Mutational analysis of the fingers domain of human immunodeficiency virus type 1 reverse transcriptase.

(1)

0022-538X/92/127533-05$02.00/0

Society

for

Microbiology

Mutational Analysis of the Fingers Domain of Human

Immunodeficiency Virus

Type 1

Reverse

Transcriptase

PAUL L. BOYER, ANDREA L. FERRIS, AND STEPHEN H. HUGHES* ABL-BasicResearch Program, NCI-Frederick Cancer Research and

Development

Center,

P.O. Box

B,

Frederick,

Maryland

21702-1201

Received 6 April 1992/Accepted 18 August 1992

UsingBspMI cassette vectors, we have constructed a series of mutations in human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) that cause specific amino acid substitutions within the polymerase domain.The RNA-dependent DNA polymerase, DNA-dependent DNA polymerase, and RNase H activities of themutant RTs were assayed. Theelucidation of the structure of HIV-1 RT makes it possible to determine the locationsof specific mutations in the three-dimensional structure of HIV-1 RT[E.Arnold, A.Jacobo-Molina, R.G. Nanni, R. L. Williams, X. Lu, J. Ding, A. D. Clark, Jr., A. Zhang, A. L. Ferris, P. Clark, A. Hizi, and S. H. Hughes, Nature

(London)

357:85-89, 1992; L. A. Kohlstaedt, J. Wang, J. M. Friedman, P. A. Rice, and T.A. Steitz, Science256:1783-1790,1992].Themutations described in this report are between amino acids 25 and 81, within the "fingers" domain of RT (Kohlstaedt et al., Science 256:1783-1790, 1992). It has been suggested that this domain may play a role in positioning the template. Although the fingers domain does not contain the active site for polymerization, several of the mutations within this domain disrupt polymerase

activitywithout

significantly

affecting RNase Hactivity.

Human immunodeficiency virus type-1 (HIV-1) is the

etiological agent of AIDS. HIV-1 virions contain reverse transcriptase (RT), the enzyme that converts the single-stranded viral RNA genomeinto double-stranded

DNA

(31,

32). HIV-1 RT is the target of drugs such as AZT

(zidovu-dine) and dideoxyinosine. Despite extensive study, further characterization of HIV-1 RT is necessary for the design of

moreeffective drugs.

In virions, HIV-1 RT exists as a heterodimer with two subunits of 66 and 51 kDa. The 66-kDa subunit contains both the RNA-dependent DNA polymerase domain and the RNaseH domain. The polymerase domain is composed of

approximately the first 430 amino acids in HIV-1 RT; the RNase H domain consists of the 130 amino acids at the

carboxy terminus. The smaller subunit is a proteolytic

cleavage product of the 66-kDa protein or of a larger

precursor and contains approximately the sequences that

comprisethe DNApolymerase domain in thelargersubunit

(4, 6, 9, 17, 20).

Thepolymerase domainsof HIV-1 RT and HIV-2 RTwere

initiallyanalyzedbystudyinginsertion anddeletion mutants

(7-10, 25).Mostof the insertion and deletion mutations that

disruptthepolymerase activityofeitherHIV-1 RTorHIV-2 RT also disrupt the RNase H activity. Similarly, insertion and deletionmutantsinthe RNase H domains of HIV-1 RT and HIV-2 RT often affectpolymerase activity. These data led to the proposal that elements in both domains are required for the proper foldingof each domain (8-10, 25).

There is biochemical evidence which provides support for

this

conjecture.

Hostomsky

et al.

(12) expressed

the

poly-merase and RNase Hdomainsseparately. Neitherofthese

separatelyexpressedproteinshad ahighlevelofenzymatic activity.SignificantRNase Hactivitywasrestored when the

two domains were mixed; however, the

activity

of the

polymerasedomainwasnotenhanced(12).Thepolymerase

domain of HIV-1 RT has been further characterized by

* Correspondingauthor.

analyzingpoint mutants.Pointmutantshave beengenerated

bysite-directed mutagenesis(3,15,16, 21,22)orbyisolation from drug-resistant strains of HIV-1 (5, 18, 19, 24, 26, 28,

30). Most of the point mutants created by site-directed

mutagenesis have targeted evolutionarily conserved amino acidresidues(2, 23) and haveidentified anumber of amino acid residues critical for theactivityof HIV-1 RT.Analyses of the RTs from drug-resistant strains of HIV-1 revealed

changes at amino acid positions that arenot evolutionarily conserved.

Wehaveprepareda series ofBspMIcassettes thatallow mutations to be introduced into the polymerase domain of the 66-kDa form of HIV-1 RT(3).Thecassettes are deriva-tives oftheplasmidHIV-1RT, which induces theexpression

of the 66-kDa RT in Escherichia coli (9, 13). We have

previouslydescribeduseof theBspMIcassettes to create20

amino acid substitutions within the polymerase domain

between aminoacids 55 and 186. The 20 mutant RTswere

assayed forRNA-dependentDNApolymeraseand RNaseH

activities. Seventeen of the mutants havevarious levels of RNA-dependent DNA polymerase activityand retain

mod-erate orhighlevels of RNase Hactivity.Twoof the mutants,

Pro-55--+Glyand Ser-156--3Ala, eliminate RNaseHactivity

but retain a significant level of polymerase activity, while

mutant Pro-150--)Glycompletelyabolishes both activities. Wehave made 33 additional amino acidsubstitutions and

rearrangements

within the HIV-1 RT polymerase domain between amino acids 25 and 81. TheRNA-dependent DNA

polymerase, DNA-dependentDNApolymerase,and RNase H activity levelswere assayed for all of the mutants. The

recentlypublished structure of HIV-1 RT (14) shows that these mutations lie

within

the

"fingers"

domain. It has been

suggestedthat thisdomain hasarole in

binding

the

template.

Results and discussion. Thirty-three amino acid substitu-tions and rearrangements were made in the

polymerase

domain of HIV-1 RT between aminoacids 25 and 81

(Table

1).Most ofthechangesare atamino acids thatareconserved inawide range ofretroviral RTs

(2,

23).

Themutations are

primarily singleamino acid

substitutions,

but someinvolve 7533

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TABLE 1. Summaryofresults ofRNA-dependentDNApolymerase, DNA-dependentDNApolymerase,and RNase Hassaysa

Activity

Clone Aminoacidchange

Polymerase (%) In situ DDDP In situ RNase H

None Pro-25-*Gly Leu-26-->Ser Thr-27-->Ala Glu-29-*Asp Lys-30--+Arg Ile-31--Ser

Ile-31-*Ser/Leu-34--Ser Leu-34--Ser

Glu-36--ValIMet-41--+Ser

Ile-37--3Ser Glu-40--*Ala Met-41--*Ser Gly-45-*Leu Lys-46-*Arg Ile-47- Leu

Ile-47--+Leu/Pro-52-->Gly Pro-52-*Gly

Pro-55--Gly Tyr-56--Trp

Pro-55--Tyr/Tyr-56---Pro Asn-57-*Gln

Asn-57-->Asp Thr-58-*Ile Pro-59--)Gly Val-60-*Ser

Pro-59--Val/Val-60--*Pro

Phe-61-+Trp

Phe-61-->Tyr Ile-63Ser Lys-64--+Arg Lys-65--+Arg Lys-66-*Arg

Asp-67---Asn/Lys-70--3Arg Ser-68--->Ala

Thr-69- Ile Lys-70--*Arg Trp-71--+Tyr

Arg-722±Lys

Asp-76-*Glu Arg-78-ALys

Arg-78-- Lys/Asn-81--+Gln Asn-81--*Gln

100 30 20 60 100 100 50 <5 <5 <5 15 100 <5 20 50 100 35 45 45 50 <5 <5 <5 40 35 80 <5 80 100 45 50 <5 90 100

100

50 100 70 45 <5 50 <5 <5

++

++ ++

++

+++

++ +

ND+

+++

++

+++

++

+++

++ ++

aLocation(s) and the amino acid substitution(s) areshown for eachmutant. Previously describedmutants(3)areunderlined.TheRNA-dependentDNA

polymerase activity of themutantswasassayed inextractsasdescribed in thetext.Theamountof HIV-1 RTpolymerase activityseenwith thewild-type RT

wasset at100%, and the results for themutantRTswerenormalizedtothis value. The in situDNA-dependent DNA polymerase (DDDP)assayandthe in situ RNase Hassay weredone aspreviously described (3, 29). For bothassays,activity is expressedasapproximately equivalenttowild-type activity (+++), moderate(+ +), weak (+),orundetectable(-). ND,notdetermined.

multiple substitutions orchanges in the order of the amino

acids. We have previously described 20 mutations intro-duced into theHIV-1 RT polymerase domain between amino

acids55 and 186 (3). These mutationswere located in four areas designated group I through group IV, based on a

schemeoriginally proposed by Larder etal. (16). Since the structureof HIV-1 RTisnowavailable,wewill discuss the mutations with reference to the subdomains in which they

reside. Thethree-dimensional structureofHIV-1 RT shows that the polymerase domain of the 66-kDa subunit is com-posed of four subdomains that, taken together, resemble a

right hand (1, 14). The active site for polymerase lies in the palm, and the thumb holds the double-stranded primer/

template. The role of the fingers subdomain is less certain; however, it has been suggested that the fingersmayserveto

hold and appropriately position the template strand. The

connection domaintowhich RNaseHis attachedappearsto haveprimarilyastructural role. These four subdomainsare

also present in the 51-kDa subunit; however, they are arranged in a much different configuration. As a

conse-quence, the 51-kDa subunit can play no direct role in the polymerization reaction. Reconstitution studies have shown

that mutations in the 51-kDa subunit do not significantly

affect thepolymerase activity of the 66-kDa subunit (11, 21).

Ahomodimer oftwo 66-kDa subunits alsoappearsto be enzymatically active (27). Although the structure of this homodimeric enzyme has not been solved, it has been suggested that this formshouldresemble the66-kDa/51-kDa heterodimer, with the RNase H domain of the 66-kDa

subunitthat takes the placeof the 51-kDa subunit partially HIV-1 RT

6133/6134

6135/6136

6137/6138

6139/6140 6141/6142

6143/6144 6145/6146

6145/6146* 6151/6152 6147/6148 6149/6150 6151/6152* 6153/6154

6155/6156

6157/6158 6159/6160 6159/6160*

2852/2853

5873/5874 5869/5870

2854/285

5871/5872 5875/5876 2424/2425 2426/2427 5877/5878 2428/2429

5879/5880

2430/2431 2432/2433 2434/2435

2436/2432

4859/4860 6187/6188 6189/6190 4857/4858

2438/2439 244012441 2800/2801 2802/2803

2804/2805 2804/2805*

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cc.

97.4

66.2

kd-42.7 kd

31.0kd _

FIG. 1. Level of expression of mutant HIV-1 RTs inE. coli. Bacteriacontaining the parental HIV-1 RT cloneoramutantHIV-1 RTweregrownfor 12to16 hat 37°C with agitation. The bacteria

werecollectedby sedimentation inamicrocentrifuge and thenlysed

in1x Spear's buffer. The proteinextractswerefractionatedon an

SDS-9.0% polyacrylamide gel and visualized by staining with

Coomassie brilliant blue. The arrow on the right indicates the predicted size (66 kDa) of the parental HIV-1 RT protein. The level

of RTexpression is approximately equivalentamongthe different E.

coli strains. Theposition of migration ofsomeof themutantRTs (e.g., 6159/6160) varies slightly from that of wild-type HIV-1 RT.

unfolded and exposed to proteolytic cleavage. We have assumed in our discussions that the primary effect of the

mutations that we have made is exerted from within the

66-kDa subunit that is active inpolymerizationand that the

presenceof the mutation in the other subunit should havea

less significant effect. Although the available data support

thissuggestion,it hasnotbeendirectlytested for each of the

mutantsthatwewilldescribe,and it remainspossiblethatat

least in some cases, the mutation exerts part or all of its effect throughthealteration in the second subunit.

The mutations were constructed by using HIV-1 RT BspMI cassettes (3). Each mutation was validated by dideoxynucleotide sequencing, andthe mutantHIV-1 RTs

werevisualizedonsodiumdodecylsulfate

(SDS)-polyacryl-amidegels.As shown inFig. 1,theamountof RTproduced bythemutantsis similartothe level ofwild-typeHIV-1RT, althoughthepositionofmigrationofsomeof themutantRTs variesslightly.The levels ofRNA-dependentDNA

polymer-ase,DNA-dependentDNApolymerase,and RNase H activ-itiesweredetermined for each of themutantRTs(Table 1).

The resultswereusedtogroupthe amino acid substitutions into four categories: substitutions that are functionally si-lent, substitutions thatprimarilyaffect polymerase activity,

substitutions that primarily affect RNase H activity, and

substitutions that affect both polymerase and RNase H

activities.

Fourteensingleamino acid substitutions and threedouble

amino acid substitutions have been introducedin theregion

between amino acids 25 and 52. The sequence PLTXEKI (aminoacids 25to31)is conservedin lentiviralRTs;there is apartialconservation inawiderrangeof retroviral RTs(2, 23).The amino acids in this motifwerealteredindividually.

The motif containstwohydrophobic aminoacids, aleucine

atposition26 andanisoleucineatposition31.Each of these

was replaced with the uncharged hydrophilic amino acid serine. Mutant HIV-1 RTs with either Leu-26-*Ser or Ile-31-+Ser had diminished polymerase activitybut nearly full RNase H activity. The mutant Leu-26--Ser had

approxi-mately

20%of thewild-typelevel ofRNA-dependent DNA

polymerase

activity (Table 1)

butwas inactive in the

DNA-dependentDNApolymeraseassay.If thefingersdomainhas

a role in binding of template (14), it is possible that this mutation could selectively affect the enzymes interactions with RNA and DNAtemplates.

Two othermutantsin the PLTXEKImotif, Pro-25--*Gly

andThr-27--+Ala,have diminishedpolymerase activityanda

slight decrease in RNase H activity. We considered the

possibility

that these mutations affect the

ability

of the enzyme to bind or

appropriately position

the

primer/tem-plate. It is believed that the

polymerase

domain and not RNase H hastheprimaryrole inbindingtheprimer/template (1, 14). Since these mutants are reasonably active in the RNase H assay,

primer/template

should be bound to the

polymerase domain. The amino acid substitutions

Glu-29--Asp andLys-30--*Arg do not

significantly

affect either

polymerase

orRNase H

activity.

Itisnotclear whether the

replaced

aminoacid isnot

important

for

polymerase

activity

or whether the

replacement

amino acid is an effective substitute for the

original

amino acid. Further mutational

analysis

should

distinguish

between these

possibilities.

Other

changes

made in conserved amino acids include

Glu-40--+Ala and Ile-47--Leu. These substitutions did not

significantly

affect either RNase Hor

polymerase

activity.

The Ile-47--*Leu mutation doesnot

significantly

change the

nature of the

original

side group, and leucine appears to substitute

effectively

for isoleucine at this

position.

The Glu-40--*Ala

mutation, however, replaces

the

charged,

acidic side group of

glutamic

acid with the

hydrophobic

side

groupof alanine. The RTsofmousemammarytumor

virus,

Mason-Pfizer

monkey virus,

and HIV-1 all have a

glutamic

acidat

equivalent positions.

Mostof the otherretroviral RTs haveeither

arginine

or

lysine.

HIV-2RT also hasa

lysine

at this

position. Although

the

evolutionary

data suggest thata

hydrophilic

amino acid is necessaryatthis

position, putting

alanineatthis

position

in HIV-1 RT hasnomeasurable effect on ourinvitro

polymerase

or RNase H assays.

The Leu-34--*Ser and Met-41--*Ser mutations

replace

a

hydrophobic

amino acid with the

hydrophilic

amino acid serine. In both cases, thesubstitution

significantly

decreases oreliminates both

polymerase

and RNaseHactivities. The mutant

Gly-45--*Leu

hasasimilar effect. When the

hydrogen

of

glycine

is

replaced

with the

bulky

side group of

leucine,

activities are

significantly

reduced. The Ile-37--+Sermutant has an effect similar to that of Leu-26---Ser. Ile-37--+Ser retains 15%of the

wild-type

levelof

RNA-dependent

DNA

polymerase

activity

(Table 1)

but has no detectable

DNA-dependent

DNA

polymerase activity.

The mutations

Lys-46--*Arg,

Ile-47--+Leu/Pro-52--

Gly,

and

Pro-52---*Gly

decrease

polymerase

activity

but have different effectsonRNase H

activity. Lys-46--*Arg

has full RNase H

activity.

Pro-52--+Gly

retains 45% of

wild-type

polymerase

activity

buthas

only

minimalRNase H

activity.

We

previously

described a

nearby

mutant,

Pro-55--*Gly,

which also retains45%of

wild-type

polymerase

activity

but has no detectable RNase H

activity

(3) (Table

1).

We

suggested previously

that this

region

of the

polymerase

domain would be in contactwith the RNase Hdomain

(3).

However, the structure of HIV-1 RT suggests a different

possibility:

thattheexact

positioning

ofthe

primer/template

isaffected

by

the

fingers.

Ifthis view is correct, amutation

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in the fingers domain which still allows properinteractions with the polymerase active site could affectthe position the primer/template and prevent proper interactions with the RNase H active site. Interestingly, the RNase H activity is partially rescued in the double mutant Ile-47--+Leu/Pro-52-*Gly (Table 1).

Twelve single amino acid substitutions and four double amino acid substitutions or rearrangements that have been generated between amino acids 55 and 81 arelisted in Table 1.The prolineatposition 55 and the asparagineatposition 57 arerequiredfor full polymerase and RNase H activities (3) (Table 1). We made a number of amino acid substitutions and rearrangements between amino acids 55 and 60 to characterize this regionmorecompletely. Several retroviral RTs have thearomatic amino acid tyrosine ortryptophan at positions equivalent to position 56 in HIV-1 RT, while the other RTs have either cysteine or glycine at this position. Theclosely related RT of HIV-2 has tyrosineatposition 56. When the tyrosine in HIV-1 RT is replaced with a tryp-tophan(Tyr-56--3Trp), approximatelyhalfof thepolymerase activityremains and RNase H activity isunaffected.

Appar-ently,thelargersidegroupoftryptophancauses adistortion

that decreases the polymerase activity. We tested whether

the order of the amino acids at positions 55 and 56 was important. The mutant Pro-55--+Tyr/Tyr-56- Pro is a rear-rangement of the normal HIV-1 RT amino acid sequence. This mutation eliminates both polymerase and RNase H activities. When the asparagine at position 57 is replaced

with the closely related amino acid glutamine, almost all of

thepolymerase activityis lost and the RNase Hactivity is diminished(3) (Table 1). Both activities areeliminated when asparagine is replacedwith aspartic acid.

Threeothermutationsinthe region from amino acids 55to 60, Thr-58-dle, Pro-59--3-Gly, and Val-60--Ser, alter

con-served amino acids. Most RTs have threonine or serine at

the equivalent position, and HIV-2 RT hasthreonine. Sub-stitutingahydrophobic isoleucine for the hydrophilic

threo-nine doesnotaffect the RNase H activity ofHIV-1 RTbut does decrease the polymerase activity to40% of the wild-typelevel. Theprolineataminoacid59 is highlyconserved. Replacing thisproline withaglycine affects polymerase but notRNaseHactivity. Atpositions equivalenttoposition 60,

mostretroviral RTs have ahydrophobic amino acid, either valine,isoleucine,orleucine. HIV-2 RT has threonineatthis

position. The mutationVal-60--+Ser has onlyaminor effect on the HIV-1 RT polymerase activity. Apparently this positioncan accommodate different amino acidsand is not

crucial to the structure or function of the enzyme. A

rear-rangement mutant was constructed at positions 59 and 60.

Themutation Pro-59--*Val/Val-60--+Proreversestheposition

of the proline at position 59 and the valine at position 60. Substitution of individual amino acids showed that the prolineatposition60isrequiredfor full polymerase activity,

butthevalineatposition 59 isnotrequired (Table1). Amino acidsubstitutionsateitherposition didnotaffect theRNase

Hactivity. Both thepolymerase and RNase H activitiesare

eliminated when the order of the two amino acids is

re-versed.

We have previously described mutations in a motif

(IKKK) atamino acids 63to66(3). We have constructeda

number of additional amino acid substitutions near this

motif. Two of these substitutions are of particular interest

because they have been identified in virus strains that are

resistant tothe drug AZT (5, 15, 18, 30). The single amino acid substitution Lys-70--*Arg and the double substitution

Asp-67--+Asn/Lys-70--+Arg have been linked to AZT

resis-tance. Although the lysine residue at amino acid 70 is conserved in various RTs, including HIV-2 RT, neither the single mutation Lys-70--+Arg nor the double mutation Asp-67--Asn/Lys-70---*Arg reduced the levels of either polymer-ase or RNase H activity in our assays (Table 1). We have made amino acid substitutions at positions 68 and 69, be-tween twoof the positions involved in AZT resistance. Most RTs, including HIV-1 RT, have serine at position 68,

al-though threonine or asparagine is found in some RTs and

HIV-2 RT has a lysine at this position. The mutation Ser-68- Ala haslittleor noeffectonpolymeraseorRNase H activity. Position 68 canapparently accommodate a number ofamino acids besides serine and isprobably notcrucial for the structure orfunction of HIV-1 RT. Most retroviral RTs have aglycine at the position equivalent to amino acid 69 in HIV-1 RT. HIV-1 RT has a threonine at this location, while the RTs from Moloney murine leukemia virus and HIV-2 have an asparagine residue at the equivalent position. The Thr-69-*Ile mutation replaces the hydrophilic threonine with thehydrophobic isoleucine and results in a loss of half of the polymerase activity.

The remaining six substitutions are between amino acids 71 and 81. These amino acids lie near the junction of the fingers and palm domains. At the position, equivalent to amino acid 71 of HIV-1 RT, most RTs have a tryptophan residue. The RTs of Rous sarcomna virus and Moloney murine leukemia virus both have a tyrosine at theequivalent position. The HIV-1 RT mutant Trp-71 -Tyr has a slight decreasein polymerase activity, indicating that a tryptophan residue is preferred to tyrosine at this position. The amino acid residues at positions 72, 76, 78, and 81 are all well conserved in retroviral RTs. The individual mutations Arg-72-*Lys, Asp-76-+Glu, and Arg-78--*Lys each result in significant decreases in polymerase activity, with littleorno measurable effect on the RNase H activity. The Asn-81- Gln mutant shows a significant reduction in both poly-merase and RNase H activities.

This research was sponsored in part by the National Cancer Institute under contract NO1-CO-74101 with ABL and by the National Institute of GeneralMedical Sciences.

We aregrateful to our colleagues, in particular Edward Arnold and Amnon Hizi forhelpful discussions, Marilyn Powers for the oligonucleotides, and HildaMarusiodisforpreparation of the manu-script.

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