAGAOkCCATATAACMMCTTTCATTCGATACGAAATGGACAGCCTTCGTT_.6120 TTGTTATTTCTTTTAGTTGAGGTCCTTAACAATTTGGTGAAATTGCGTGTGGATTTCCGCTTTTACCTAGTCTTGGTATATTGTTTTGAAAGTAAGCTATGCTTTACCTGTCGGAAGCAA S D

S R

L

Y

D

P

F

N

K

0 D

L

S 0 P

F

K V

F

S

0

L

14

N

K

T

A

1275

E N P Y

N

TCGGATTCCAGGCTCTACATCCCAGACTTCATTAACATCAAACTGCAAGACTTTATTTCTGATCCCTTTAAAGTTATAAGTCAGCTAAT GAGATGCCTTACATT_0240 AGCCTAAGGTCCGAGATGTAGGGTCTGAAGTAATTGTAGTTTGACGTTCTGAAATAAAGACTAGGGAAATTTCAATATTCAGTCGATTACTTGTTTCGTTGGTTGCTCTACGGAATGTAA E

0 A

T

L

K W L

T

K

L

0

N

N K

F

C L S A

S E

G K E A

F

P

F

N

Y

A

A

1315

R

L

CAAGCAGAGATAACTCTCAAATGGTTAACAAAATTAATACACGACATCAACAAATTCTGCCTGTCAGCTATAAGCGAATTCGGTA,AAGAAGCAATCCCATTCAATTACGCAGCCCTACGA_6360 GTTCGTCTCTATTGAGAGTTTACCAATTGTTTTA,ATTATGTGCTGTAGTTGTTTAAGACGGACAGTCr.ATATTCGCTTAAGCCATTTCTTCGTTAGGGTAAGTTUTGCGTCGGGATGCT C

L L

1

0

ACKLLLP 8 LLLNY A 1. 1L F158 A PW08L 8Y

0

I80V

08

115S

1

V F A K1 18£

8

C

N

T

K

Y

V

E

E

N

K

C N E

V

T

E

V

T K

k

N

K

P

L

T

L K Q L v 1355

L

K

CACGAAAAACATTCCAAAACTGACTAAGCTCTTGAAGCAGTTAGTT_64M

CTAAACCTCATAGTCTAGTTGTGGTTTATGCATCTTTAACTTTTGTTTCAGTAAACGTTGCTTTGCCAGCTTTTGTGCTTTTTGTAAGGTTTTGACTGATTCGAGAACTTCGTCAATCAA P

S

0

E Y Q

L

GATTTGGAGTATCAGATCAACACCAAATACGTAGA"TTGAAAACAAAGTCATTTGCAACGAAACGGTC K

R

V

A

G G 0 E

Q

Y

Q

T

L

N

N

K

L

S

S

E

S N 0 0

T

T

Y

E

K

E Q

L

K

K

E

F

Y

1395

E

CCTAAACGCGTTGCCGGTGGACAGGAACAGTATCAAACTCTCATGAACAAGATTCTCAGTTCCGA"CTTCTATGCAACAGACCTACGAAAAAGAACAACTCAAAAAAGAAYACTTTGAG_6600 GGATTTGCGCAACGGCCACCTGTCCTTGTCATAGTTTGAGAGTACTTGTTCTAAGAGTCAAGGCTTTGAAGATACGTTGTCTGGATGCTTTTTCTTGTTGAGTTTTTTCTTATGAAACTC

V TNT YSP11186 6 8L8L1 VIIL

T195

A Y9

1

V N N V A S F K L A F N F P 0 0 L 0 N V E R L E K F K S L P K S 0 P F E K F 1435 ATCGTCAATAATGT CGCCAGCT TCAAGT TAGCATTCAACT T TCC T CAGCAACTACAAAATGT GGAACGGCTAATAGAGAAGT TCAAATCGCTGCCCAAMGCCAGCCATT TGAM"T T T_6720

Y L

K I8TADA IS8TSL

11F 0110

0

1T 0

AIDEI

A

23516

A9T

TAGCAGTTATTACAGCGGTCGAAGTTCAATCGTAAGTTGAAAGGAGTCGTTGATGTTTTACACCTTGCCGATTATCTCTTCAAGTTTAGCGACGGGTTTTCGGTCGGTAAACTTTTTAAA P

I 8 0 1 0 0 1 0 0 6 8 8 8

DLOADEIIDLE8APDOROO8S8SRLEG 11

N

L

275

E

0

0

0

0

tS 111181 09N01 F

A

19

10

8

9

8

K

8

91T 180Rit1

315

0

L

L

S D

S

L

N

E N Y

T

N

G

R A L

L

L

N

F

T

A

A

Q

N

Y

0 N

T

L

L K

1475

GGCGTCCTTTTACTAGATGAAAGACTGAGTGAGTTGTGTCTTTTA,ATATAATTGCCAGACGCGCGTGAGGAGTTGAAGTATTGCCGTCGCGTTTTGATGTAGGTCTTGTGTGAAGACTTT A

aW O 1 8 D 0 0

N

CCGCAGGAAAATGATCTACTTTCTGACTCACTCAACACAGAAAATTATATTAACGGTCTGCGCGCACTCCTCAACTTCATA,ACGGCAGCGCAAAACTACATCCAGRACACACTTCTGA"_6540 F

a

a Q

F

G T

N

F

V

A

N

V

K

S

P

0

L

Y

A

R

L

E

R

R

E

R

0

V

F

Y

1515

TTYCATCC."TTGDATTYACTSCAGTAGCGAATGTCAAACCTATTTCGGATTTATATGCCCGCCTCAGGATTGAACGGGAACGTCAAGTGTTCTAC_6960

CAGTGGGCGGTCTTT GT CGTTGTTTTAAAGTAGGGCTAACYAATGAGTCATCGCTTACkGTTTGGATAAAGCCTAAATATACGGGCGGAGTCCTAACTTGCCCTTGCAGTTCACAAGATG Q V N S V

N

L

V D

0

T

G V P

L 0

F

N

F

N

N

N A

V

K

F

F

1555

S L N Y K Q

CMGTAAACTCCGTCTTTGGAACTCACCTAATCGTTGACGATACAGGAGTTCCGCTGCAATTCCATAACATCTTTAACAACGCCATAGTAAAGTTTTTCTCCCTGAACTACAAGCAGATT_7m GTTCATTTGAGGCAGAAACCTTGAGTGGATTAGCAACTGCTATGTCCTCAAGGCGACGTTAAGGTATTGTAGAAATTGTTGCGGTATCATTTCAAAAAGAGGGACTTGATGTTCGTCTAA E

L

1

8

aT

C

a8

T

E

T 18811C011T118 11 8 MLR'

0

K1

11A

0350

N V P E D T P R CATGTAC GTACATGGTCTTCTGTGG

pY 819D80800810£9oSP AIY

F

L

R D

G 7

L

V

S S 0 Y

K

L

L

S V C K S

F

N

L

0 a

F

W E

T

N

L D L G

1595

TTGGTATCTAGCCAATATAAACTCCTGTCCGTATGTAAATCTTTCATAATGATACTACAGCAGTTCTG-GAGAATATAATCACCCTGGACCTAGGC_7200 TAGATCGGTTATATTTGAGGACAGGCATACATTTAGAAAGTATTACTATGATGTCGTCAAGACCCTCTTATATTAGTGGGACCTGGATCCG

0 N

F

K

R E

V N

P

L

L

K

L

F

Y

C

T

0

A

W

T

A

S E D S 7

V

5

1635

CCITACCTCAGAGACGGTACACAAAATTTTAAAAGAGAACTCATACCGATCGTGAACTTGAAATTATTTATATACTGCAYAACACAAGCCTGGACAGCCTCTGAAGACAGTACTGTTTCC_7320 GGAATGGAGTCTCTGCCATGTGTTTTAAA,ATTTTCTCTTGAGTATGGCTAGCACTTGAACTTTAATAAATATATGACGTATTGTGTTCGGACCTGTCGGAGACTTCTGTCATGACAAAGG

P0 L 8186EEElK08

1081907

T

A

F

E L P

K 0 F

T

L

L

L

C S N P E

Y

L

Y

G C L

S N S 7

D

L

V

N

S

L

A K S

1675

ACAGCGTTCGAATTACCCATCAAGCAATTCACACTTTTGATACTATGCTCACATCCTGAATACCTCTACGGCTGCCTCAGTCATTCCACAGATCTGGTTATAAATTCATTAGCCAAAAGC_7440 TGTCGCAAGCTTAATGGGTAGTTCGTTAAGTGTGAAAACTATGATACGAGTGTAGGACTTATGGAGATGCCGACGGOLGTCAGTAAGGTGTCTAGACCAATATTTAA,GTAATCGGTTTTCG

I0 K N S L Y N T F V V S N N P P E K P N N L N R N C 0 T Q L U a P A K L N 1715 ATA CTCTATAACACATTCGTCGTTAGCCACAACCCTCCCGAGAAGCCCATGCATTTAATGAGAAATATATGTATCGACACCCAGCTATGGCAACCCGCCAAACTTATG_nW TATCTGTTTTTG TATTGTGTAAGCAGCAATCGGTGTTGGGAGGGCTCTTCGGGTACGTAAATTACTCTTTATATACATAGCTGTGGGTCGATACCGTYGGGCGGTTTGIkATAC 0

A

V

1A V F KP

T

0

6 ES

P T AL

T

N 80898098 1T 0

36SS

T 1D

K D T F 0 Q T F F T Q L C P K N E K F F Y L T A F L L P Y K F N N Y W 0 1755 AAAUTACCTTCCAACAAACATTTTTTACACAACTGT TTTTTTATCTATC7CACTGCCTTTTTAATTCTACCCTACAAATTTATGAACTATATCTGGATTCAA_7680 TTTCTATGGAAGGTTGTTTGTAAAAA,ATGTGTTGACACGGGGTTTTTGCTTTTCAAAAAATAGATAGAGTGACGGAAAAATTAAGATGGGATGTTTAAATACTTGATATAGACCTAAGTT

Y

K P A

F

T

0

R S Y 0 N L

K 0

L

C S E Y V N 0 N K

T

T

S S V T

P

N

E P D T 1795

TACAAACCAGCCATTTTYACCCAAAGATCTTATCAAAACCTAATTAAGGACTYATGCTCCGAATACGTACACCAAAATAAGATCACTACGTCCTCGGTAACTCCCCACGAACCAGACACA_78W

ATGTTTGGTCGGTAAAAATGGGTTTCTAGAATAGTTTTGGATTAATTCCTGAATACGAGGCTTATGCATGTGGTTTTATTCTAGTGATGCAGGACCCATTGAGGGGTGCTTGGYCTGTGT

K S G E R 7 S K T V N K A 0 N T P T L T R L 0 A Q E Y V F D Y L Y S F L 1835 ATAAAATCCGGAGAAAGGATCACCTCAAAAATCACCGTACA CACACCGACACTCACTCGCCTGCAGGCTCAGGAATATGTCTTCGACTACATTCTTTACTCTTTCTTG_7920 TATTTTAGGCCTCTTTCCTAGTGGAGTTTTTAGTGGCATGTGTTTCGTGTTTTGTGTGGCTGTGAGTGAGCGGACGTCCGAGTCCTTATACAGAAGCTGATGTAAGAAATGAGAAAGAAC I I I S

T 808190

D01 K 0P R G15

G

A

C

S

A

0

L N

0

8

0

715

G Y

T

E N

T

F

A N Y

D

T

E

K

T

Y

L

L

C N R

N

L

E N

V

L

N

D

K D

F Q S V L R A

1875

ACAGGGTATGAAATGACATTCGCTATGTACATCGATACCATCGAAAAAACATACCTACTTTGCATGAGACACCTGGAAAACGTACTCCACGATAAAGACTTCCAATCTGTCCTCAGAGCA_8%0 TGTCCCATACTTTACTGTAAGCGATACATGTAGCTATGGTAGCTTTTTTGTATGGATGAAACGTACTCTGTGGACCTTTTGCATGAGGTGCTATTTCTGAAGGTTAGACAGGAGTCTCGT

R T F D V E N S L F S V 0 L D K V S Y L N N T N R A 1915 L K 0 S W T K N D y CGCACAT TC""TCGACTACATCCTGAAACAGTCATGGACTAAAAACATAGTCGAAUTTCACTCTTTTCAGTACAGCTCGATAAAATCGTCTCCTACCTCAACCACACAAACAGAGCG_8160

GCGTGTAAGCTGTAGCTGATGTAGGACTTTGTCAGTACCTGATTTTTGTATCAGCTTGTAAGTGAGAAAAGTCATGTCGAGCTATTTTAGCAGAGGATGGAGTTGGTGTGTTTGTCTCGC T

P

N

P

L

L

F k Y D N E V V N V Y L P P N S T N P K K V A F

Y

K

N

P

F

N

F

19SS

ACACCCAATATTCCTTTGATATTATTTAATTATGACAACGAAGTTGTCAACGTTTATCTACCGCCGATGTCCACCAATCCAAAAAAAGTAGCCTTTTACATCAAAAATCCTTTCCACTTT_BM TGTGGGTTATAAGGAAACTATAATAAATTAATACTGTTGCTTCAACAGTTGCAAATAGATGGCGGCTACAGGTGGTTAGGTTTTTTTCATC P

V

0 E

Y

E

A T

N

L

S

F

N

L

Y

P K T

T

D

L

N

0

L

P

P

N

N

TGTAGTTTTTAGGAAAGGTGAAA

T

E

S

T

R

P

G K 0 T

1995

CCCGTGCAAGAATACGAAGCGACAAATTTAATTTCCTTCCATCTCTACCCAAAAACAACAGATATTTTAAACCAACTTCCGCCCAACAATACTGAGTCTACTCGCCCGGGTAAACAAACC_4400 GGGCACGTTCTTATGCTTCGCTGTTTAAATTAAAGGOtAGGTAGAGATGGGTTTTTGTTGTCTATAAAATTTGGTTGAAGGCGGGTTGTTATGACTCAGAYGAGCGGGCCCATTTGTTTGG S E T L T T K N L S E P K F K K P A V T G L N P K S Q S L S T 0 T N V P E T 2035 TCTGAAACTTT CACCAAAAACCTCTCCGAACCAAAGTTTAAAAAGCCTGCCGTTACTGGACYTATGCCCAAGAGCCAATCGATCATCCTATCCACTGATACCAACGTACCGGAAACC_8520 AGACTTTGAAATTGGTGGTTTTTGGAGAGGCTTGGTTTCAAATTTTTCGGACGGCAATGACCTGAATACGGGTTCTCGGTTAGCTAGTAGGATAGGTGACTATGGTTGCATGGCCTTTGG N E F S E S N 7 T S K L K S 04 2075 S P D V K A N 7 A S A A K 0 V T L A R E K AGTCCAGACGTAAAAGCTAACACGGCCAGCGCAGCAATAA""TGTCACTCTAGCTCGAGAA""T CAACGAGTTCTCCGAATCGATAAATACAACTAT T TCAAAATTAAAATCTATG_8"0

TCAGGTCTGCATTTTCGATTGTGCCGGTCGCGTCGTTATTTTCTACAGTGAGATCGAGCTCTTTTTTAGTTGCTCAAGAGGCTTAGCTATTYATGTTGATAAAGTTTTAATTTTAGATAC

TY

L

2077

TATCTGTAACATTCAACACACAGTCATACTCCTTTTATAAATGTTTATTATCCAAACATACGAAATAAATGAAGCTT_8717

ATAGACATTGTAAGTTGTGTGTCAGTATGAGGAAUTATTTACA"TAATAGGTTTGTATGCTTTATTTACTTC"A

FIG. 7. Nucleotide sequence and translation of the HHV-6 clone

pZVH14 sequences showing open reading frames of greater than 100

amino acids with initiator methionine residue.

5602

. :. ~ .

J. VIROL.

NOTES

1380 1390 1350 1360 1370 HHV-6 NTKNIPKLTKLLKQLVPKRVAGGQEQYQTLMNKILSSETSMQQTYEKEQLKKEYFEIVNN : ....... ::::: .......... .: : .:::. .

HCMV-HFRFO RVDMVQAVEAVWQRLEPGRVAGGAARHQKVQELLQRLGQTLGDLELQETLATEYFALLHG 1010

1020

1030

1040

1050

1060

1450 1410 1420 1430 1440 1400 HHV-6 VASFKLAFNFPQQLQNVERLIEKFKSLPKSQPFEKFPQENDLLS-----DSLNTENYING

HCMV-HFRFO IQTFSYGLDFRSQLEKIRDLRTRFAELAKRRG-TRLSNEGVLPNPRKPQATTSLGAFTRG 1070

1080

1090

1100

1110

1120

1490 1500 1510 1470 1480 1460 HHV-6 LRALLNFITAAQNYIQNTLLKQWAVFQQQNFIPIDYSVANVKPISDLYARLR---IERER :........ ::: HCMV-HFRFO LNALERHVQLGHQYLLNKLNGSSLVYRLEDIPSVLPATHETDPALIMRDRLRRLCFARHH 1180 1160 1170 1130 1140 1150 1550 1540 1560 1520 1530 HHV-6 QVFYQVNSVFGTHLIVDDTGVPLQFHNIFNNAIVKFF ------- SLNYKQIHVPEDTPRL

HCMV-HFRFO DTFLEVVDVFGMRQIVTQAGEPIHLVTDYGNVAFKYLALRDDGRPLAWRRRCSGGGLKNV 1200

1190

1210

1220

1230

1240

1600 1620 1580 1590 1610 1570 HHV-6 VSSQYKLLSVCKSFIMILQQFWENIITLDLGPYL-RDGTQNFKRELIPIVNLKLFIYCIT

HCMV-HFRFO VTTRYKAITVAVAVCQTLRTFWPQISQYDLRPYLTQHQSHTHPAETHTLHNLKLFCYLVS 1300 1260 1270 1280 1290 1250 1630 HHV-6 QAWTASEDS

HCKV-HFRFO TAWHQRIDT 1310 1830

1840

1850

1860

1870

1880

HCMV-HFRFO LDYALGSWLFGIPVCLGVHVADLLKGQRILVARHLEYTSRDRDFLRIQRSRDLNLSQLLQ 1550

1560

1570

1580

1590

1600

1940 HHV-6 QSWTKNIVEHSLFSVQLDKIVSYLNHTNRATPNIPLILFNY-DNEVVNVYLPPMSTNPKK

1890

1900

1910

1930

1920

HCMV-HFRFO DTWTETPLEHCWIQAQIRRLRDYLRFPTRLE-FIPLVIYNAQDHTVVRVLRPPSTFEQDH 1610

1620

1630

1640

1650

1660

1950 HHV-6 VAFYIKNPFH-FPV

HCMV-HFRFO SRLVLDEAFPTFPL 1680 1670

FIG. 8. Alignment of the most-conserved regions of the HHV-6 pZVH14 RF5- and HCMV UL48 (HFRFO)-encoded putative large tegument peptides.

protein at position 652 was highly conserved among the other four human herpesviruses, although HHV-6 contained a serine residue at position 654 instead of the threonine found in HCMV, HSV-1, EBV, and VZV gH proteins (34) (Fig. 5). Like the gH proteins of the other four herpesviruses (EBV, HCMV, VZV, and HSV), the peptide sequence of HHV-6 gH has a short carboxyl-terminal cytoplasmic region and four cystine residues near the potential transmembrane region. Analysis of clone pZVH14, an 8.7-kb HindIII fragment clone of HHV-6 (19), showed that it contained 8,717 nucleotides with a G+C content of 39.9%. Seven possible reading frames were found, as depicted in the termination codon scan shown in Fig. 6. Five potential reading frames (RF1 to RF5) of 300 bp or more were located in the right-hand reading frame, and three (LF1 to LF3) were located in the opposite strand. Peptides of 100 amino acid residues or more containing initial methionine residues were found in all of the frames except LF1 and LF3 (Fig. 6). A search of the GenBank data base with the DNA sequence found no significant matches. By using the predicted translation products of these reading frames and the PIR data base, a significant score was found between the RF5 peptide (bp 2413 to 8647 [Fig. 6]) and the predicted peptide of the HCMV UL48 open reading frame (previously termed the HFRF0 open reading frame) (7, 22). The regions with the most significant scores are shown in Fig. 7. The HHV-6 peptide was 2,077 residues in length and had a calculated molecular mass of 235,000 Da and an estimated pl of 6.5. A computer comparison (Fig. 8) using the LFASTA program showed a homology score of 14.8% over the carboxyl-terminal regions of these peptides, with an optimized score of 620. The solid diagonal line in Fig. 9 suggests that the homology extends throughout the peptides. No significant homology was noted

2200

200

>HCMVUL48.pep FIG. 9. Homology between the putative large tegument proteins of HHV-6 (clone pZVH14, RF5) and HCMV (>HCMVUL48.pep) as revealed by LFASTA analysis.

with peptides of other human herpesviruses. Searches performed with the HCMV peptide also showed no significant homology scores with peptides of the other herpesviruses (22). The UL48 gene product is thought to be the HCMV large tegument protein, since its size is comparable to the large tegument protein encoded by the BOLF-1 gene of EBV (22). There are AT-rich regions upstream of the coding sequences, at positions 1747 to 1756 (Fig. 2) for the putative HHV-6 gH-coding region in pZVB43 and at positions 2331 to 2337 and 2388 to 2398 (Fig. 7) for the tegument-coding region in pZVH14 RF5, which may serve as promoters. Potential polyadenylation sites of the AATAAA consensus were found at position 3913 of gH and at position 8704 of pZVH14. A consensus TATAAA is located just upstream of this sequence at 8676. Analysis of the nucleotide sequences of two molecular clones of HHV-6 and of the predicted amino acid sequences of two large open reading frames in these clones suggests that they correspond to the putative gH and the large tegument protein genes of HHV-6, on the basis of their sequence similarities to the HCMV gH and large tegument gene products. Recent sequence determination of the major capsid antigen region of HHV-6 (isolate U110) (11, 23) has shown a close genetic relationship between HHV-6 and HCMV, both in the extent of their homology scores and in the overall shared orientation of specific open reading frames. The data presented here extend the homology to two additional genes of HHV-6. The homology of the HHV-6 gH is observed throughout the human herpesviruses. The finding of extensive homology between the HCMV tegument (UL48) peptide and the pZVH14 RF5 putative tegument peptide (Fig. 9) is consistent with previous studies which revealed a close genetic relationship of HHV-6 and HCMV, especially in that both the HCMV and HHV-6 gene products lack detectable homology to the respective gene products of other human herpesviruses (11, 23). Because HHV-6 is lymphotropic and mainly infects a

VOL. 65, 1991

subset of T and B cells in culture (1-3, 28, 31, 49), it has been

proposed (28) that HHV-6 be classified as a gammaherpesvirus (8) along with EBV, a B-cell-tropic virus, and herpesvirus saimiri, a T-cell-tropic virus (41). This is in contrast to HCMV, a member of the betaherpesvirus group (41), which productively infects host species-derived fibroblasts. Although the complete genetic structure of HHV-6 has not yet been determined, previous Southern blot hybridization studies (11) and DNA sequence analysis of the putative gH and tegument proteins reported here and the major capsid antigen region reported previously (23) indicate a closer phylogenetic relationship of HHV-6 to HCMV than to other human herpesviruses, except that a GGGTTA repeat sequence is shared by the DNA of HHV-6 and that of Marek's disease herpesvirus (21). These results support the suggestion that HHV-6 be tentatively classified as a betaherpesvirus rather than a gammaherpesvirus or perhaps that it not yet be subclassified. The role of the HHV-6 putative gH and tegument protein in virus architecture, viral infection, and host immune response remains to be ascertained. The gH gene product of HCMV is thought to function in fusion events early in the process of infection. Certain monoclonal antibodies against HCMV gH protein capable of neutralizing viral infection have been described elsewhere (9). It is reasonable to assume that the putative gH protein described in this report plays a similar role in HHV-6 infection. The detailed information presented here may facilitate the development of molecular and immunologic tools for delineating the events in virus infection and studying aspects of host immune response. We are very grateful to S. Arya of the Laboratory of Tumor Cell Biology, National Cancer Institute, Bethesda, Md., for his helpful comments and support in preparation of the manuscript. We also acknowledge helpful suggestions from N. Balachandran of the University of Kansas, Kansas City. REFERENCES 1. Ablashi, D. V., P. Lusso, C.-L. Hung, S. Z. Salahuddin, S. F. Josephs, T. Llana, B. Kramarsky, P. Biberfeld, P. D. Markham, and R. C. Gallo. 1988. Utilization of human hematopoietic cell lines for the propagation and characterization of HBLV (human herpesvirus 6). Int. J. Cancer 42:787-791. 2. Ablashi, D. V., S. Z. Salahuddin, S. F. Josephs, N. Balachandran, G. R. F. Krueger, and R. C. Gallo. 1991. Human herpesvirus-6. In Vivo 5:193-200. 3. Ablashi, D. V., S. Z. Salahuddin, S. F. Josephs, F. Imam, P. Lusso, R. C. Gallo, C. Hung, J. Lemp, and P. D. Markham. 1987. HBLV (or HHV-6) in human cell lines. Nature (London) 329:207. 4. Bertram, G., N. Dreiner, G. R. F. Krueger, D. V. Ablashi, and S. Z. Salahuddin. 1989. Serological correlation of HHV-6 and EBV infections in infectious mononucleosis, p. 361-364. In D. V. Ablashi, A. Faggioni, G. R. F. Krueger, J. S. Pagano, and G. R. Pearson (ed.), Epstein-Barr virus and human disease. The Humana Press Inc., Clifton, N.J. 5. Biberfeld, P., B. Kramarsky, S. Z. Salahuddin, and R. C. Gallo. 1987. Ultrastructural characterization of a new human B lymphotropic DNA virus (human herpesvirus 6) isolated from patients with lymphoproliferative disease. JNCI 79:933-941. 6. Carrigan, D. R., K. K. Knox, and M. Tapper. 1990. Suppression of human immunodeficiency virus type 1 replication by human herpesvirus-6. J. Infect. Dis. 162:844-851. 7. Chee, M. S., A. T. Bankier, S. Beck, R. Bohni, C. M. Brown, R. Cerney, T. Horsnell, C. A. Hutchison III, T. Kouzarides, J. A. Martignetty, and B. G. Barrell. 1990. Analysis of the protein coding content of the sequence of the human cytomegalovirus strain AD169. Curr. Top. Microbiol. Immunol. 154:127-169. 8. Chee, M. S., G. L. Lawrence, and B. G. Barrell. 1989. Alpha-, beta- and gamma-herpesviruses encode a putative phospho-

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