Nucleotide sequence of a region of the herpes simplex virus type 1 gB glycoprotein gene: Mutations affecting rate of virus entry and cell fusion

VIROLOGY 137, 185-190 (1934)

Nucleotide Sequence of a Region of the Herpes Simplex Virus Type 1 gB Glycoprotein Gene: Mutations Affecting Rate of Virus Entry and Cell Fusion DAVID J. BZIK, BARBARA A. Fox, NEAL A. DELUCA,~

AND STANLEY PERSONS

Molecular and CeU Biology Program, The Pennsylvania State University, University Park, Pennsylvania 16802

Received February 16, 1984 accepted May 4, 198.4 The tsB5 isolate of herpes simplex virus type I (HSV-1) enters host cells more rapidly than does KOS, an independent isolate of HSV-1, and this rate-of-entry determinant is located between prototypic map coordinates 0.350 and 0.360 (1). The nucleotide sequence of strain tsB5 has now been determined between prototypic map coordinates 0.347 and 0.360. Comparison of the tsB5 sequence to the homologous KOS sequence revealed that the rate-of-entry difference between these two HSV-1 strains may be due to the single amino acid difference observed within these sequences (0.350 to 0.360). A cell fusion determinant in tsB5 is located between coordinates 0.345 and 0.355 and to the left of the rate-of-entry determinant (1). Nucleotide sequence analysis revealed a second amino acid difference between tsB5 and KOS at coordinate 0.349. The cell fusion determinant was tentatively assigned to this location.

Herpes simplex virus type 1 (HSV-1) specifies and incorporates into virion envelopes and infected cell membranes four major glycoproteins designated gB, gC, gD, and gE (2). The nucleotide sequences specifying gB (3), gC (4), and gD (5) were recently determined. Only gB is known to be essential for viral growth. It probably promotes virus entry by fusion between the viral and plasma membrane envelopes. Temperature-sensitive mutants for gB produce noninfectious virions which lack gB when grown at the nonpermissive temperature (6, 7). The infectivity of these virions is enhanced if inoculated cultures are exposed to the chemical fusagen polyethylene glycol (7, 8). DeLuca et al. (1) demonstrated that HSV-1 strain tsB5 virions enter host cells more rapidly (fast-entry phenotype) than do HSV-1 strain KOS virions (slow-entry phenotype). Generation, and restriction endonuclease analysis, of tsB5 X KOS recombinants revealed that a 1Present address: Dana-Farber Cancer Institute, Harvard University, 44 Binney Street, Boston, Mass. 02115. r Author to whom requests for reprints should be addressed.

determinant located between prototypic map coordinates 0.350 and 0.360 affects the rate of virus entry into host cells (I). This entry domain lies entirely within coding sequences specifying glycoprotein gB (0.348 to 0.366). Early electron microscope studies have proposed endocytosis and fusion as the mechanism of HSV-1 entry (9-11). The fusion mechanism of entry is supported by the biochemical evidence that glycoprotein gE is transferred from the virion envelope to the plasma membrane during infection in the absence of protein synthesis (12). Consistent with fusion as the mechanism of entry is the observation that a measurable amount of virus-induced cell fusion characterizes HSV-1 infections (u), and that syncytial (SW) mutants that cause extensive cell fusion can be readily isolated (14-16).

The hypothesis that the HSV-1 glycoprotein gB promotes cell fusion is based on studies using tsB5, a mutant of strain HFEM that is temperature dependent for virus growth, production of gB, and fusion of Vero and HEL cells. The syn lesion and the ts lesion of tsB5 are separable by recombination (1,17). The syn defect in tsB5 185

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is located between 0.345 and 0.355 map units (I), and the ts defect in tsB5 is located between 0.360 and 0.368 map units (1, 18). The rate-of-entry determinant of tsB5 (map coordinates 0.350 and 0.360) is genetically separable from both the SW lesion and the ts lesion (1). Bond et al. (19) suggested that the syz and ts lesions of tsB5 are in the same gene because the sun phenotype of tsB5 is expressed in mixed infections with sun+ virus at 34” but not at 33”. In the present study we determined the nucleotide sequence of tsB5 between prototypic map coordinates 0.347 and 0.360. We compared this nucleotide sequence to the homologous sequence in KOS to determine the specific locations of the rateof-entry and svn determinants in tsB5. The plasmid pTEF contains the EcoRI fragment of tsB5 that is homologous to the 200

bp

0.3#45 Ba (a)

1

Ba

EcoRI F fragment of HSV-1 strain KOS (0.317 to 0.421 map units) inserted into the unique EcoRI site in pBR325. The unique 2.25-kb BamHI/SuZI fragment contained within pTEF, representing homologous DNA sequences between prototypic map coordinates 0.345 and 0.360 in KOS, was cloned into BumHI/SuZI-cleaved pBR322. After transformation, ampicillin-resistant and tetracycline-sensitive colonies were picked and tested for the presence of the recombinant plasmid, the relevant plasmid was designated pTBG-BSl. A restriction map for tsB5 between map coordinates 0.345 and 0.360 was constructed by single and pairwise double digests of pTBG-BSl (Fig. la). A restriction map for HSV-1 KOS DNA for the same enzymes is included for comparative purposes (Fig. la). For the enzymes tested, four restriction site differences were identified between map co-

0.350 Sm

Be

I Sm

BI sm

-

II

0.375 PI

Pv

Pv

I

I PV

I Pv

Pn

--

PvSm

I Sm

0.3fio Bs

PaSm

I

sm SI

I I

I I

PsRsm

Sm St

<

.

(b) FIG. 1. Restriction mapping and strategy used for tsB5 DNA sequencing between 0.360 and 0.347 map units. (a) A complete restriction map of tsB5 (below line) and KOS (above line) between 0.345 and 0.360 map units The enzymes used were BumHI (Ba), B&E11 (Bs), P&I (Ps), PvuII (Pv), Sal1 (Sl), and SmoI (Sm). Restriction endonucleases and T4 DNA ligase were obtained from Bethesda Research Laboratories or from New England Biolabs and were used as recommended by the manufacturer. DNA fragments were resolved in horizontal agarose gels for restriction site mapping. Gels were made and run as previously described @O). Electrophoresis buffer (TBE) contained 89 mM Tris (pH 8.3), 89 mM boric acid, and 2.5 mM EDTA. RR1 grown in Luria broth or on Luriabroth plates (.??I)was used for plasmid cloning experiments. To select for cells containing plasmids, ampicillin or tetracycline was used at 25 pg/ml. The procedure of Birnboim and Doly (.%) was used for the isolation of plasmid DNA and Ml3 double-stranded, replicative-form DNA. For large-scale preparations, the DNA was further purified by equilibrium banding in 50% (w/w) CsCl and 150 pg/ml ethidium bromide. Ethidium bromide was removed by n-butanol extraction and the DNA was dialyzed against cold TE (pH 8.0) (,?3). (b) MlI/I-ISV-1 clones constructed for DNA sequence determination of HSV-1 (tsB5). Endpoints of sequence analysis are indicated by solid dots (5’ ends) and arrowheads (3’ ends). The plasmid pTEF, described by DeLuca et al (I), served as the source of HSV-1 tsB5 DNA. Defined restriction fragments of HSV-1 tsB5 DNA were inserted into the cloning/sequencing vectors M13mp8 and M13mp9 (a, %). Following ligation and transformation @6) of JM103 cells, the progeny Ml3 phage were plated under selective conditions and putative recombinants (clear plaques on X-gal) were picked (27). JM103 was grown in V broth or on V plates (28). Recombinant phage were grown and the replicative-form DNA from each recombinant was analyzed for the presence of the correct size insert.

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ordinates 0.349 and 0.356 between the two strains. Restriction endonuclease-generated fragments derived from pTBG-BSl were cloned into the bacteriophage Ml3 (Fig. lb) for DNA sequence analysis. The extent of sequence information obtained from each clone is shown in Fig. lb. A total of 1857 nucleotides were sequenced, beginning at the Sal1 site at 0.360 and extending to 0.347 map units. The DNA sequence beginning at the Sal1 site at 0.360 and extending to the 3’ end of the coding sequences specifies the 583 Cterminal amino acids out of the total number of 903 for the gB of the KOS strain (3). Using the same reading frame, the nucleotide sequence and predicted amino acid changes are given for tsB5 (Fig. 2). Only two amino acid differences were predicted to result from a total of 12 base substitutions in the coding region. One of these is a GC 5 AT change at nucleotide 695 which gives rise to a val to ala amino acid change; the second is due to a transition at nucleotide 1610 which causes an arg to his change. The location of the predicted amino acid differences in strain tsB5 is shown relative to the structure of the strain KOS gB gene. The 10 remaining nucleotide changes were not predicted to result in any amino acid changes within gB. Relative to the 12-base substitutions within the 1749 base pairs of the gB coding region sequenced, five changes (three substitutions and a two-base addition/deletion) were found in the 108 3’ noncoding bases sequenced. It has been shown previously that the rate-of-entry difference between KOS and tsB5 maps within 0.350 and 0.360 map units, corresponding to sequences from the common Sal1 site (nucleotide 1) to the tsB5specific SmaI site (nucleotide 1566) in Fig. 2. There is a single amino acid difference predicted between the two strains within this region, due to the change at nucleotide 695. The base substitution predicts an amino acid of ala (GCC) for tsB5 and vu2 (GTC) for KOS at amino acid 552 of the gB peptide (Fig. 3). Both the amino acids are chemically similar, and the rate-of-entry difference between tsB5 and KOS virions is also very small at 34” and 37”. The

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difference in rate of entry was maximized by lowering the assay temperature to 30” (DeLuca, unpublished). We conclude that this amino acid difference accounts for the rate-of-entry difference between the two strains. This conclusion assumes an amino acid identity between tsB5 and its parent strain HFEM at this residue. We only know that there is no rate-of-entry difference between tsB5 and HFEM (unpublished observation). Finally, we note that this nucleotide change gives a B&E11 restriction site (GGZ’CACC) in KOS that is not present in tsB5. The available data on entry of different HSV-1 isolates show that mp, MP, and tsB5 enter host cells more rapidly than does strain KOS (1). MP (a mutant derived from mp), mp, and at least one HSV-2 strain also lack the B&E11 site at 0.355 (not shown) and enter fast, relative to KOS. Therefore, the presence of the B&E11 site may be diagnostic for the slow-entry phenotype. The DNA sequences between 0.350 and 0.360 are not the only DNA sequences affecting the rate of entry because gCstrains enter host cells slightly faster than do gC+ strains (I). We cannot ascertain the importance of DNA sequences between 0.350 and 0.360 relative to those that affect gC production in virus entry at this time. tsB5 forms syncytial plaques in HEL and Vero cells. Most HSV-1 strains induced limited but measurable cell fusion, and the syncytial (syn) defect is expressed as a large increase in the extent of cell fusion (13). There may be as many as four HSV1 genes affecting cell fusion (31), and only one of these maps near gB (32). The syn mutation in tsB5 was mapped to 0.345 to 0.355 map units and was shown to be separable by recombination from, and to the left of, the entry determinant (1). If the syn mutation resides in gB, then it must be caused by the nucleotide change at 1610. This change is predicted to cause a his (CAC) insertion in tsB5 relative to arg (CGC) at amino acid residue 857 (Figs. 2, 3) in KOS. It is interesting that the amino acid change that appears to cause the syn mutation in gB is located on the cytoplasmic side of the plasma membrane.

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1-GTC GAC GGC TTC TAC GCG CGC GAC CTC KC 67-CTG

ACC AAG GCC CGG GCC ACG GCG CCG ACC ACC CGG AK-342

CTC ACG ACC CCC AAG TTC ACC GTG GCC TGG GAC TGG GTG CCA AAG CGC CCG TCG GTC TGC ACC-364

133-ATG

A ACC AAG TGG CAG GAG GTG GAC GAG ATG CTG CGC TCC GAG TAC GGC GGC TCC TX

199-TCC

GAC GCC ATA TCC ACC ACC TTC ACC ACC AAC CTG ACC GAG TAC CCG CTC TCG CGC GTG G4C CTG-408

CGA TTC TCC-386

X5-GGG

GAC TGC ATC GGC AAG GAC GCC CGC GAC GCC ATG GAC CGC ATC TTC GCC CGC AGG TAC AAC GCG-430

331-ACG

CAC ATC AAG GTG GGC CAG CCG CAG TAC TAC CTG GCC AAT GGG GGC T T T CTG ATC GCG TAC CAG-452

397-CCC

CTT CTC AGC AAC ACG CTC GCG GAG CTG TAC GTG CGG GAA CAC CTC CGA GAG CAG AGC CGC AAG-474

463-CCC

CCA AAC CCC ACG CCC CCG CCG CCC GGG GCC AGC GCC AAC GCG TCC GTG GAG CGC ATC AAG Act-496

529-ACC

TCC TCC ATC GAG TTC GCC :GG CTG CAG T T T ACG TAC AAC CAC ATA CAG CGC CAT GTC AAC GAT-518

59%ATG

A T T G GGC CGC G T T GCC ATC GCG TGG TGC GAG CTG CAG AAT CAC GAG CTG ACC CTG TGG AAC GAG-540

661-CCC

Val T CGC AAG CTG AAC CCC AAC GCC ATC GCC TCG GCC ACC GTG GGC CGG CGG GTG AGC GCG CGG ATG-562 ala

727~CTC

GGC GAC GTG ATG GCC GTC TCC ACG TGC GTG CCG GTC GCC GCG GAC AAC GTG ATC GTC CAA AAC-584

793-TCG

ATG CGC ATC AGC TCG CGG CCC GGG GCC TGC TAC AGC CGC CCC CTG GTC AGC T T T CGG TAC GAA-606

859-GAC

G CAG GGC CCG T T G GTC GAG GGG CAA CTG GGG GAG AAC AAC GAG CTG CGG CTG ACG CGC GAT GCG-628

925-ATC

GAG CCG TGC ACC GTG GGA CAC CGG CGC TAC TTC ACC TTC GGT GGG GGC TAC GTG TAC TTC GAG-650

991-GAG

TAC GCG TAC TCC CAC CAG CTG AGC CGC GCC GAC ATC ACC ACC GTC AGC ACC TTC ATC 'SAC CTC-672

1057-AAC

ATC ACC ATG CTG GAG GAT CAC GAG T T T GTC CCC CTG GAG GTG TAC ACC CGC CAC GAG ATC AAG-694

1123~GAC

AGC GGC CTG CTG GAC TAC ACG GAG GTC CAG CGC CGC AAC CAG CTG CAC GAC CTG CGC TTC GCC-716

1189-GAC

ATC GAC ACG GTC ATC CAC GCC GAC GCC PAC GCC GCC ATG ll+

GCG GGC CTG GGC GCG TTC TTC-738

1255~GAG

GGG ATG GGC GAC CTG GGG CGC GCG GTC GGC RAG GTG GTG ATG GGC ATC GTG GGC GGC GTG GTA-760

1321-TCG

GCC GTG TCG GGC GTG TCC TCC TTC ATG TCC AAC CCC T T T GGG GCG CTG GCC GTG GGT CTG TTG-782

1387-GTC

CTG GCC GGC CTG GCG GCG GCC TTC l-K

1453-ATG

T AAG GCC CTG TAC CC6 CTA ACC ACC AAG GAG CTC AAG AAC CCC ACC AAC CCG G4C GCG TCC GGG-826

1519~GAG

A GGC GAG GAG GGC GGC GAC T T T GAC GAG GCC AAG CTA GCC GAG GCC CGG GAG ATG ATA CGG TAC-848

T GCC T T T CGC TAC GTC ATG CGG CTG CAG AGC AAC CCC-804

1585-ATG

ar9 G G G GCC CTG GTG TCT GCC ATG GAG CAC ACG GAA CAC AAG GCC AAG AAG AAG GGC ACG AGC CGT CTG-870 his

1651-CTC

AGC GCC AAG GTC ACC GAC ATG GTC ATG CGC AAG CGC CGC AAC ACC AAC TAC ACC CAA G T T CCC-892

1717~AAC

MA 'SAC GGT GAC GCC GAC GAG GAC GAC CTG TGACGGGGGGTTTGTTGTAPPAACCACGGGTGTTAMCC

C T 1793-GCATGTGCATCllTTGGTTTGTTTGTTTGGTCAGCCTTTTGTGTGTG YT

GGAAGAAAGAAAAGGG A

FIG. 2. Nucleotide sequence of taB5 DNA between 0.330 and 0.34’7 map units and comparison to the homologous KOS DNA sequence. DNA sequencing using dideoxy nucleotide analogs was originally described by Sanger et al (29). The protocol used for DNA sequencing was previously described (24). Single-stranded recombinant phage DNA was prepared as described previously (24). Klenow fragment of DNA polymerase I was obtained from Boehringer-Mannheim Biochemicals and was used as recommended by the manufacturer. The l’l-base universal Ml3-specific primer was used to prime DNA sequencing reactions and was obtained from Collaborative Research. All deoxy and dideoxy nucleotides were obtained from P-L Biochemicals. [a-VjDeoxynucleotide triphosphate (dATP, >f500 Ci/mmol) was obtained from New England Nuclear. Denaturing polyacrylamide gels were used to resolve DNA fragments created in DNA sequencing experiments. Denaturing polyacrylamide gels were made and run as previously described (SO). The denaturing polyacrylamide

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signal

rate

of &try

addition

(552)

GmC++ Val ti

GDC Ala

(KOS)

IlSB5)

siles

membrane

189 spanning

internal

-syn G357, CgJCOCQJC c, His Arg (KOS) (tsB5)

FIG. 3. Location of taB5-specific amino acids within gB. Schematic representation of the HSV-1 (KOS)-specifiedgB gene showing the predicted signal sequence, the locations of N-linked saccharideaddition sites (I), a possible membrane-spanning region, and the portion of the protein expected to lie internal to the plasma membrane (3). Amino acid numbers are shown below the schematic from the initiator ATG codon (1) to the C-terminal CTG codon (903). The locations of two amino acid differences between strains KOS and taB5 are depicted.

The syn mutation is probably not caused by the numerous base changes in the 108 nucleotides immediately 3’ to the gB coding sequence because this appears to be a noncoding region. However, it is possible that the cell fusion mutation occurs between 0.347 and 0.345 map units of tsB5 (beyond the nucleotides that were sequenced) because data for KOS in this region indicate a possible coding sequence for another protein transcribed from left to right and having a stop codon and poly(A) addition signal at 0.347 (to be published). There are three reasons for concluding that the syn mutation in tsB5 is within the gB coding sequences. (i) Fusion produced by recombinants between tsB5 and MP correlated with the presence of gB (6). (ii) Genetic studies (19) suggest that the syn and ts mutations in tsB5 are in the same gene.

(iii) Virus entry probably occurs by a fusion mechanism (12,.33) and tsB5 virions grown at the nonpermissive temperature are noninfectious (8). Note added in prooj Using a plasmid containing only gB coding sequences of tsB5, a syn marker was transferred to intact KOS DNA. The KOS-specific B&E11 restriction site at coordinate 0.355 was not present in Southern blots of DNA from some of the syz recombinants (unpublished results). We conclude that the syn determinant in tsB5 is due to the amino acid change at residue 857. ACKNOWLEDGMENT This work was supported by the Ernest W. Peters Memorial grant from the American Cancer Society. REFERENCES 1. DELUCA, N., BZIK, D. J., BOND, V. C., PERSON, S., and SNIPES, W., Virology 122, 411-423 (1982).

gel dimensions were 35 X 42 X 0.035 cm, and the gels were either 6 or 10% acrylamide in order to resolve different fragment sizes. At the conclusion of electrophoresis, the gels were exposed to Xray film, usually for 15 to 24 hr. The nucleotide sequence of tsB5 DNA (noncoding strand, 5’ to 3’) beginning at the Sal1 site is shown. Nucleotide numbers are shown in the left margin, beginning with the first nucleotide of the Sol1 site. KOS DNA-specific nucleotides (8) are shown above the tsB5 nucleotides. Amino acid numbers (codon triplet numbers) are shown in the right margin (KOS gB ATG initiation codon = 1). If the nucleotide difference was predicted to cause an amino acid substitution in gB then the amino acid changes are also shown. The predicted membrane-spanning region and polyadenylation signal for the gB gene are underlined. A reexamination of sequencing gels revealed that nucleotides AC (2899 and 2810) of the gB sequence of KOS were reversed (3)). They should read CA, as is correctly shown above for nucleotides 1302 and 1313 for taB5. The amino acids reported previously for these positions were glz/ (GGA) and Zeu (CTC) for amino acids 754 and ‘755; they become gly (GGC) and ile (ATC).

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