Human Immunodeficiency Virus Type 1 Can Use Different tRNAs as

JOURNAL OF VIROLOGY, Oct. 1995, p. 6021–6029 Vol. 69, No. 10

0022-538X/95/$04.0010

Human Immunodeﬁciency Virus Type 1 Can Use Different tRNAs as

Primers for Reverse Transcription but Selectively Maintains

a Primer Binding Site Complementary to tRNA

Lys

JOHN K. WAKEFIELD, ANDREA G. WOLF, AND CASEY D. MORROW*

Department of Microbiology, University of Alabama at Birmingham, Birmingham, Alabama 35294

Received 27 March 1995/Accepted 22 June 1995

The initiation of human immunodeﬁciency virus type 1 (HIV-1) reverse transcription occurs at a site in the

viral RNA genome which is designated the primer-binding site (PBS). The HIV-1 PBS is an 18-nucleotide

sequence that is complementary to the 3*-terminal 18 nucleotides of tRNA

Lys

, which is used as the primer for

reverse transcription. All HIV-1 isolates sequenced to date contain a PBS complementary to tRNA

Lys

, sug-

gesting that other cellular tRNAs might not function as primers for reverse transcription. To investigate this

possibility, we have substituted the HIV-1 PBS with sequences predicted to be complementary to the 3*-

terminal nucleotides of tRNA

1,2

Lys

, tRNA

Ile

, and tRNA

His

, which previous studies have identiﬁed to be packaged

into HIV-1 virions along with tRNA

Lys

. We demonstrate that infectious viruses which utilized tRNA

1,2

Lys

, tRNA

Ile

and tRNA

His

in reverse transcription can be recovered. However, the appearances of viruses with PBSs

complementary to these alternate tRNAs were delayed compared with the wild type. After extended in vitro

culture, viruses containing the PBSs complementary to these different tRNAs reverted back to the wild-type

PBS complementary to tRNA

Lys

. Furthermore, only the ﬁrst 9 nucleotides of the 18 nucleotide PBSs were

sufﬁcient for HIV-1 to utilize the alternate tRNA primers in reverse transcription, demonstrating that HIV-1

does not require the complete 18-nucleotide PBS to utilize these tRNA primers for reverse transcription. These

results suggest that factors other than complementarity between the PBS and the primer tRNA contribute to

the selectivity of tRNA

Lys

to initiate HIV-1 reverse transcription.

A hallmark of retrovirus replication is the conversion of the

single-stranded RNA genome into a double-stranded DNA

proviral intermediate. This process, which is referred to as

reverse transcription, is carried out by an RNA-dependent

DNA polymerase termed reverse transcriptase (RT) which is

encoded by the viral pol gene (4, 41). Retroviruses use a cel-

lular tRNA as the primer for the initiation of reverse transcrip-

tion. A sequence in the viral RNA genome designated the

primer-binding site (PBS) is complementary to the 39-terminal

18 nucleotides of the primer tRNA molecule. The RT initiates

DNA synthesis by extension of the 39-OH of the tRNA mole-

cule which is bound to the PBS (38, 42).

Retroviral virions contain all of the necessary viral proteins

(e.g., RT) as well as the cellular tRNA primer used for reverse

transcription of the viral genome. The tRNA primer used for

reverse transcription is believed to be incorporated into the

virion during assembly and positioned on the PBS. Although

retroviruses differ with respect to the tRNA primer used to

initiate reverse transcription, the primer tRNA utilized by a

given group of retroviruses is highly conserved. For example,

all human immunodeﬁciency viruses (HIV) use tRNA

Lys

initiate reverse transcription. However, the type and relative

abundance of tRNA species found in retroviral virions have

been shown to vary greatly, depending on the cell type from

which the virion was obtained (14, 15, 17, 19, 20, 27, 28, 34).

Analysis of HIV type 1 (HIV-1) virions obtained from trans-

fection of proviral genomes into COS cells revealed that

tRNA

1,2

Lys

and tRNA

Ile

were present at levels comparable to

those of tRNA

Lys

(19). The same study also indicated that

tRNA

His

was present within HIV-1 virions expressed from

chronically infected Vero cells.

During reverse transcription, the PBS is regenerated when

the RT copies the ﬁrst 18 nucleotides of the still-attached

tRNA primer during plus-strand strong-stop DNA synthesis

(16, 39, 40). The sequence of the PBS, then, reﬂects what

tRNA was used to initiate reverse transcription. Since a dis-

tinguishing feature of all HIV-1 proviruses sequenced to date

is that the PBSs are complementary to tRNA

Lys

, the initiation

of HIV-1 reverse transcription would be predicted to selec-

tively use only tRNA

Lys

, even though other tRNAs are present

in the virions at relatively high levels such as tRNA

1,2

Lys

, which

also closely resembles tRNA

Lys

in primary structure (29). To

directly test the possibility that various tRNAs present in

HIV-1 virions can be used in reverse transcription, we have

constructed proviral genomes in which the wild-type PBS

complementary to tRNA

Lys

was mutated to be complemen-

tary to the 39-terminal nucleotides of tRNA

1,2

Lys

, tRNA

Ile

, and

tRNA

His

. The proviral genomes containing the mutant PBSs

were transfected into COS-1 cells to generate mutant viruses,

which were used to infect SupT1 cells. The results provide new

insights into HIV-1 reverse transcription by demonstrating that

although these alternative tRNAs can be used in initiation,

HIV-1 has evolved to selectively use tRNA

Lys

to initiate re-

verse transcription and maintain a PBS complementary to

tRNA

Lys

MATERIALS AND METHODS

Materials. Unless otherwise speciﬁed, all chemicals were purchased from

Sigma Chemical Co. Restriction endonucleases and DNA modiﬁcation enzymes

were obtained from New England Biolabs. The Taq polymerase was purchased

from BRL-Gibco, and the reagents for PCR were obtained from Perkin-Elmer

Cetus. Radioactive nucleotides and the reagents for enhanced chemilumines-

cence were purchased from Amersham Corp. [

S]methionine (Translabel) was

obtained from ICN. Tissue culture media and reagents were obtained from

BRL-Gibco. The synthetic DNA oligonucleotides used for mutagenesis and PCR

* Corresponding author. Phone: (205) 934-5705. Fax: (205) 934-

1580.

6021

were prepared by the Cancer Center Oligonucleotide Synthesis Facility at the

University of Alabama at Birmingham.

Tissue culture. COS-1 cells were maintained in Dulbecco’s modiﬁed Eagle’s

medium supplemented with 5% fetal bovine serum and 13 G-MSG (sodium

selenite [0.0067 mg/liter], insulin [10 mg/liter], and transferrin [5.5 mg/liter]) at

378Cina5%CO

atmosphere. SupT1 cells were grown in RPMI supplemented

with 10% fetal bovine serum.

DNA manipulation and mutagenesis. General laboratory procedures were

used for DNA manipulation, plasmid preparation, and subcloning essentially as

described elsewhere (25). Single-stranded DNA of M13mp18PBS used for oli-

gonucleotide site-directed mutagenesis was prepared as described elsewhere

(43). M13mp18PBS contains HIV-1 proviral sequences between the HpaI and

PstI sites of pHXB2gpt including the PBS region (32). The DNA oligonucleotides

used to create the desired mutations in the HIV-1 PBS region were as follows:

(i) pHXB(Ile), CGCTTTCAATCCCCGTACGGGCCACCA CTTCTA; (ii)

pHXB(Lys

1,2

), CGCTTTCAAGCCCCACGTTGGGCGCCACTT; (iii) pHXB

(Ile15), CCCTTTCGCTCCCCTTTCAAAGGACAGCAGGGCCACCA; (iv)

pHXB(Lys

1,2

15), CCTTTCGCGCCCCTTTCAAAGGACAGCATGGGCGC

CA; (v) pHXB(His15), CCTTTCGCATCCGTTTCAAAGGACAGCAACGG

CACCACTTCTAGAG; (vi) pHXB(Gln

15), CCTTTCGCATCCTTTTCAAA

GGACAGCACCCCAGCCACTTCTAGAG. The PBS mutations pHXB(Ile15)

and pHXB(Lys

1,2

15) were constructed by using the appropriate mutagenic oli-

gomers on single-stranded M13mp18 phage DNA containing the Ile [M13mp18

(Ile)] or Lys

1,2

[M13mp18(Lys

1,2

)] mutations, respectively. Screening and subse-

quent identiﬁcation of the M13 plaques containing the desired mutations were

performed by Sanger dideoxy DNA sequencing (36) with Sequenase (U.S. Bio-

chemicals). Each mutant PBS was subcloned into pHXB2gpt, a plasmid encoding

the entire HIV-1 provirus ﬂanked by unique HpaI(59) and XbaI(39) restriction

sites (30, 31). Brieﬂy, replicative-form DNA containing the PBS mutation was

isolated and digested with HpaI and BssHII, resulting in an 868-bp fragment

encompassing the PBS region. The 868-bp fragment was subcloned between the

HpaI and BssHII sites of pHXB2gpt, and the resulting mutant proviral plasmids

were screened by restriction digests and conﬁrmed by direct dideoxy DNA

sequencing (36).

DNA transfections and analysis of viral replication. Transfection of proviral

plasmid DNA into COS-1 cells by using DEAE-dextran was carried out as

previously described (43). To test for viral replication and infectivity, SupT1 cells,

which express CD4 and support high-level replication of HIV-1, were either

cocultured with transfected COS-1 cells or infected with cell-free virus pelleted

from transfected COS-1 supernatants as previously described (43). Following

coculture, the SupT1 cells were removed by low-speed centrifugation (1,000 3 g)

and further cultured with fresh medium and additional SupT1 cells. For cell-free

infections, SupT1 cells (10

cells per ml) were infected with known amounts of

virus as measured by p24 antigen (50 ng/ml). After the virus was allowed to

adsorb for 24 h, SupT1 cells were pelleted (1,000 3 g), washed with phosphate-

buffered saline (pH 7.0), and further cultured for as long as 3 weeks. SupT1

cultures were monitored visually for the formation of multicell syncytia, and, at

various intervals postcoculture or postinfection, supernatants were collected, and

cultures were split and refed with fresh SupT1 cells. Supernatant samples were

ﬁltered through a 0.45-mm-pore-size nitrocellulose syringe ﬁlter (Nalgene) and

analyzed for p24 antigen by enzyme-linked immunosorbent assay (ELISA

[Coulter Laboratories]).

Metabolic labeling of transfected cultures and immunoprecipitation. The

procedures for metabolic labeling of COS-1 cells were as previously described

(43). Brieﬂy, the cells were washed once with Dulbecco’s modiﬁed Eagle’s me-

dium and incubated in methionine-cysteine-free Dulbecco’s modiﬁed Eagle’s

medium for 1 h, and then 100 mCi of [

S]Translabel was added for 2 h. HIV-1

proteins were detected by incubating labeled cellular extracts with pooled sera

from AIDS patients for 24 h, with constant rocking at 48C. Immunoprecipitates

were processed essentially as described elsewhere (43), and the proteins were

resolved by sodium dodecyl sulfate-10% polyacrylamide gel electrophoresis

(SDS-PAGE). Following ﬂuorography with sodium salicylate, the dried gels were

autoradiographed with Kodak X-AR ﬁlm.

Western blot (immunoblot) analysis. Supernatants of COS-1 cells transfected

with either wild-type or mutant proviral genomes were collected 60 h posttrans-

fection and ﬁltered through a 0.45-mm-pore-size syringe ﬁlter. Viral particles

were pelleted through a 20% sucrose cushion by centrifugation for2hat28,000

rpm in an SW41 rotor. Pelleted virions were resuspended in gel sample buffer

and were subjected to SDS-PAGE with SDS-10% polyacrylamide gels. The

proteins were electrophoretically transferred to nitrocellulose membrane for 2 h

at 100 V. Blotted nitrocellulose ﬁlters were blocked for1hatroom temperature

with phosphate-buffered saline (pH 7.0)–0.1% Tween containing 5% nonfat dry

milk and then incubated overnight at 48C with pooled sera from AIDS patients.

Following incubation with primary antibody, the blots were extensively washed

with phosphate-buffered saline (pH 7.0) for 30 min and incubated with a goat-

anti-human secondary antibody conjugated to horseradish peroxidase for2hat

room temperature. The blots were again extensively washed with phosphate-

buffered saline (pH 7.0) and subjected to enhanced chemiluminescence (Amer-

sham) as described by the manufacturer.

PCR ampliﬁcation and sequencing of PBS-containing proviral DNA. Se-

quence analysis of the proviral PBS sequences resulting from infection of SupT1

cells with wild-type and mutant viruses was carried out by a previously described

protocol (43). Brieﬂy, approximately 10 mg of total cellular DNA from infected

SupT1 cultures was subjected to PCR under reaction conditions previously

described (43) to amplify integrated proviral DNA encompassing the PBS. The

HIV-1-speciﬁc oligonucleotide primers used to PCR amplify the PBS region

were as follows: primer 1, 59-GGCTAACTAGGGAACCCACTGC-39 (nucleo-

tides 42 to 63); primer 2, 59-AGTGAATTCCTCCTTCTAGCCTC CGCTAG

TC-39 (nucleotides 309 to 330). (Note that the numbers indicating the nucleotide

sequences which hybridize to the PCR primers are based on the nucleotide

numbering of the pHXB2gpt proviral genome. In addition, the underlined nucle-

otides in primer 2 indicate the additional sequences at the 59 end which corre-

spond to an EcoRI restriction endonuclease site.) PCR-ampliﬁed DNA was

digested with HindIII and EcoRI; the HindIII site is found at nucleotide 77 of the

HIV-1 proviral genome, and the EcoRI site was engineered into primer 2.

Digested PCR products were subcloned between the EcoRI and HindIII sites

present in the polylinker region of M13mp18 replicative-form DNA. Ligation

products were transformed into competent Escherichia coli (DH5aF9) and plated

on a lawn of E. coli (DH5aF9) containing isopropyl-b-

D-thiogalactopyranoside

and 5-bromo-4-chloro-3-indolyl-b-

D-galactoside (X-Gal [U.S. Biochemicals]) as

previously described (43). Single-stranded DNAs prepared from individual recom-

binant phage clones were sequenced by Sanger dideoxy DNA sequencing (36).

RESULTS

Construction of HIV-1 proviral genomes with PBSs comple-

mentary to tRNA

Ile

and tRNA

1,2

Lys

. In a previous study, we

constructed a mutant proviral genome containing a PBS com-

plementary to the 39-terminal 18 nucleotides of a yeast

tRNA

Phe

molecule (43). Although transfection of this provirus

[pHXB2(Phe)] into COS-1 cells resulted in the production of

wild-type levels of virus particles, the virus was noninfectious in

SupT1 cells. Sequence differences in the 39 acceptor stems of

yeast and mammalian tRNA

Phe

species may explain why

HIV-1 was unable to use a PBS complementary to yeast

tRNA

Phe

. Therefore, we wanted to determine if HIV-1 reverse

transcription could be primed by tRNAs known to be present

within the virion. In recent studies, Li et al. demonstrated that

infectious viruses could be recovered with PBSs complemen-

tary to human tRNA

Phe

and tRNA

1,2

Lys

(23). Since tRNA

Ile

has

been found in HIV-1 virions (19), we wanted to determine if

viruses with a PBS complementary to tRNA

Ile

would also be

infectious. To address this possibility, site-directed mutagene-

sis was used to substitute the wild-type PBS with the 18 nucle-

otides predicted to be complementary to the 39-terminal nucle-

otides of human tRNA

Ile

. As a positive control, a proviral

genome containing a PBS complementary to tRNA

1,2

Lys

was also

constructed. The mutant constructs are referred to as

pHXB2(Ile) and pHXB2(Lys

1,2

), respectively, and are pre-

sented in Fig. 1B as they would appear in the viral RNA

genome.

Expression of viral proteins from proviral genomes contain-

ing mutant PBSs. The 59 nontranslated region of the HIV-1

genome has many cis-acting elements such as the transactiva-

tion response region (nucleotides 1 to 59) and the splice-donor

site (nucleotide 289), as well as the putative C encapsidation

signal (nucleotides 300 to 319) (3, 10, 22) (Fig. 1A). Since the

HIV-1 PBS is also located at nucleotides 183 to 200 within the

59 nontranslated region, the substitutions in the PBS may affect

viral gene expression (26). To determine if substitution of the

wild-type PBS complementary to tRNA

Lys

with sequences

complementary to tRNA

Ile

or tRNA

1,2

Lys

affects the expression

of viral proteins, the intracellular expression of viral proteins in

COS-1 cells transfected with these mutant proviral genomes

was analyzed by immunoprecipitation (Fig. 2A). Similar levels

of gag gene products (Pr55

gag

and p24) and env gene product

(gp160) were detected in COS-1 cell lysates transfected with

the various proviral constructs. To determine whether a sub-

stituted PBS affects levels of extracellular particles, superna-

tants of transfected COS-1 cells were analyzed by Western

blotting by using pooled sera from AIDS patients to identify

viral proteins. The virion-associated viral proteins detected in

6022 WAKEFIELD ET AL. J. VIROL.

supernatants from cells transfected with the wild type were

similar to those from cells transfected with proviruses contain-

ing the mutant PBSs (Fig. 2B). Finally, we assayed the super-

natants for soluble p24 antigen by an ELISA. Similar amounts

of p24 antigen were detected in the supernatants from the cells

transfected with the different proviral constructs, indicating

that mutations in the PBS did not affect release of virus par-

ticles (Fig. 2C).

Replication potential of proviral genomes containing sub-

stituted PBS. To determine whether HIV-1 can utilize PBSs

complementary to the 39-terminal 18 nucleotides of tRNA

species different from that of tRNA

Lys

, SupT1 cells were either

cocultured with COS-1 cells transfected with the mutant or

wild type proviral constructs or were infected with equal

amounts of cell-free virus (50 ng of p24 per ml) obtained from

transfected COS-1 supernatants. Samples of culture superna-

tants were collected at various times (postcoculture or postin-

fection), and replication of both the wild-type and mutant

viruses was measured by quantitating the levels of p24 antigen.

Cell-free virus derived from COS-1 cells transfected with

proviral genomes possessing PBSs complementary to either

tRNA

Ile

(Ile) or tRNA

1,2

Lys

(Lys

1,2

) replicated less efﬁciently in

SupT1 cells than the wild-type virus [Lys,3(WT)], with a 3-day

delay in the appearance of wild-type virus in the culture su-

pernatants (Fig. 3). In agreement with previous results, viruses

derived from proviral genomes containing a PBS complemen-

tary to yeast tRNA

Phe

(Phe) failed to replicate (43). The over-

all patterns for the replication kinetics were similar when co-

culture of COS-1 and SupT1 cells was used to initiate virus

infection (data not shown).

Sequence analysis of the PBS from proviruses after ex-

tended in vitro culture. The model of retroviral reverse tran-

scription predicts that the PBS is regenerated during plus-

strand strong-stop viral DNA synthesis. The RT copies the ﬁrst

18 nucleotides of the tRNA used to initiate reverse transcrip-

tion which is still attached to the 59 end of the minus-strand

viral cDNA (16, 39, 40, 42). Therefore, the PBS present in the

proviral form will be complementary to the tRNA species used

to initiate minus-strand synthesis. To determine which tRNA

species was used to initiate reverse transcription, PCR was

used to amplify the PBS region from proviruses derived from

FIG. 1. The 59 nontranslated region of the HIV-1 genome and PBS mutants.

(A) Schematic diagram of the 59 nontranslated region in the HIV-1 genome

showing the locations of relevant cis-acting elements. The PBS is positioned at

nucleotides 183 to 200 within the 59 nontranslated region between the viral U5

and the leader RNA sequences (30, 31). Also located within the 59 nontranslated

region is the transactivation response region (TAR), the splice-donor site (SD),

and one of the putative encapsidation signals (C). (B) Nucleotide sequence of

the wild-type and substituted PBSs. The wild-type HIV-1 PBS, which is comple-

mentary to the 39-terminal 18 nucleotides of a cellular tRNA

Lys

molecule, is

designated pHXB2(WT). Two mutant proviral genomes, designated pHXB2(Ile)

and pHXB2(Lys

1,2

), were constructed such that the wild-type PBS was substi-

tuted with a sequence predicted to be complementary to the 39-terminal 18

nucleotides of either tRNA

Ile

or tRNA

1,2

Lys

, respectively. The nucleotide differ-

ences between the wild-type and mutant PBSs are denoted in the boxes.

FIG. 2. Intracellular expression of viral proteins and analysis of released

virion particles. (A) COS-1 cells were transfected with either the wild type or the

various mutant proviral genomes and were metabolically labeled 24 h posttrans-

fection with [

S]Translabel for 2 h, and then HIV-1-speciﬁc proteins were

immunoprecipitated with pooled sera from AIDS patients. The immunoprecipi-

tated proteins were analyzed by separation on an SDS-10% polyacrylamide gel,

which was followed by ﬂuorography and autoradiography. Lanes: 1, mock trans-

fected; 2, pHXB2(WT); 3, pHXB2(Ile); 4, pHXB2(Lys

1,2

); 5, pHXB2PBS(phe).

The molecular mass markers and the relevant HIV-1 proteins are noted. (B)

Western blot analysis of virions released from transfected COS-1 cells. Super-

natants of COS-1 cells transfected with the indicated proviral DNAs were ﬁltered

through a 0.45-mm-pore-size syringe ﬁlter, and the virus particles were pelleted

through a 20% sucrose cushion by centrifugation in a SW41 rotor at 28,000 rpm

for 2 h. The viral pellets were resuspended in gel sample buffer, electrophoresed

through an SDS-10% polyacrylamide gel, and electrophoretically transferred to

nitrocellulose membrane for Western blotting. Lanes: 1, mock transfected; 2,

pHXB2(WT); 3, pHXB2(Ile); 4, pHXB2(Lys

1,2

); 5, pHXB2PBS(phe). The mo-

lecular mass markers and the relevant HIV-1 proteins are noted. (C) Superna-

tants of COS-1 cells transfected with wild-type (Lys,3) or proviral genomes

containing PBSs complementary to tRNA

Ile

(Ile), tRNA

Lys1,2

(Lys

1,2

), or

tRNA

Phe

(Phe). At 48 h posttransfection, the culture supernatants were analyzed

for released virus particles by quantitation of p24 antigen (Coulter). The bar

graph illustrates the mean values of p24 antigen detected from six independent

transfections, with standard deviations represented by error bars.

VOL. 69, 1995 PBS-tRNA INTERACTION IN HIV-1 REVERSE TRANSCRIPTION 6023

infection of viruses containing PBS substitutions. The proviral

PCR products were subcloned into M13 replicative-form

DNA, and the PBS region contained in the individual recom-

binant M13 phage clones was sequenced. Table 1 shows the

PBS sequences of proviruses derived from infected SupT1 cells

at two different time points: early (day 4) and late (day 8)

postinfection. At day 4 postinfection, all of the proviruses se-

quenced maintained PBSs that were complementary either to

tRNA

Ile

or tRNA

1,2

Lys

, but not the wild-type sequence. However,

at day 8 postinfection, all but one of the proviruses sequenced

possessed a wild-type PBS complementary to tRNA

Lys

. These

results suggest that HIV-1 can utilize alternate tRNA primers

to initiate reverse transcription if a PBS complementary to that

particular tRNA species is present in the viral RNA genome.

Utilization of alternate tRNA primers for HIV-1 reverse

transcription does not require complete PBS. In previous stud-

ies, we and others have demonstrated that HIV-1 does not

require a complete 18-nucleotide PBS to initiate reverse tran-

scription (13, 23, 32, 43). We have exploited this feature to

demonstrate that only the ﬁrst six nucleotides of the PBS are

required to initiate reverse transcription (32). In the present

study, we have constructed proviral genomes in which the ﬁrst

nine nucleotides correspond to alternate tRNAs. To distin-

guish between the PBS sequence present in the input genomes

from the PBS sequences of proviruses derived from reverse

transcription, mutant proviral genomes were constructed in

which the PBS region was altered so that proviruses which

were generated as a result of reverse transcription would pos-

sess a 6-bp deletion 39 to the PBS as depicted in Fig. 4. These

constructs are based on our previous results which demon-

strated that an insertion downstream of a mutated PBS of ﬁve

nucleotides identical to the last ﬁve of the wild-type PBS could

be used to facilitate the second template transfer event of

reverse transcription (43). As a consequence, complementary

binding of these ﬁve nucleotides, which are deleted in the

mutated form of the PBS, with the last ﬁve nucleotides of the

plus strand PBS sequence would result in a deletion of the

intervening six nucleotides. For the present studies, ﬁve nucle-

otides corresponding to the last ﬁve of either the Ile or Lys

1,2

PBS were inserted 39 of a mutated form of these PBSs, respec-

tively. The ﬁrst 9 nucleotides of the mutated form of the PBSs

are complementary to either tRNA

1,2

Lys

or tRNA

Ile

, while the

last 9 of the 18 nucleotides have been substituted with non-

complementary nucleotides (Fig. 5). Therefore, if either a

tRNA

Ile

or tRNA

1,2

Lys

initiates reverse transcription, in addition

to regenerating the authentic 18-nucleotide Ile or Lys

1,2

PBS,

a 6-bp deletion 39 of this PBS would be present in the inte-

grated proviruses. The advantage of this strategy is that it

FIG. 3. Kinetics of replication of viruses containing substituted PBSs. Equal

amounts of virus (approximately 50 ng/ml) isolated from COS-1 cells transfected

with either pHXB2(WT) [Lys,3(WT)], pHXB2(Ile) (Ile), pHXB2(Lys

1,2

) (Lys

1,2

), or

pHXB2PBS(phe) (Phe) were used to infect 10

SupT1 cells in RPMI medium

containing 10% fetal bovine serum. At 24 h postinfection, the virus-containing

medium was removed by low-speed centrifugation (1,000 3 g) and the SupT1

cells were washed once in phosphate-buffered salne (pH 7.0), which was followed

by further culturing in fresh medium and new SupT1 cells. Samples of culture

supernatants were removed at speciﬁed times postinfection and assayed for

soluble p24 antigen. The origins of the samples obtained for infection of SupT1

cells with wild-type and mutant viruses are as marked.

TABLE 1. Sequence analysis of the proviral PBSs derived from pHXB2(Ile) and pHXB2(Lys

1,2

)

Provirus sample

PBS sequence Frequency

Input sequence

TGG TGG CCC GTA CGG GGA TTG AAA

(Ile)

vHXB(Ile) (day 4 postinfection) TGG TGG CCC GTA CGG GGA TTG AAA

8/8

(Ile)

vHXB(Ile) (day 8 postinfection) TGG CGC CCG AAC AGG GAC z

TG AAA 5/5

(Lys,3)

Input sequence

TGG CGC CCA ACG TGG GGC TTG AAA

(Lys

1,2

)

vHXB2(Lys

1,2

) (day 4 postinfection) TGG CGC CCA ACG TGG GGC TTG AAA 9/9

(Lys

1,2

)

vHXB2(Lys

1,2

) (day 8 postinfection) TGG CGC CCG AAC AGG GAC TTG AAA 8/10

(Lys,3)

TGG CGC CCG AAC AGG GAC C

TG AAA 1/10

(Lys,3)

TGG CGC CCA ACG TGG GGC TTG AAA

1/10

(Lys

1,2

)

PBS regions PCR ampliﬁed and sequenced from genomic DNAs isolated from SupT1 cells infected with viruses derived from transfection of the corresponding

plasmids.

Frequencies of the DNA sequences of the PBS region obtained from independent M13 phage clones.

The input sequence refers to the initial mutation in the PBS region.

Nucleotide deletion.

Nucleotide difference from original proviral sequence.

6024 WAKEFIELD ET AL. J. VIROL.

allows identiﬁcation of integrated proviral genomes which have

been generated as a result of reverse transcription (Fig. 5). To

extend our analysis, two additional mutant HIV-1 proviral ge-

nomes were constructed which contained PBSs predicted to

be complementary to mammalian tRNA

His

and an E. coli

tRNA

Gln

, which are designated pHXB2(His15) and pHXB2

(Gln

15), respectively (Fig. 5). We were especially interested

in the viral genomes with PBS complementary to tRNA

His

since this tRNA has been identiﬁed in viruses from chronically

infected Vero cells (19). E. coli tRNA

Gln

has been shown in

vitro to be used by the HIV-1 RT to prime DNA synthesis (21).

However, we anticipate, that a proviral genome with a PBS

complementary to E. coli tRNA

Gln

would be noninfectious

because of the sequence differences in the 39 acceptor stem

between mammalian and E. coli tRNA

Gln

In preliminary experiments, we again found that these mu-

tations in the PBS did not affect the intracellular expression of

the viral proteins. The amounts of p24 antigen in the superna-

tants of COS-1 cells transfected with the proviruses containing

the mutant PBSs were similar to amounts found in COS-1 cells

transfected with the wild-type genome (data not shown). Virus

infection was assayed following coculture with SupT1 cells by

measuring soluble p24 antigen levels in the supernatant. Al-

though there was a delay compared with the wild type, infec-

tious virus was recovered following transfection of pHXB2

(Ile15) and pHXB2(Lys

1,2

15). Interestingly, infectious virus

was seen from the transfection of pHXB2(His15), although its

appearance was considerably delayed compared with the oth-

ers. Infectious virus was not recovered from transfection of

pHXB2(Gln

15), even after prolonged in vitro culture of as

long as 3 months (Fig. 6).

To determine the status of the PBSs of the viruses derived

from transfection of pHXB2(Ile15), pHXB2(Lys

1,2

15), and

pHXB2(His15), PCR was used to amplify the PBS regions

from integrated proviruses. Sequence analysis of the PBS re-

gions derived from Ile15 viruses early in postcoculture (day 5)

revealed that in 50% (8 of 16) of the recombinant M13 clones

sequenced, a PBS complementary to tRNA

Ile

was regenerated

with a 6-bp deletion 39 of the PBS (Table 2). When proviral

FIG. 4. Model for the second template transfer of Ile15 reverse transcrip-

tion and the predicted sequence of the proviral PBS region. (A) Steps involved

in the second template transfer event of reverse transcription (16, 40, 42).

Plus-strand strong-stop viral DNA synthesis (thick line) initiates from an RNA

primer that remains following incomplete digestion by the viral RNase H activity

at the polypurine tract located just upstream of the U3 region in the viral

genome. DNA synthesis continues until the RT copies the ﬁrst 18 nucleotides of

the still-attached primer tRNA used to initiate minus-strand cDNA synthesis,

regenerating the plus-strand copy of the PBS (denoted by the uppercase PBS).

Removal of the primer tRNA by the RNase H activity allows the plus-strand PBS

to bind with the minus-strand copy of the viral PBS (lowercase pbs). Binding of

the plus- and minus-strand PBSs (illustrated in box) facilitates the template

transfer of the plus-strand strong-stop DNA to the 39 end of the minus-strand

cDNA (thin line), which is required for completion of full-length proviral DNA.

U3, R, and U5, long terminal repeat. (B) Predicted base pairings of the plus- and

minus-strand PBSs during the initial reverse transcription reaction of viral ge-

nomes containing an Ile15 PBS. When tRNA

Ile

is used to initiate HIV-1 reverse

transcription, the plus-strand copy of the PBS will be complementary to the

39-terminal 18 nucleotides of tRNA

Ile

. It is important to note that during the

initial reverse transcription reaction, the minus-strand PBS will contain the

complementary sequences of the mutated Ile15 PBS as well as the 5-nucleotide

insertion corresponding to the last 5 nucleotides of the full-length PBS comple-

mentary to tRNA

Ile

, which are located downstream of this PBS (see Fig. 5).

Although the ﬁrst 9 nucleotides of the plus- and minus-strand PBSs form base

pairs in a complementary manner, similar complementarity does not exist for the

last 9. However, insertion of the 5 nucleotides (which are a cccct in the minus

strand) provides the complementary sequences which can base pair with the last

5 nucleotides of the plus-strand PBS, allowing elongation of the plus strand by

the RT to continue. Utilization of the 5 nucleotides to facilitate the second template

transfer will cause a looping out of the intervening nucleotides including the 6

nucleotides (aacttt) between the mutated Ile15 PBS and the 5-nucleotide insertion.

Previous studies from this laboratory have validated this aspect of the model (43).

The non-base paired nucleotides will be removed by the host cell DNA strand repair

machinery after the full-length proviral DNA integrates into the host chromosome.

scription host cell DNA strand repair. Repair of the minus strand will result in a

provirus containing an authentic 18-nucleotide Ile PBS along with a deletion of the

adjacent 6 nucleotides (TTGAAA) 39 of the regenerated PBS.

FIG. 5. Depiction of the PBS sequences with 39 nucleotide insertions. The

four mutated PBS sequences found in the proviral constructs are depicted. The

ﬁrst 9 nucleotides of the PBSs are complementary to 39-terminal 9 nucleotides of

the corresponding primer tRNAs (shown in boxes). The second 9 nucleotides are

designed such that they are not complementary to the primer tRNAs. The arrows

indicate the insertion of the 5 nucleotides, which correspond to the last 5 nucle-

otides of the authentic substituted PBS, that are positioned 6 bp downstream

from the end of the PBS (CCU). Upon successful reverse transcription, the

entire PBS will be complementary to the 39-terminal 18 nucleotides of the

initiating tRNA as well as the 6-nucleotide deletion. PBS sequences complemen-

tary to the 39-terminal 18 nucleotides of tRNA

His

and tRNA

Gln

are as follows:

59-UGG UGC CGU GAC UCG GAU (His) and 59-UGG CUG GGG UAC

GAG GAU (Gln

), respectively.

VOL. 69, 1995 PBS-tRNA INTERACTION IN HIV-1 REVERSE TRANSCRIPTION 6025

PBSs that resulted from the reverse transcription of proviral

genomes containing a Lys

1,2

15 PBS were sequenced, 8 of 14

proviruses had a PBS complementary to tRNA

1,2

Lys

along with a

6-bp deletion 39 of the PBS (Table 3). Taken together, these

results demonstrate that both tRNA

Ile

and tRNA

1,2

Lys

were used

as primers to initiate HIV-1 reverse transcription, since regen-

eration of a complete 18-nucleotide PBS complementary to

tRNA

Ile

or tRNA

1,2

Lys

requires elongation from these alternate

species. Even with the original PBS complementary to tRNA

Ile

or tRNA

1,2

Lys

, the virus was able to utilize tRNA

Lys

for initiation

of reverse transcription, as evidenced by the isolation of PBS

complementary to tRNA

Lys

, which also contained the 6-bp

deletion. Analysis of the proviruses after 10 and 12 days of in

vitro virus culture revealed that the viruses containing the PBS

complementary to tRNA

Lys

were predominant, indicating that

the viruses containing the PBS complementary to tRNA

Lys

had

an advantage in replication over viruses containing the PBS

complementary to tRNA

1,2

Lys

or tRNA

Ile

The virus derived from transfection of cells with pHXB2

(His15) exhibited a strikingly different infection proﬁle. Early

in culture (prior to 25 days), the levels of virus were undetect-

able by the p24 antigen capture assay (Fig. 6). At day 30 of in

vitro culture, the level of p24 antigen in the culture was higher

than the background (uninfected SupT1 cells). Surprisingly,

PCR ampliﬁcation and DNA sequencing of the PBS revealed

that a complete 18-nucleotide PBS complementary to the 39-

terminal 18 nucleotides of tRNA

His

, along with a 6-nucleotide

deletion adjacent 39 to the PBS, was present at a higher fre-

quency than the PBS complementary to tRNA

Lys

(Table 4).

The results of this analysis clearly demonstrate that HIV-1 can

utilize tRNA

His

in reverse transcription. By day 37 of culture,

the levels of p24 antigen had increased substantially; PCR

ampliﬁcation and sequence analysis revealed that the PBSs of

the viruses were now complementary to tRNA

Lys

. The initial

delayed appearance and slow virus growth appeared to be the

result of the PBS being complementary to tRNA

His

. Once

tRNA

Lys

was utilized to initiate reverse transcription from the

PBS complementary to tRNA

His

, the revertant would contain

a PBS complementary to tRNA

Lys

(generated from reverse

transcription) resulting in a virus with wild-type replication

kinetics.

DISCUSSION

In this study, we have determined whether changing the PBS

of HIV-1 can result in viruses utilizing different tRNAs in

reverse transcription. Proviral genomes were initially con-

structed which substituted the wild-type PBS with sequences

complementary to the 39-terminal 18 nucleotides of either

tRNA

Ile

or tRNA

1,2

Lys

, which are known to be present within

virions expressed from COS cells (19). Transfection into

COS-1 cells of these proviral genomes [pHXB(Ile) and pHX-

B(Lys

1,2

), respectively] resulted in the production of infectious

virus competent to replicate in SupT1 cells. DNA sequence

analysis of the PBS region PCR ampliﬁed from integrated

proviruses revealed that the mutant PBSs were maintained

early in infection, suggesting the use of tRNA

Ile

and tRNA

1,2

Lys

in HIV-1 reverse transcription. To conﬁrm that alternate

tRNAs could substitute for tRNA

Lys

, proviral genomes were

constructed which contained a mutated form of PBSs comple-

mentary to tRNA

Ile

, tRNA

1,2

Lys

, tRNA

His

, or a tRNA

Gln

mole-

cule. These proviral genomes were constructed such that if the

speciﬁc tRNA had been used to prime initiation from these

PBSs, in addition to regenerating the entire 18-nucleotide PBS

complementary to the initiating tRNA, a 6-nucleotide deletion

39 of the regenerated PBS would occur. Full-length PBSs com-

FIG. 6. Kinetics of the appearance of infectious virus. Proviral genomes

containing mutant PBSs were transfected into COS-1 cells, which was followed

24 h later by coculture with SupT1 cells (5 3 10

). After 48 h of coculturing, the

SupT1 cells were isolated by low-speed centrifugation and were further cultured

with fresh SupT1 and media (day 0). At designated times postcoculture, samples

of supernatants were collected and analyzed for p24 antigen. The origins of the

samples are as marked and refer to the initial mutations in the PBS.

TABLE 2. Sequence analysis of the PBSs of HIV-1 proviruses derived from pHXB2(Ile15)

Provirus sample PBS sequence

Frequency

Input sequence (Ile15)

TGG TGG CCC TGC TGT CCT TTG AAA GGGGA GCG AAA GGG

vHXB(Ile15) (day 6 postcoculture) TGG CGC CCG AAC AGG GAC ... ... .CG AAA GGG GAT CCA 8/16

(Lys,3)

TGG TGG CCC GTA CGG GGA ... ... GCG AAA GGG GAT CCA 8/16

(Ile)

vHXB(Ile15) (day 10 postcoculture) TGG CGC CCG AAC AGG GAC ... ... .CG AAA GGG GAT CCA 12/14

(Lys,3)

TGG TGG CCC GTA CGG GGA ... ... GCG AAA GGG GAT CCA 2/14

(Ile)

Sequences of the PBS regions PCR ampliﬁed from genomic DNAs isolated from SupT1 cells infected with viruses derived from pHXB2(Ile15). Note that the PBS

sequence located in the square represents the ﬁrst nine nucleotides that are complementary to the 39-terminal nine nucleotides of tRNA

Ile

. The ﬁve nucleotides inserted

39 of the mutated PBS are underlined. Dots reﬂect the numbers and positions of the deleted nucleotides.

Frequencies of the DNA sequences of the PBS region obtained from independent M13 phage clones.

The input sequence refers to the initial mutation in the PBS region of pHXB2(Ile15).

6026 WAKEFIELD ET AL. J. VIROL.

plementary to either tRNA

Ile

, tRNA

1,2

Lys

, or tRNA

His

accompa-

nied by the 6-bp deletion were detected in the proviral ge-

nomes, conﬁrming the use of these alternate tRNAs in the

initiation of HIV-1 reverse transcription. After extended cul-

ture, viruses were detected with wild-type PBS sequences com-

plementary to tRNA

Lys

The results of this study clearly demonstrate that HIV-1 can

utilize tRNA primers other than tRNA

Lys

in reverse transcrip-

tion. In one recent study, Li et al. mutated the PBS of HIV-1

such that it was complementary to that of human tRNA

1,2

Lys

tRNA

Phe

(23). Transfection of the proviral genomes with these

mutant PBSs gave rise to infectious virus with delayed kinetics

of appearance compared with those of the wild type. We have

extended their results by analyzing PBSs complementary to

additional tRNAs, tRNA

Ile

, and tRNA

His

, which have been

found to be present within HIV-1 virions (19). Viruses with

PBSs complementary to tRNA

1,2

Lys

, tRNA

Ile

, and tRNA

His

were

infectious. At early times postinfection, low levels of p24 an-

tigen in the culture supernatant for the cultures infected with

viruses with the substituted PBSs were seen. The cultures did

not have the giant, multinucleated cells (syncytia) characteris-

tic of wild-type infection of SupT1 cells. Sequence analysis of

the proviral PBS region from high-molecular-weight DNA at

this time revealed that the PBSs were complementary to the

substitute primer (i.e., tRNA

1,2

Lys

, tRNA

Ile

, or tRNA

His

). In our

experimental design, we also made use of the fact that comple-

mentarity between the plus- and minus-strand copies of the

PBS sequence is required for transfer of plus-strand strong-

stop DNA to complete the synthesis of the full-length double-

stranded provirus. By placing a 5-nucleotide sequence comple-

mentary to the last ﬁve nucleotides of the plus-strand PBS, we

can facilitate this step in reverse transcription (43) (Fig. 4).

Following reverse transcription and integration, the proviruses

will contain a complete PBS complementary to the 39-terminal

sequences of the tRNA used to initiate minus-strand synthesis.

The six nucleotides located 39 of the minus-strand PBS, which

are not base paired with the plus-strand PBS, would be re-

moved by the host DNA repair mechanisms. Deletion of the 6

bp 39 to the PBS, then, serves as a genetic marker to identify

only PBSs generated as a result of reverse transcription. One of

the important points of this study was that our results unequiv-

ocally demonstrate that the alternate tRNAs were used to

initiate HIV-1 reverse transcription, since regeneration of the

entire 18-nucleotide PBS sequences complementary to

tRNA

1,2

Lys

, tRNA

Ile

, and tRNA

His

, as well as the accompanying

6-nucleotide deletion, could be explained only by utilization of

these respective tRNAs as primers for reverse transcription.

Another signiﬁcant point is that it is clear that complete

complementarity between the entire 18-nucleotide HIV-1 PBS

and the 39-terminal nucleotides of tRNA

Lys

was not necessary

to initiate reverse transcription. Even with our initial construc-

tions of the pHXB2(Lys

1,2

15), pHXB2(Ile15), and pHXB2

(His15) proviral clones, only the ﬁrst 9 nucleotides of the PBSs

were complementary to tRNA

1,2

Lys

, tRNA

Ile

, and tRNA

His

, re-

spectively. Since the ﬁrst 3 nucleotides of each PBS are the

same (TGG), our results show that the remaining 6 nucle-

TABLE 3. Sequence analysis of the PBSs of HIV-1 proviruses derived from pHXB2(Lys

1,2

15)

Provirus sample PBS sequence

Frequency

Input sequence (Lys

1,2

15)

TGG CGC CCA TGC TGT CCT TTG AAA GGGGC GCG AAA GGG

vHXB(Lys

1,2

15) (day 6 postcoculture) TGG CGC CCG AAC AGG GAC ... ... GCG AAA GGG GAT CCA 6/14

(Lys,3)

TGG CGC CCA ACG TGG GGC ... ... GCG AAA GGG GAT CCA 8/14

(Lys

1,2

)

vHXB(Lys

1,2

15) (day 12 postcoculture) TGG CGC CCG AAC AGG GAC ... ... GCG AAA GGG GAT CCA 14/16

(Lys,3)

TGG CGC CCA ACG TGG GGC ... ... GCG AAA GGG GAT CCA 2/16

(Lys

1,2

)

Sequences of the PBS regions PCR ampliﬁed from genomic DNAs isolated from SupT1 cells infected with viruses derived from pHXB2(Lys

1,2

15). Note that the

PBS sequence located in the square represents the ﬁrst nine nucleotides that are complementary to the 39-terminal nine nucleotides of tRNA

1,2

Lys

. The ﬁve nucleotides

inserted 39 of the mutated PBS are underlined. Dots reﬂect the numbers and positions of the deleted nucleotides.

Frequencies of the DNA sequences of the PBS region obtained from independent M13 phage clones.

The input sequence refers to the initial mutation in the PBS region of pHXB2(Lys

1,2

15).

TABLE 4. Sequence analysis of the PBSs of HIV-1 proviruses derived from pHXB2(His15)

Provirus sample PBS sequence

Frequency

Input sequence (His15)

TGG TGC CGT TGC TGT CCT TTG AAA CGGAT GCG AAA GGG

vHXB(His15) (day 30 postcoculture) TGG TGC CGT GAC TCG GAT ... ... GCG AAA GGG GAT CCA 19/24

(His)

TGG CGC CCG AAC AGG GAC ... ... GCG AAA GGG GAT CCA 5/24

(Lys,3)

vHXB(His15) (day 37 postcoculture) TGG CGC CCG AAC AGG GAC ... ... GCG AAA GGG GAT CCA 10/10

(Lys,3)

Sequences of the PBS regions PCR ampliﬁed from genomic DNA isolated from SupT1 cells infected with viruses derived from pHXB2(His15). Note that the PBS

sequence located in the square represents the ﬁrst nine nucleotides that are complementary to the 39-terminal nine nucleotides of tRNA

His

. The ﬁve nucleotide inserted

39 of the mutated PBS are underlined. Dots reﬂect the numbers and positions of the nucleotides deleted.

Frequencies of the DNA sequences of the PBS region obtained from independent M13 phage clones.

The input sequence refers to the initial mutation in the PBS region of pHXB2(His15).

VOL. 69, 1995 PBS-tRNA INTERACTION IN HIV-1 REVERSE TRANSCRIPTION 6027

otides were sufﬁcient for the utilization of these alternate

tRNAs in the initiation of reverse transcription. Studies are

ongoing to deﬁne the minimum number of complementary

nucleotides required for the initiation event.

After extended culture times, we noted a rapid rise in the

levels of p24 in the culture supernatant which correlated with

an increase in syncytia and cytopathicity. Analysis of the PBS

regions from proviruses which started with PBSs complemen-

tary to tRNA

1,2

Lys

, tRNA

Ile

, or tRNA

His

revealed the presence of

the PBS complementary to tRNA

Lys

. This means that for re-

version to wild-type PBS, a tRNA

Lys

must have been used to

prime reverse transcription from the PBSs complementary to

either tRNA

Ile

, tRNA

1,2

Lys

, or tRNA

His

. In order for this to

happen, the 39-terminal 18 nucleotides of tRNA

Lys

must bind

to the different PBSs to initiate reverse transcription. This is in

agreement with earlier studies which have demonstrated that

tRNA

Lys

could prime reverse transcription from PBSs contain-

ing either deletions or nucleotide substitutions (13, 32, 43). It

is possible that infectious proviruses could not be recovered

from proviruses with a PBS complementary to tRNA

Gln

be-

cause there was not sufﬁcient complementarity with tRNA

Lys

That is, including guanine-uridine base pairing (G-U), only

67% (12 of 18) of the nucleotides of the PBS complementary

to tRNA

Gln

are complementary to the 39-terminal nucleotides

of tRNA

Lys

. However, the interaction between tRNA and the

PBS is undoubtedly complex, because the number of comple-

mentary nucleotides between tRNA

Lys

and the PBSs comple-

mentary to tRNA

Ile

and tRNA

His

is only slightly higher (14 of

18 [77%]). It is possible, then, that there are speciﬁc base pair

interactions between the tRNA

Lys

and PBS that are required

for the initiation of reverse transcription. Future studies will be

required to identify which nucleotides in the HIV-1 PBS are

important for the interaction with tRNA

Lys

Finally, reversion of the PBSs back to wild-type PBS pro-

vides an explanation for why only PBSs complementary to

tRNA

Lys

have been found in all of the HIV-1 proviruses iden-

tiﬁed to date. It appears that HIV-1 has evolved such that it

preferentially selects and utilizes tRNA

Lys

in the initiation of

reverse transcription. The reversion to a PBS complementary

to tRNA

Lys

cannot be explained solely by the availability of the

tRNA in the virion. Previous studies have shown that the levels

of tRNA

1,2

Lys

and tRNA

Ile

are relatively high in HIV-1 virions

expressed from COS cells; in fact, the levels of tRNA

1,2

Lys

are

comparable to, if not greater than, those of tRNA

Lys

(19). It is

possible, then, that regions of the HIV-1 viral RNA genome

outside the PBS are important for efﬁcient utilization of alter-

nate tRNAs in reverse transcription. Previous studies of avian

retroviruses suggested that maintenance of stem-loop struc-

tures involving the U5, PBS, and leader sequences was critical

for efﬁcient initiation of reverse transcription (1, 2, 8, 11, 12).

It is also possible that sequences within the tRNA such as the

TCC (1) and the anticodon loops (21) are important for the

proper positioning of the primer tRNA at the PBS. In support

of this idea, a recent study by Isel et al., demonstrated in vitro

that a region located upstream of the PBS, termed the A-rich

loop, interacts with the tRNA

Lys

used in reverse transcription

(18). Selectivity may also be mediated by viral proteins such as

the HIV-1 RT, which has been shown in vitro to speciﬁcally

bind tRNA

Lys

even in the presence of higher levels of compet-

itor tRNAs (5–7, 9, 33, 35, 37, 44). Recent studies have sug-

gested that primer tRNA selection may actually occur in the

context of the Gag-Pol precursor protein (24). Thus, antiviral

agents targeted to prevent interaction of tRNA

Lys

with virion

proteins or disruption of the positioning of tRNA

Lys

at the PBS

will likely signiﬁcantly impair replication. Although it is clear

that the implementation of these strategies will require further

development, the results of the present studies provide a clear

rationale to consider the tRNA-PBS interaction a target of

strategies to inhibit HIV-1 replication.

ACKNOWLEDGMENTS

We thank Beatrice Hahn, Jeff Engler, and Etty Benveniste for help-

ful comments and Dee Martin for preparation of the manuscript.

C.D.M. expresses his thanks to Mark A. Richardson for early encour-

agement.

Culture of HIV was carried out at the UAB AIDS Center Virus

Core Facility (AI-27767). Oligonucleotides were prepared by the DNA

Oligonucleotide Cancer Center Core Facility (NCI-CA13148). The

UAB AIDS Center Molecular Biology Core (AI-27767) provided help

with PCR. J.K.W. was supported by training grant GM-08111. A.G.W.

was supported by training grant AI-07493. This study was supported by

a Public Health Services grant (AI-34749) from the NIH (C.D.M.).

REFERENCES

1. Aiyar, A., D. Cobrinik, Z. Ge, H.-J. Kung, and J. Leis. 1992. Interaction

between retroviral U5 tRNA and the TCC loop of the tRNA

Trp

primer is

required for efﬁcient initiation of reverse transcription. J. Virol. 66:2464–2472.

2. Aiyar, A., Z. Ge, and J. Leis. 1994. A speciﬁc orientation of RNA secondary

structures is required for initiation of reverse transcription. J. Virol. 68:611–

618.

3. Aldovini, A., and R. A. Young. 1990. Mutations of RNA and protein se-

quences involved in human immunodeﬁciency virus type 1 packaging result

in production of noninfectious virus. J. Virol. 64:1920–1926.

4. Baltimore, D. 1970. RNA-dependent DNA polymerase in virions of RNA

tumor viruses. Nature (London) 226:1209–1211.

5. Barat, C., S. F. J. Le Grice, and J.-L. Darlix. 1991. Interaction of HIV-1

reverse transcriptase with a synthetic form of its replication primer,

tRNA

lys,3

. Nucleic Acids Res. 19:751–757.

6. Barat, C., V. Lullien, O. Schatz, G. Heith, M. T. Nugeyre, F. Gruninger-

Leitch, F. Barre-Sinoussi, S. F. J. Le Grice, and J.-L. Darlix. 1989. HIV-1

reverse transcriptase speciﬁcally interacts with the anticodon domain of its

cognate primer tRNA. EMBO J. 8:3279–3285.

7. Barat, C., O. Schatz, S. Le Grice, and J.-L. Darlix. 1993. Analysis of the

interactions of HIV1 replication primer tRNA

lys3

with nucleocapsid protein

and reverse transcriptase. J. Mol. Biol. 231:185–190.

8. Baudin, F., R. Marquet, C. Isel, J.-L. Darlix, B. Ehresmann, and C. Ehres-

mann. 1993. Functional sites in the 59 region of human immunodeﬁciency

virus type 1 RNA form deﬁned structural domains. J. Mol. Biol. 229:382–

397.

9. Bordier, B., L. Tarrago-Litvak, M.-L. Sallafranque-Andreola, D. Robert, D.

Tharaud, M. Fournier, P. J. Barr, S. Litvak, and L. Sarih-Cottin. 1990.

Inhibition of the p66/p51 form of human immunodeﬁciency virus reverse

transcriptase by tRNA

lys

. Nucleic Acids Res. 18:429–436.

10. Cann, A. J., and J. Karn. 1989. Molecular biology of HIV: new insights into

the virus life cycle. AIDS 3(Suppl.):S19–S34.

11. Cobrinik, D., A. Aiyar, Z. Ge, M. Katzman, H. Huang, and J. Leis. 1991.

Overlapping retrovirus U5 sequence elements are required for efﬁcient in-

tegration and initiation of reverse transcription. J. Virol. 65:3864–3872.

12. Cobrinik, D., L. Soskey, and J. Leis. 1988. A retroviral RNA secondary

structure required for efﬁcient initiation of reverse transcription. J. Virol.

62:3622–3630.

13. Das, A. T., S. E. C. Koken, B. B. Oude-Essink, J. L. B. van Wamel, and B.

Berkhout. 1994. Human immunodeﬁciency virus uses tRNA

Lys,3

as primer

for reverse transcription in HeLa-CD4

cells. FEBS Lett. 341:49–53.

14. Erikson, E., and R. L. Erikson. 1971. Association of 4S RNA with oncorna-

virus RNA. J. Virol. 8:254–256.

15. Faras, A. J., A. C. Gerapin, W. E. Levinson, J. M. Bishop, and H. M.

Goodman. 1973. Characterization of the low-molecular weight RNAs asso-

ciated with the 70S RNA of Rous sarcoma virus. J. Virol. 12:334–342.

16. Gilboa, E., S. W. Mitra, S. Goff, and D. Baltimore. 1979. A detailed model

of reverse transcription and tests of crucial aspects. Cell 18:93–100.

17. Harada, F., G. G. Peters, and J. E. Dahlberg. 1979. The primer tRNA for

Moloney murine leukemia virus DNA synthesis: nucleotide sequence and

aminoacylation of tRNA

Pro

. J. Biol. Chem. 21:10979–10985.

18. Isel, C., R. Marquet, G. Keith, C. Ehresmann, and B. Ehresmann. 1993.

Modiﬁed nucleotides of tRNA

Lys,3

modulate primer/template loop-loop in-

teraction in the initiation complex of HIV-1. J. Biol. Chem. 268:25269–

25272.

19. Jiang, M., J. Mak, A. Ladha, E. Cohen, M. Klein, B. Rovinski, and L.

Kleiman. 1993. Identiﬁcation of tRNAs incorporated into wild-type and

mutant human immunodeﬁciency virus type 1. J. Virol. 67:3246–3253.

20. Jiang, M., J. Mak, M. A. Wainberg, M. A. Parniak, E. Cohen, and L.

Kleiman. 1992. Variable tRNA content in HIV-1

IIIB

. Biochem. Biophys.

Res. Commun. 185:1005–1015.

6028 WAKEFIELD ET AL. J. VIROL.

21. Kohlstaedt, L. A., and T. A. Steitz. 1992. Reverse transcriptase of human

immunodeﬁciency virus can use either human tRNA

Lys,3

or Escherichia coli

tRNA

Gln,2

as a primer in an in vitro primer-utilization assay. Proc. Natl.

Acad. Sci. USA 89:9652–9656.

22. Lever, A., H. Gottlinger, W. Haseltine, and J. Sodroski. 1989. Identiﬁcation

of a sequence required for efﬁcient packaging of human immunodeﬁciency

virus type 1 RNA into virions. J. Virol. 63:4085–4087.

23. Li, X., J. Mak, E. J. Arts, Z. Gu, L. Kleiman, M. A. Wainberg, and M. A.

Parniak. 1994. Effects of alterations of primer-binding site sequences on

human immunodeﬁciency virus type 1 replication. J. Virol. 68:6198–6206.

24. Mak, J., M. Jiang, M. A. Wainberg, M.-L. Hammarskjo¨ld, D. Rekosh, and L.

Kleiman. 1994. Role of Pr160

gag-pol

in mediating the selective incorporation

of tRNA

Lys

into human immunodeﬁciency virus type 1 particles. J. Virol.

68:2065–2072.

25. Maniatis, T., E. F. Fritsch, and J. Sambrook. 1982. Molecular cloning: a

laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor,

N.Y.

26. Nagashunmugam, T., A. Velpandi, C. S. Goldsmith, S. R. Zaki, V. S. Kaly-

anaraman, and A. Srinivasan. 1992. Mutations in the primer binding site of

the type 1 human immunodeﬁciency virus genome affects virus production

and infectivity. Proc. Natl. Acad. Sci. USA 89:4114–4118.

27. Peters, G., F. Harada, J. E. Dahlberg, A. Panet, W. A. Haseltine, and D.

Baltimore. 1977. Low-molecular-weight RNAs of Moloney murine leukemia

virus: identiﬁcation of the primer for RNA-directed DNA synthesis. J. Virol.

21:1031–1041.

28. Peters, G. G., and C. Glover. 1980. tRNAs and priming of RNA-directed

DNA synthesis in mouse mammary tumor virus. J. Virol. 35:31–40.

29. Raba, M., K. Limburg, M. Burghagen, J. R. Katze, M. Simsek, J. E. Heck-

man, U. L. Rajbhandary, and H. J. Gross. 1979. Nucleotide sequence of

three isoaccepting lysine tRNAs from rabbit liver and SV40-transformed

mouse ﬁbroblasts. Eur. J. Biochem. 97:305–318.

30. Ratner, L., A. Fisher, L. Linda, J. Agodzinski, H. Mitsuya, R.-S. Liou, R. C.

Gallo, and F. Wong-Staal. 1987. Complete nucleotide sequences of func-

tional clones of the AIDS virus. AIDS Res. Hum. Retroviruses 3:57–69.

31. Ratner, L., W. Haseltine, R. Patarca, K. J. Livak, B. Starcich, S. F. Josephs,

E. R. Doran, J. A. Rafalski, E. A. Whitehorn, K. Baumeister, L. Ivanoff, S. R.

Petteway, Jr., M. L. Pearson, J. A. Lautenberger, T. S. Papas, J. Ghrayeb,

N. T. Chang, R. C. Gallo, and F. Wong-Staal. 1985. Complete nucleotide

sequence of the AIDS virus, HTLV-III. Nature (London) 313:277–284.

32. Rhim, H., J. Park, and C. D. Morrow. 1991. Deletions in the tRNA

Lys

primer-binding site of human immunodeﬁciency virus type 1 identify essen-

tial regions for reverse transcription. J. Virol. 65:4555–4564.

33. Robert, D., M.-L. Sallafranque-Andreola, B. Bordier, L. Sarih-Cottin, L.

Tarrago-Litvak, P. V. Graves, P. J. Barr, M. Fournier, and S. Litvak. 1990.

Interactions with tRNA

lys

induce important structural changes in human

immunodeﬁciency virus reverse transcriptase. FEBS Lett. 277:239–242.

34. Rosenthal, L. J., and P. C. Zamecnik. 1973. Amino acid acceptor activity of

the ‘‘70S-associated’’ 4S RNA from avian myeloblastosis virus. Proc. Natl.

Acad. Sci. USA 70:1184–1185.

35. Sallafranque-Andreola, M. L., D. Robert, P. J. Barr, M. Fournier, S. Litvak,

L. Sarih-Cottin, and L. Tarrago-Litvak. 1989. HIV RT expressed in trans-

formed yeast cells. Biochemical properties and interactions with bovine

tRNA

Lys

. Eur. J. Biochem. 184:367–374.

36. Sanger, F., S. Nicklen, and A. R. Coulson. 1977. DNA sequencing with

chain-terminating inhibitors. Proc. Natl. Acad. Sci. USA 74:5463–5467.

37. Sarih-Cottin, L., B. Bordier, K. Musier-Forsyth, M.-L. Andreola, P. J. Barr,

and S. Litvak. 1992. Preferential interaction of human immunodeﬁciency

virus reverse transcriptase with two regions of primer tRNA

lys

as evidenced

by footprinting studies and inhibition with synthetic oligoribonucleotides. J.

Mol. Biol. 226:1–6.

38. Taylor, J. M. 1977. An analysis of the role of tRNA species as primer for

transcription into DNA of RNA tumor virus genomes. Biochim. Biophys.

Acta 473:57–71.

39. Taylor, J. M., and T. W. Hsu. 1980. Reverse transcription of avian sarcoma

virus RNA into DNA might involve copying of the tRNA primer. J. Virol.

33:531–534.

40. Temin, H. 1981. Structure, variation and synthesis of retrovirus long terminal

repeat. Cell 27:1–3.

41. Temin, H. M., and S. Mitzutani. 1970. RNA-directed DNA polymerase in

virions of Rous sarcoma virus. Nature (London) 226:1211–1213.

42. Varmus, H., and R. Swanstrom. 1982. Replication of retroviruses, p. 369–

512. In R. Weiss, N. Teich, H. Varmus, and J. Cofﬁn (ed.), Molecular biology

of tumor viruses: RNA tumor viruses, 2nd ed. Cold Spring Harbor Labora-

tory Press, Cold Spring Harbor, N.Y.

43. Wakeﬁeld, J. K., H. Rhim, and C. D. Morrow. 1994. Minimal sequence

requirements of a functional human immunodeﬁciency virus type 1 primer

binding site. J. Virol. 68:1605–1614.

44. Weiss, S., B. Konig, H.-J. Muller, H. Seidel, and R. S. Goody. 1992. Synthetic

human tRNA

Lys,3

(UUU) and natural bovine tRNA

Lys,3

(SUU) interact with

HIV-1 reverse transcriptase and serve as speciﬁc primers for retroviral

cDNA synthesis. Gene 111:183–197.

VOL. 69, 1995 PBS-tRNA INTERACTION IN HIV-1 REVERSE TRANSCRIPTION 6029