The nucleotide sequence, 5' end, promoter domain, and kinetics of expression of the gene encoding the herpes simplex virus type 2 latency-associated transcript.

(1)

0022-538X/91/105619-05$02.00/0

The

Nucleotide

Sequence,

5' End,

Promoter

Domain,

and Kinetics

of

Expression of the

Gene

Encoding

the

Herpes

Simplex

Virus

Type

2 Latency-Associated

Transcript

PHILIP

R.

KRAUSE,

JEFFREY M. OSTROVE, AND STEPHEN E. STRAUS*

Medical Virology

Section, Laboratory of ClinicalInvestigation, NationalInstitute of

Allergy

and

Infectious

Diseases,

Building

10,

Room

IIN228,

Bethesda,

Maryland

20892

Received5April1991/Accepted 20June 1991

The sequence of the herpes simplex virus type 2 (HSV-2) latency-associated transcript (LAT) region

resembles

that of HSV-1 only where theLATs overlapICPOand in the putativepromoterregion. Otherwise,

the LAT 5' ends, kinetics ofexpression, and promoter elements are mostly conserved between HSV-1 and

HSV-2.

Theremaining differences betweentheLATs could contribute toeach virus's distinctive pattern of

reactivation.

After replication at the site of initial mucocutaneous

inoculation, herpes simplex virus types 1 and

2

(HSV-1 and

HSV-2) establish lifelong latent infections of the sensory

neurons

serving those tissues.

Periodically, the viruses

re-activate to cause symptomatic or asymptomatic recurrences.

The patterns of latency and reactivation in natural human

infection differ for HSV-1 and HSV-2 (11). HSV-1

reacti-vates

more

frequently and efficiently from the trigeminal

ganglia

than

from

the

sacral ganglia; the opposite is true of

HSV-2.

The

latency-associated transcripts (LATs) are the only

gene

products

detectable during

HSV latency (2-5, 21, 22).

They are

transcribed from within the long repeats, opposite

in direction to and

overlapping

the 3' end

of the

immediate-early

transcript encoding infected-cell protein 0 (ICPO). In

HSV-1,

1.85- to 2.0-kb and 1.35- to 1.5-kb RNAs (the "major

LATs")

(9, 10, 16, 19, 20,

23-26) are

spliced

from an

8.3-kb

"minor

LAT"

transcript

(7), whose promoter

lies 600 to 800

bases upstream

of

the

major LATs (1, 6, 28). Evidence

obtained

by using

mutant

viruses

bearing deletions

in the

LAT

and its

promoter

region

suggests

that the LATs

permit

or

stimulate

reactivation from

the latent

state

(8,

12, 13,

18).

No

influence

of the HSV-1 LATs

on

the

establishment

or

maintenance

of

latency has been

demonstrable.

Latent

HSV-2

transcribes

a

single

LAT 2.2

to

2.3 kb

long (2, 3, 15).

It

is

not

known

whether

or

how the HSV-2 LAT

is

proc-essed,

but

minor

LAT

transcription

has been

found

to

extend

downstream of the HSV-2 LAT

(15).

The

HSV-2

LAT

promoter

element has

not

yet

been

identified

or

char-acterized.

In

this

article,

we

report

data

regarding

the DNA sequence

of

the

HSV-2

LAT

and

its upstream

region,

the

specific

5'

end

of

the

HSV-2

LAT,

identification of promoter elements

possibly responsible

for

HSV-2 LAT

transcription,

and

investigations of

the

kinetics of

expression

of the HSV-2

LAT.

We

hypothesize

that

a

better

understanding

of these

details may

yield insights

into the differences between

recur-rence

patterns

of HSV-1

and

HSV-2.

The

HSV-2 LAT

region

was

subcloned from

plasmid

pGR90

(which

contains

the HSV-2 strain 333

HindIll

JM

fragment and

was

obtained from

Gary

Hayward)

and

se-*

Corresponding

author.

quenced (Fig. la). Figure lb highlights the strong

organiza-tional and sequence homology between HSV-1 and HSV-2 in

the

region of the HSV-1 LAT promoter and in the area

corresponding

to

the

3'

coding

sequences

for

ICPO.

In

the

HSV-2 region homologous to the HSV-1 LAT

promoter

(bases -900 to -700), consensus elements

for

several

potentially

important regulatory elements are found.

These

include

a

TATA

box,

upstream

CAAT elements,

a

cyclic

AMP

(cAMP)-responsive element-binding site,

Spl-binding sites, and a potential ICP4-Spl-binding site (on the

negative strand).

The

HSV-1 LAT promoter also

contains

these

elements in similar locations relative to the TATA

element.

To

identify

the

HSV-2 LAT promoter, regions upstream

of

the

HSV-2 LAT were

screened for

promoter

activity

in

an

in

vitro

transient

expression

system by

using the

chloramphen-icol

acetyltransferase (CAT)

gene

as a

reporter gene.

Frag-ments to

be tested for promoter

activity

were

cloned

into

a

plasmid

vector

upstream

of

the gene for CAT and

trans-fected

into Vero

cells. Protein

was

extracted after

48 h,

and

the extent

of

acetylation of radiolabelled

chloramphenicol

was

compared

for

the

different

constructs

(Fig. 2). All

HSV-1 and HSV-2 LAT

fragments

tested

showed

promoter

activity. Thus,

promoter

activity

is

found in the HSV-2

sequences

which

are

homologous

to

the

HSV-1

LAT

pro-moter.

Direct

comparison of

the

activities of

the HSV-1

and

HSV-2-LAT promoters under

varying

and

physiologic

cir-cumstances

will

be needed

to

delineate

the

functional

rele-vance

of

their

similarities

and

differences.

At

a

position

762 bases downstream

of

the TATA element

(starting

at

base

-3),

the decamer

CAGGTAGGTT

(itali-cized

in

Fig.

1)

is

conserved

between the

two

viruses.

The

HSV-1

major

LAT

start

site lies

on

the

second

G in this

conserved

splice

donor

consensus

sequence.

Moreover,

a

splice

acceptor

consensus

sequence

is also

conserved

be-tween

the

viruses,

1.95 kb downstream

of the HSV-1 LAT 5'

end and 2.2 kb downstream

of

this site in HSV-2. These

conserved

sequences suggest

that the

HSV-2

major

LAT is

also

an

intron

spliced

from

a

larger

precursor,

as

is the

case

with HSV-1

(7).

Primer

extension

analysis (9) (Fig. 3a)

was

performed

to

localize

the

HSV-2 LAT

start

site.

A

discrete

reaction

product of

125 bases is

seen

in

HSV-2-infected

Vero

cell

extracts

and is absent from the

uninfected-cell

extract.

The

5619

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Sac I I

a CCGCGGGC ATCCGGCGGC CGGCCCCACG CCCTTCCATT AAGCACTCCC

Spi

-900 ACGTTGGGGG GGGGGCGCGC CAGCTGAGTG CTCTGCGGTT GCGGGCGCCG TGCCCGGAGA TCCATTAAGCCGCCGGAGAGCCCGAGCCCC GCCCGCGTGT cAMP respelement TATA Spl <ICP4 site

-800 TGCTGTGGGCATTTCTGCTGCGTCATCCCT GTCTTTATAAAACCGGGGGC GGCGGCAGCA ACGAACACAGGGGCCCGCCGCCGATCGAGAGGGACTCCGG -700 AGAAGGAAGG CTGCTCCGCG CACCGGCGCG CCCTTCTCCTCTCCCCTCCCTACCTCCCCCTCTCTTCCCCCTTTTTTCCC CCGCCTCCCG TCTTCTTCCG

Spl Not I

-600 CGCCTCCGAG GGTCCGCCTCTGCCTCGGGGACCCCCGGGC GGGCCGGGGCTCTGGCCGCCGAGGTGCGCCCCGGCCGGAGGGGCCCCCGCACCTCGGCGG Spl

-500 CCGCCCCCTC CGGCGCCGCG CGTTCGCGAAAGGCGCGAAA GGGGCCCCCGGAGGCTTTTT TCGATTCCCGGCCGCGGGTCCCGGGTAGCCGCCCGGCGCC -400 GGTCGGAAGG CGTCCCCCGCCCGGCGGTCC GGCCCGGGCCCCCGGCGGAG CGCGGGGGCCCCGGGGCCCC GGGCCGCGCCGGCGGCGTTTCCGCGTTCCG -300 TTTCTTCTCCCTCCCGGCCG CCCCGCTCCC GGGCCCGACCCTCGCCCCTTCCCTTCTCCTCGTCTTCCCC CGTCCCGCCG CGCCCCTTCC CTCTTCCTTC -200 TCTCTCTCTGTCTCGCTGTC TCGCTCTCCTCACATTTCCC CCCCCCCCCC CGCCGCCGCC GCCGCCCTCTGCCCGCGTCCCACCGAGACGCCGCGCCGCG -100 TGAGCCGTCC GCCCGGGCGACCCAGGCTCCGGGGGGGGGG CGCGCCTGCG TGTGTCTCGTGTGAGAGAGCGCGCCCCTCG AACGCCGCGC GTTCTCGCAG

LATstart site

0 GTAGGTAGGGTCGTACAG GTGAGCTTCT GCTGAGGCGG CGGGAGAGGG GGGGGCGGGCGGAAGAGAGA GAGAGGCAGG GGTTGGGGGAAAACTGTTCT

100 TCCTCCCCCTTTCAAGAAAC ACGAGGCGGG GGTCCCAGAA AGGGCAGGCA GGTCAGCCGC ACCGCCCGOCG AGCCAACCCGTATCCTTTTTTTCTAGGrGT Bam HI Bam HI

200 TTOGTrTGTTCGTI TTTTGTGTTT TGTTATTATT TTICGCGGATC CGGCGTGTTCGGATCCACCCCCCCCTTTC CCTTCCTCTT CCCTTCCACC 300 CACCCCCGTTTCCCCCCCCCCCCGTGGTGT CGTCCGGGGGCGTCGTTCCCAGGGGGGCAGGCGCGGOTCG GGCCCATACG CCCACCGCCCCCACGCGCCG 400 GTCACCCCCC CCCCAACAAC CCCAAAGGCGCGTGCCCGGC CACAGCCGTG GGrGTGGCGC CCGTCCCCTT CCTCTACCGC GTGGGCGCGGGCGGOGGGGT

Sal I

500 GGTGGTGGTAGTGGTGGCGG AAGGAAACGG GCCGGGGGGC CGGGGCCGCT AGGGAAAGGT AGGCACGCGC GCGGTGTGTCGACTTGCATGCCCCGCAAAA 600 CGCGTCGTGT CGTGTTGTGT CGTGGTGGGC CGTGTTGTGG TGGGCCGTGT GGTGTGGTGT GGTGTTGCGA ACGCGCGAGC CCCCTCGCCC CGATGGGAGT 700 CTCCOCCAG CCAGGGTAAG GAGGGCGG CGTGGCGGGC AGTGTGCGG GCGGGGTGGGGTGAGTGCGG TTGCATGCCTCGGGTCTCCTCTTCCTGCTC

ORF-1 M P R V S S S C S 800 CTCCTCCTTTCTOCCAGCCA GGGTGAGGAG GGGCGGGCGT GGCGGGCAGG TGTGCGGGCG GGGTGGCGC CGGGGCGGGG GTGGGCACGGGCGTAAGTGC

S S F L P A R V R R G G R G GQ V C G R G G R R G G G G H G R K C 900 GGGTGCATGCCTCGGGTCTT CTCTTCTCCC TCCTCCTTCC TCCCACCCGT CCCCGGGGGC AGAGGGCGTG CATGCGTTGT GATTCAACCG CCCTCGCCCC

G C M P R V F S S P S S F L P P V P G G R G R A C VV I Q P P S P 1000 CGCCCACTTTCCCCCCTCT CTATCAAAGTTCCCTGGCCC CTGGCTTCGC GCCGGTGGTGCGGCrGACCC CCCCCTCCTCCCTCCCCGAG CCAGGCGCCC

PP H F P P S L S K F P G P W L R A G G A A D P P L L P P R A R R P 1100 TCCCACTCCTGCCCACOACCCCCCGGGTCT GGCCGGCCAG ACGTGCGrGCTCTGCACGAT CGGGCCCCCC TCCCTGICAACACGGACACACTCTTTTT

P T P A H H P P G L A G Q T C V L C T I G P P S L S T R T H S F F

SpS

1200 AOCCGCCAGCCCGCCCACCCACCAAGACAGGGAGCCAGAA CGCAGGCCGGGGCCCCGGCTCTGTTCAIT ATAAAGACCAACAGGCCTOGGGGOOG

T R Q P A H P P R Q G A R T O A G A P A L F Y D K D Q Q A S G V G

GRF-2 M I K T N R P R G W G

FIG. 1. Sequence oftheHSV-2 LAT and homology with HSV-1. (a) Numbering of the HSV-2 LAT sequence is based on the 5' end of theHSV-2 major LAT (base 0). Positions of consensus sequences forregulatory elements and restriction enzymes used for cloning are indicatedabove the sequence. Translations of sequences which may encode proteins (potential ORFs 1,

2,

and 3) are indicated below the correspondingDNAsequence. A consensussequence,CAGGTAGGTT,whichincludes the LAT 5' ends shared by HSV-1 and HSV-2 LATs is italicized.A47-bp repeat sequence isunderlined. Bases which we were unable to identify after attempts using several primers are labeled

N. Becauseof the strong secondary structure of the HSV-2 DNA in the region from base -386 to base -314 (which has a G+C content of >97%), someuncertainty regarding the precise sequence between bases -319 and -314 exists. (b) The degree of homology between the HSV-1 and HSV-2 DNA sequences is shown by light (40 to 60% homology), medium (60 to 80% homology), or dark (>80% homology) stippling.The3'ends of the HSV-1 and HSV-2 LATs are drawn on the basis of theassumption that the LATs end at splice junction consensus sequences shared by the two viruses. Positions of consensus sequences for regulatory elements (cAMP-responsive element binding site [CRE],ICP4-binding site [ICP4], Spl-binding sites[Spl],TATA boxes[TATA]), restriction enzyme sites used for cloning, the long terminal repeat(TRL), long unique (UL), long internal repeat (IRL), short internal repeat

(IRs),

short unique (Us), and short terminal repeat

(TRs)

are indicatedand arebasedonthesequences of HSV-1 strain 17 syn + and HSV-2 strain 333.Protein-codingregions ofICP0andpotential ORFs (ORF-1 and ORF-2of HSV-1 and ORF-1, ORF-2, and ORF-3 of HSV-2) of the LATs are shown with thick arrows. Strong focal homology between HSV-1 ORF-2 and HSV-2 ORF-3 isindicatedby the blackportions of the arrows.

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Spl

1300 CGCTTCTCGTICCOGC= CCCCCTCCTCCTCCCTTCCCCCCCCCCA TCCCCGGCCC CCCTGCGCGGGGGAGCTGCATCAAAGGCCAACAACAAAGT A A S R A R P P S S S S L S P P P S P A P L R G G A A S K A N N K V R L L V P A P P P P P P F P P P H P R P P C A G E L H Q R P T T K

TATA TATA

1400 GTGTCAAAAG CATCACAAAA CTTrATTGTAAAATTTTTATAAATATAAAGTTTTTTc TCAAGTTTTCAACAAGGCCP.GAAAGTCCATAACAAAATGC

C Q K H H K T L L > ORF-3 M C V K S I T K L Y C K I F I N I K F F F L K F S T R P E S P > 1500 TGGTGTGrGTTGCTTGTCG GTOCCCCCCCC ACTCCCACCC CCACTTCCTGTCTCCTCCCCGTCTTCCCCCCCCCCCACTCC:CCTGC

L V C V A V R G R V R P P P L P P P L P V S S P S S P P P T S P C P

Spi Sac II

1600 CcGGGCGCC TCGICCGGTG 0 CCTTICGGCA GCAAGCCGAG TGTTAGCTCCOCCTACTCCCCGTGGCCCGCGGGGGCGTCG

R G A S A G G P V G G G F P S G S K P S V S S P Y S P W P A G A S Sac II

1700 CCGGCCGCGG GCGCGCCCTG CTCCCGAGACCACGGGTGGCGCGACCGGAGGCCGTGGAAGTCCAGCGCGC CCACCAGGGT GCCCTGGTCAAAGAGCATGT

P A A G A P C S R D H G W R D R R P W K S S A P T R V P W S K S M

1800 TGCCCACOGGGGTCATCCAG AGGCTGrTCCACTCCGACGCGGGGGGCGTC GGGTAGTCGGGGGGCCYCAC GCAGTTGCGC GCGTGCTCGGOGAGCP.GGGT

L P T G V I Q R L F H S D A G G V G > Sac II

Not I

00 GCGCGGCTC CACGCGGGGGCCGCGGCCCGCAGCAGG7CCGCCACGTTCCCCGTCTGGTCCACGAGGACCACGTAGGCCCCTATGTGGCCCGTCTCCATG

00 TCAGGACGGGCAGGG CCCCGTGACCGTCTTGTTCA CGTAAGGCGC CAGGGCCAAC GCTCGAGACC CCCGCGATGG GCAGGTAGCGCGTGACGGCG

Sac II

00 GGCGCCGGGT Ct;CGGGCCCCGGCTCGGGCCGCCCTCCGCGTGGCGCGTCTTCCTGGCACACTTCCTCG GCCCCGCGG CGCAGCAGCG CGGNNNCCGA

spliceacceptor

00 wGwA.G0tC TCGTCTCC cAGcCC ACGCGGAcGC GACGCTCCCACCAGCCCCGCCCGCAGAGGA AGAGGCGAGCAWAGGAGG AGGCGGA 00 AGAGGAGGAGGAGGCGGAAG AGGAGGAGGA GGAAGAGGCGGCGCGCGGCGACCNGCGACGAC

ORF-2

b (119,462) POTENTIALHSV-1

-688 0 OPEN READING ORF-1 1954

FRAMES: O

iFI

W<,t

HSV-1 LATs

~I

.

2''

__~~~~~~~~~~~R- RF-3

POTENTIALHSV-2J ~~ ~HSV/

I I\ M I

HOMOLOGY: -_

U>80% *60-80* 040-60%* ~ __,

TRL a-UL IR 'IRS US TRS

EE

cn~~~~n1Ci

TIo

II

toT

I

FIG. 1-Continued.

neighboring

sequence shows that the 5' end isatthe second

G of the decamer

CAGGTAGGTT,

atthesamebaseasthat

of the HSV-1LAT. Anadditional

primer

extensionreaction

identifiedthe identical 5' end 91 bases downstream of the

primer

TTCCCCCAACCCCTGCTC

(data

not

shown).

Northern

(RNA) hybridization analyses

were

performed

to determine the

temporal

orderof HSV-2 LAT

expression

during

acute infection

(Fig. 3b).

At 5

h,

a small amount of

2.2-kb LATwas detectable;

by

18h,the LAT concentration

had increased

considerably,

and a weak band

migrating

at

191

20C

21C

22C

231

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a T C G A U

C A G

>-125b--G

T A

T

-G -7;-iX T 7'

T

[image:4.612.339.531.76.376.2]

R.a}I-, t.l Sac 11-No1

FIG. 2. CATassays to identify promoter activityin sequences upstream of the LAT. The HSV-2 fragments (Fig. lb) tested

included HindIII-SalI (bases --3500 to 577), SphI-SalI (bases --2500 to577), SacII-SaIl (bases -945 to 577), and SacII-NotI

(bases -945 to -503). The comparable HSV-1 LAT promoter,

which was contained in the 281-bp HSV-1 strain F Hinfl-PvuIl

fragment, and the emptyCATvector(pCAT Basic)wereused as

positive and negative controls, respectively.

b Lf) CO- CD

C > > x

D < < C)

22kb

-approximately 6

to 8 kb was visible. This band may

repre-sent a

larger HSV-2 minor LAT.

Expression

of the

major

2.2-kb LAT

was

still

detected in the

presenceof

acyclovir

at

5

and 18

h butwas markedly decreased inamount

compared

with

untreated cells. Cycloheximide completely

inhibited

detectable

LAT

expression.

In all

lanes,

small amounts of

cross-hybridizing

28S

and

18S

rRNA are seen. Previous

experiments showed

that

transcription of

the

larger

of the

HSV-1 major

LATs is

inhibited

in

vitro

by both

acyclovir

and

cycloheximide (9).

In

this

study,

we

found that

cyclo-heximide blocked HSV-2

LAT

expression and acyclovir

did

so to agreat extent.

The failure of

acyclovir

to

completely

inhibit

HSV-2

LAT

expression could be due

to

the lower

degree

of

intrinsic

acyclovir sensitivity

of

HSV-2

compared

with HSV-1.

Nonetheless, the results remain consistent,

with

HSV-2

LAT

being transcribed

as

either

a

beta-gamma

or agamma gene. As has been

pointed

out

for

HSV-1

(17),

however, the expression of

LAT

during

latency, in the

absence

of other

HSV

transcription,

suggests

that its

syn-thesis is

regulated

in a mannerdifferent

from that of

other

HSV

transcripts.

TheHSV-2 LAT sequence

contains three

potential

open

reading

frames

(ORFs) which could encode proteins of sizes

214, 74,

and119

amino acids

with

ATGs

at

bases 784, 1267,

and

1496, respectively. The

three

strains of

HSV-1

se-quenced

inthe

region of the LATs (KOS [23],

F [27],

and

17

syn +

[14]) share

two

potential ORFs,

one

of

which (ORF-2

[Fig. lb])

bears

partial homology

to

the

third HSV-2 LAT

ORF

(ORF-3), with conservation of

38 of 44 consecutive

amino acids.

However, the HSV-2

ORF is truncatedto 119

amino acids

compared with

the273-amino-acid HSV-1LAT ORF-2. It is thus

possible that

the LATs encode partially

homologous protein products.

Excepting this modest ORF

similarity

and

the homology in

the ICPO region, the HSV-1

and HSV-2

LATs

possess

markedly different

sequences, an

observation which

does

not

exclude their

functioning as

antisense messages to

ICPO.

However,

if

LAT expression interfered with the

ICPO

message,

the

end result would

FIG. 3. Determinationofthe HSV-2 LAT5'end and kinetics of

expression. (a)RNAextracted from HSV-2 strain 333-infected Vero

cells(lane I) and uninfected Vero cells(lane U) was subjectedto

primer

extension

analysis using

the 18-mer CTCGTGTTTCTT GAAAGG.The results ofasequencingreaction(lanes T, C,G,and

A) using the identical primer are shown alongside. The indicated

baseislocated 125 bases downstreamof the 5'endof theprimer. (b) Northernhybridization analysis wasperformedonRNA extracted

from uninfectedVero cells(lane U) andat5or18 h after infection

(atamultiplicityof infection of 4.5 PFUpercell)withHSV-2 strain

333 from Vero cells (lanesI 5 andI18), Vero cells pretreatedwith

the viral DNAsynthesis inhibitor acyclovir (lanes ACV 5andACV

18), or Vero cells pretreated with the protein synthesis inhibitor

cycloheximide (lane CHX 18). The end-labelled oligonucleotide GTGGAACAGCCTCTGGATGA was used as the hybridization probe in this experiment. The asterisks denote the positions of 28S (upper) and18S (lower) rRNAsontheoriginal gel.

presumably

beareducedrather than

increased likelihood

of

virus

reactivation.

The present

study

revealed

overall

organizational identity

and focalsequence

homology

in the LAT

regions of HSV-1

and

HSV-2,

as well as similarities in the

expression of

the

LATs themselves. These

findings imply conservation of

a genome domain

important

to

the

evolutionary

success

of

these viruses.Clear differences

between

the LAT regions of

the viruses have also been

elucidated.

Further study

will be

neededtoassess

their relevance

to

the known differences in

thepatternsof reactivation

of these viruses.

Nucleotide sequence accession number.

The GenBank

ac-cession number for the sequence

presented

in

this article is

M69065.

Wegratefully acknowledge the technical assistance of Quentin Allen, Yolanda Yang, Jumana Kurawadwala, and Holly Smith. We

Cj ;|

Hinl'--Pv!,

HI

-Ale

srol!-S-. 0

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thank Gary Hayward, John Martin, Stephen Wechsler, and Jeffrey Meier for providing clones and viruses and Duncan McGeoch for sharing preliminary sequence information for HSV-2 strain HG52.

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