VIROLOGY 206, 387 394 (1995)

E n h a n c e m e n t o f H I V - 1 P r o t e i n a s e A c t i v i t y b y H I V - 1 R e v e r s e T r a n s c r i p t a s e

LAURA GOOBAR-LARSSON, *'1 B. G. MAI-rlAS LUUKKONEN,* TORSTEN UNGE,t STEFAN SCHWARTZ,* GORAN UTTER,* BROR STRANDBERG,1- AND BO OBERG*';~

*Microbiology and Tumorbiology Center (MTC), Karolinska Inst/tute, Box 280, S-171 77 Stockholm, Sweden; f Department of Molecular Biology, BMC, University of Uppsala, S-111 11, Uppsala, Sweden; and #-Medivir AB, S-141 44 Huddinge, Sweden

Received July 14, 1994; accepted October 13, 1994

HIV-1 reverse transcriptase (RT) was found to increase the activity of HIV-1 proteinase in vitro and in eukaryotic cells. The effect of RT on proteinase activity was dose-dependent and independent of pH or salt concentration. The cleavage of sequences corresponding to all the naturally occurring cleavage sites that could be tested in vitro was enhanced. The effect of RT on cleavage was greatest at the cleavage site between RT and integrase. The enhancement of viral proteinase activity by the virus RT may contribute to regulation of the order and/or efficiency of cleavage at different sites during virus replication and maturation. © 1995 Academic Press, Inc.

INTRODUCTION

HIV-1 proteinase is expressed within the Gag-Pol polyprotein (Kramer et a/., 1986), which is the precursor of all viral structural proteins and enzymes, including reverse transcriptase (RT). The proteinase is responsible for the proteolytic processing of this precursor into ma- ture proteins (Graves et aL, 1988; Mous et aL, 1988), a process that leads to the formation of infectious virus particles (Kohl et aL, 1988, G6ttlinger eta/. , 1989; Peng eta/., 1989). The proteinase is active within the Gag-Pol polyprotein since it cleaves itself out from the polyprotein precursor (Debouck et a/., 1987; Giam and Bores, 1988). However, very little is known about the activity and sub- strate specificity of the Gag-Pol precursor form of the proteinase or the effect of other viral proteins on the activity and substrate specificity of the mature form of the proteinase (which is a 22-kDa dimer) (Katoh et aL, 1989; Lapatto eta/., 1989; Meek eta/., 1989; N avia eta/., 1989; Wlodawer eta/. , 1989).

Since RT is expressed within the Gag-Pol polyprotein, it is a substrate of the proteinase, and as such it interacts directly with the enzyme. The proteinase and the RT are the main targets for anti-HIV/AIDS therapy. The interac- tion between the RT and proteinase may therefore be important not only for understanding the process of viral replication and maturation, but also for the development of antiviral drugs.

We have studied the interaction between these two enzymes by testing the effect of HIV-1 RT on HIV-1 pro- teinase activity. Incubation of purified proteinase with purified RT heterodimer efficiently enhanced proteinase

~To whom correspondence and reprint requests should be ad- dressed.

activity in vitro. The effect of RT on proteinase activity was also studied intracellularly, using a vaccinia T7 ex- pression system. Our results show that HIV-1 RT en- hances HIV-1 proteinase activity both in vitro and in cell culture. The characterization of this enhancement is pre- sented and its possible biological function discussed.

M A T E R I A L S A N D M E T H O D S

In vitro HIV-1 proteinase activity

HIV-1 Reverse transcriptase and proteinase were ex- pressed in Escherichia coi l and purified as previously described (Unge et aL, 1990; Bhikhabhai et aL, 1992; Goobar eta/., 1991). HIV-1 RT 216 and RT 235 represent- ing the first 216 and 235 N-terminal amino acids of HIV- 1 RT, respectively, were expressed in E. coi l and purified to homogeneity (Unge et al., 1994; Unge, Bhikhabhai, Osterlund, and L0vgren, manuscript in preparation),

Purified recombinant HIV-1 p24 capsid (CA) protein was from MicroGenSys (Merridan, CT, U.S.A.), and was kindly provided by Dr. F. Volvovitz (MicroGenSys) and Professor B. Wahren (Swedish Institute for Infectious Dis- ease Control). Bovine serum albumin (BSA), fraction V, No. A-4503, was from Sigma Chemical Co. (St. Louis, MO, U.S.A.).

Proteinase activity was measured as previously de- scribed (Goobar et al., 1991) using synthetic octapep- tides with sequences corresponding to the naturally oc- curring cleavage sites. Cleavage products were analyzed and quantified by reverse phase HPLC. HIV-1 proteinase was preincubated for 5 min with or without purified HIV- 1 RT at 37 ° prior to addition of substrate. RT, proteinase, and substrates were diluted in activity buffer (50 mM NaAc, pH 5.5, 10% glycerol, 2 mM Dq-i-, 1 mM EDTA, and

387 0042-6822/95 $6.00 Copyright © 1995 by Academic Press, inc. All rights of reproduction in any form reserved.

388 GOOBAR-LARSSON ET AL.

175 mM NaCI, unless otherwise indicated) and incubated at 37 ° in the same buffer for the times indicated. For measurements of proteinase activity at neutral pH, the activity buffer was 75 mM sodium phosphate, pH 7.0, 10% glycerol, 1 mM D-i-i-, and 1 mM EDTA. Reactions were stopped by addition of an equal volume (50 #1) of 0.2% trifluoroacetic acid followed by heating at 100 ° for 5 min. The proteinase concentration varied between 5 and 130 nM. The substrate used was SQNYPIVQ-NH2, corresponding to the cleavage site between HIV-1 p17 matrix protein (MA) and p24 CA, and its concentration was 0.8 mM, unless otherwise indicated. The hydrolysis of this octapeptide was linear with time up to 50% conver- sion to products.

Inhibition of HIV-1 proteinase activity

HIV-1 proteinase inhibitors U 75875 (Ashorn et aL 1990) and Re 31-8959 (Roberts et aL, 1990) were kindly provided by Dr. W. G. Tarpley (The Upjohn Co.) and Dr. I. B. Duncan (Roche Research Centre), respectively.

Inhibitors were dissolved in dimethyl sulphoxide (DMSO) and dilutions were made in activity buffer con- taining 1 M NaCI. The final DMSO concentration in the samples was 0.5%. Samples without inhibitors were ad- justed to the same DMSO concentration. Purified HIV-1 proteinase was preincubated with or without purified HIV-1 RT and inhibitors for 5 min at 37 ° and reactions were started by the addition of the substrate peptide SQNYPIVQ-NH2. Samples were incubated for 2 hr at 37 ° and reactions were stopped as described above. The reaction products were analyzed and quantified by re- versed phase HPLC as previously described (Goobar et aL, 1991).

Peptide synthesis

All peptides were synthesised using t-Boc amino acids (Bachem, Bubendorf, Switzerland) and p-methylbenzhy- drylamine resin (Fluka, Buchs, Switzerland) according to the multiple solid-phase peptide synthesis method (Houghten, 1985). Removal of protecting groups from the formyl-tryptophane and methionine sulfoxide residues was achieved by cleavage with 25% hydrogen fluoride (Tam et aL, 1983). The peptides were then cleaved from the resin with liquid hydrogen fluoride using a multives- sel apparatus (Houghten et aL, 1986). All peptides were approximately 90% pure.

Plasmid constructs

To generate the HIV-1 proteinase expressing plasmid pT7-PR, the proteinase coding sequence was PCR-ampli- fled from the HIV-1 clone pHXB2 using the oligonucleo- tides, PRATGBSS (5'-CGCGCG-CGAAATGCCTCAGATC- ACTC-FI-i-GG-3'), which introduces a translational start codon, and PRSTOP (5'-TCACTAAAAATTTAAAGTGCA- GCCAATC-3'), which introduces two translational stop

codons. The PCR product was blunt-end ligated to an EcoRV-digested and ClAP-treated pBluescript (KS-) plasmid (Stratagene, La Jolla, CA, U.S.A.).

The HIV-1 proteinase-RT producing plasmid pT7-PR- RT, was generated by POR-amplification of the protein- ase and RT coding sequences of pHXB2 using the oligo- nucleotides PRATGBSS and 6702 (5'-GGATCCCTATAG- TAT-FFFCCTGATFCCAGCACTGAC-3'). The PCR product was blunt-end ligated to the EcoRV-digested and ClAP- treated pBluescript (KS-) plasmid. The p55 Gag produc- ing plasmidpT7-55 Gag was constructed by POR-ampli- fying the Gag region of pHXB2 using oligonucleotides 2311 (5'-GGCTTGCTGAAGCGCGCACGGCAAGAGG-3') and 7492 (5'-TTA-I-FGTGACGAGGGGTCGTTGCC-3'). The PCR product was blunt-end ligated into the EcoRV-di- gested and ClAP-treated pBluescript (KS-) plasmid.

Intracellular HIV.-1 proteinase activity

All experiments were carried out in HeLa cells grown in Dulbecco's modified Eagle's medium (Gibco) supple- mented with 10% fetal calf serum (Flow Laboratories, Scotland, LIK), 5.3 mM L-glutamine (Flow Laboratories, Scotland, UK), and 0.1 mg/ml of each streptomycin (Sigma Chemical Co.) and benzylpenicillin (ASTRA, Swe- den). Cells were seeded 24 hr before transfection in 60- mm plates (Falcon), (Becton-Dickinson, N J, U.S.A.) at a density of 5 x 10 ~ cells/plate.

For measurements of intracellular HIV-1 proteinase ac- tivity, HeLa cells were infected with the recombinant vac- cinia virus vTF7-3 expressing the bacteriophage T7 RNA polymerase (Fuerst et al., 1986), generously provided by Dr. B. Moss. Virus was titrated to the maximal dilution which would give reproducible expression of p55 Gag when transfected with 4 #g/plate of Gag-expressing plasmid. Two hours after infection cells were transfected with iolasmids by calcium phosphate coprecipitation. The amount of DNA transfected was adjusted to 16 #g/plate in all samples with pBluescript as carrier DNA.

Cells were transfected with 4 #g/plate pT7-55 Gag alone or cotransfected with different amounts (0.063- 0.25 fig/plate) of pT7-PR or pT7-PR-RT. Six hours after transfection, the cells were washed twice with pho s- phate-buffered saline and new medium was added. Twenty-four hours after transfection, the cells were har- vested in 25 mM Tris, pH 7.4, 75 mM NaCI, 0.5% Triton X-100, 0.5% Na-deoxycholate, 0.05% SDS, 0.07 TIU/ml Aprotinin (Sigma Chemical Co.). After three freeze/thaw cycles, cell extracts were centrifuged at 12,000 g for 15 min at 4 ° and supernatants transferred to new tubes. Sample amounts were adjusted for protein content prior to sodium dodecyl sulphate-polyacrylamide gel electro- phoresis (SDS-PAGE) and Western blot.

SDS-PAGE and Western blot

SDS-PAGE and Western blots were performed ac- cording to Sambrook et al. (1989). Samples were sepa-

lOO j

8O

60

EFFECT OF HIV-1 REVERSE TRANSCRIPTASE ON HIV-1 PROTEINASE 389

0 100 200 300 400 500

RT conc. (nM)

~. 128 nM P R • 37nMPR JI, 5nMPR

FtG. 1. The concentration-dependent effect of HIV-1 RT on HIV-1 proteinase (PR) activity was measured at different concentrations of HIV-1 proteinase. Activity is expressed as % cleavage of substrate peptide SQNYPIVQ-NHz (0.8 mM) after 2-hr incubation at 37 ° with the proteinase in the absence or presence of RT. Values at 128 nM PR correspond to the mean + SD of two independent determinations run in duplicate. Values at 37 and 5 nM PR are the mean + SD of duplicates from single experiments.

rated on 12% acrylamid, 0.32% bis-acrylamid gels at 150 V for 1 hr in a Bio-Rad Mini-Protan II apparatus and transferred to nitro-cellulose filters (Schleicher & Schuell, Dassel, Germany) for 1 hr at 300 mA. After blocking with 10% nonfat dry milk for 1 hr at 37 ° , filters were rinsed briefly and washed for 3 x 5 min in phosphate-buffered saline with 0.3% Tween 20. Membranes were incubated with 7500-fold diluted anti-p24 CA monoclonal antibody EF7 (Hinkula et aL, 1990), a generous gift from Dr. J. Hinkula (Swedish Institute for Infectious Disease Con- trol) for 1 hr at 37 ° , washed as described above, and incubated for 1 hr at 37 ° with 15,000-fold diluted horse radish peroxidase-conjugated rabbit immunoglobulins to mouse immunoglobulins (Amersham, Sweden). After washing as previously described, proteins were detected by enhanced chemiluminescence (Amersham, Sweden) and exposure to X-ray film (Fuji, RX).

RESULTS

Enhancement of HIV-1 proteinase activity by HIV-1 RT

Purified HIV-1 reverse transcriptase was found to in- creases HIV-1 proteinase activity in vitro in a dose-de- pendent manner (Fig. 1). The concentration of RT giving maximal effect was dependent on the concentration of proteinase (Fig. 1), suggesting a direct interaction be- tween the proteinase and RT. At the highest proteinase concentration used (128 riM), maximal effect was ob-

served at roughly equimolar concentrations of RT and proteinase (Fig. 1). Although the substrate concentration is rate limiting at 80% cleavage (Fig. 1), optimal effect was observed at the same proteinase/RT ratio even at higher substrate concentrations, in the linear range for substrate dependence (data not shown). No degradation of substrate peptides was observed when they were in- cubated with RT alone. Likewise, no degradation of RT or proteinase was observed when they were incubated together in the absence of substrate, measured by HPLC (data not shown). RT was preincubated with proteinase for 5 min at 37 ° to allow for association of the proteins prior to addition of substrate. The effect of RT on protein- ase activity was also observed when reactions were started by the addition of substrate immediately after mixing ice-cold RT and proteinase (data not shown).

Proteinase activity was completely inhibited by the high affinity HIV-1 proteinase inhibitors U 75875 (Ashorn et aL, 1990) and Ro 31-38959 (Roberts et aL, 1990), both in the presence and absence of RT. Figure 2 shows the results obtained with Ro 31-38959. U 75875 gave similar results.

Proteinase activity was measured for incubation times between 10 rain and 2 hr; during this time interval protein- ase activity was linear both in the presence and absence of RT (Fig. 3). No degradation of proteinase was detect- able by silver staining of SDS-PAGE of incubation mix- tures, when proteinase was incubated without RT for 2 hr at 37 ° (data not shown).

The effect of pH and ionic strength on enhancement of HIV-1 proteinase activity by HIV-1 RT

The optimal conditions for proteinase activity in vitro include a high salt concentration (Wondrak et aL, 1991)

30

• -RT [ ] +RT

20'

10'

0 I~M 0,1 p.M 1 p,M

Ro 31-8959 concentration

FtG. 2. Inhibition of HIV-1 proteinase activity by Ro 31-8959 in the abscence (-RT) or presence (+RT) of HIV-1 RT (250 nM). Proteinase activity is expressed as % cleavage of substrate peptide SQNYPIVQ- NH2 (2 mM). The proteinase concentration was 66 nM.

390 GOOBAR-LARSSON ET AL.

30

2O

_ O - R T

O • + R T

10

0 0 50 100 150

time (min)

FIG. 3. HIV-1 proteinase activity expressed as % cleavage of substrate peptide SQNYPIVQ-NH2 (0.8 mM) after different incubation times at 37 ° in the absence (-RT) or presence (+RT) of HIV-1 RT (500 nM). The proteinase concentration was 17 nM. Values are the mean + SD of duplicates of one of three independent determinations that gave similar results,

and a low pH (Darke et aL, 1989; Ido et al., 1991). At neutral pH and physiological salt concentration, condi- tions encountered in the infected cell, proteinase activity in vitro is very low.

In order to investigate if the enhancement of protein- ase activity by RT could occur at neutral pH, and if it was dependent on the salt concentration, we compared the proteinase activity in the presence and absence of RT at different pH's and salt concentrations (Fig. 4). The effect of RT on proteinase activity at low salt concentrations and low pH is much larger than the increase in protein- ase activity observed at high salt concentrations in the absence of RT at the same pH (Fig. 4). Both effects are roughly additive, indicating that they are independent of each other. The fact that RT can enhance proteinase activity at 1 M NaCI (Fig. 4), suggests a strong interac- tion between the proteinase, or the substrate peptide, and RT.

Approximately the same degree of cleavage was mea- sured at neutral pH (7.0) in the presence of RT, as at low pH (5.5) in the absence of RT, when measured at low ionic strength (/ = 0.18 for pH 7.0 and / = 0.24 for pH 5.5) (Fig. 4).

Specificity of the enhancement of HIV-1 proteinase activity by HIV-1 RT

To test the specificity of the enhancement of protein- ase activity by RT, we compared the effect of RT with that of HIV-1 p24 CA, BSA, and two fragments of HIV-1 RT corresponding to the first 216 N-terminal amino acids (RT 216) or 235 N-terminal amino acids (RT 235) at equi- molar concentrations. P24 CA had no significant effect on proteinase activity up to 500 nM (Fig. 5A). A significant

increase of activity was observed at 1 #M p24 where a plateau was reached. Even at saturating p24 CA concen- trations its effect on proteinase activity was lower than that of RT. If the effect of RT on proteinase activity in vitro was a bulk effect due to an increase in protein concentration in the assay, the higher molar concentra- tion needed for p24 CA to obtain a measurable effect could be explained by its lower molecular weight. This would, however, not explain the differences found at sat- urating p24 CA concentrations. To elucidate if the differ- ences between p24 CA and RT were due to the differ- ences in molecular weight we tested the effect of two fragments of RT. RT 216 has a slightly lower molecular weight than p24 CA, approximately 23,000 Da, whereas R-i- 235 has a slightly larger molecular weight than p24 CA, approximately 25,000 Da. As shown in Fig. 5A, all three proteins had consistently different effects than RT on proteinase activity at protein concentrations below 1 #M. At protein concentrations of 1 #M or above RT 216, RT 235, and p24 CA reached the same level of saturation which was lower than the level reached with RT. We cannot exclude the possibility that there is a bulk effect at the higher protein concentrations used here; however, the differential effect of p24 CA, RT 216, and RT 235 at concentrations below 1 #M shows that there is a specific effect of RT and its fragments in this molar range.

An increase in proteinase activity in the presence of BSA has been previously reported (Jordan et al., 1992). The effect of saturating BSA concentrations on protein- ase activity was much lower than that of RT (Fig. 5B).

To determine if the effect of RT on proteinase activity was substrate dependent, we compared the proteinase cleavage of different substrates in the presence and ab-

100

80 ' -

6 0 -

q~

40,

20 ,

• -RT [] + R T

0 . . . . . . . .

Z ~ Z Z

E m ~

u~ o ~

FIG. 4. HIV-1 proteinase activity expressed as % substrate (SQNYP- IVQ-NH2 0.8 mM) cleaved after 2-hr incubation at 37 ° at the pHs and NaCI concentrations indicated, in the presence (-I- RT) or absence (-RT) of HIV-1 RT (107 nM). The HIV-1 proteinase concentration was 56 nM. Values are the mean + SD of duplicates of one of three determinations which gave similar results.

EFFECT OF HIV-1 REVERSE TRANSCRIPTASE ON HIV-1 PROTEINASE 391

A

6O

50

4O

30

o

2o

lO-

T p / / ~ +p24 f / ~ it + RT216

~ , " ' ~ J~ • + RT 235

! =

lOOO 2o00

Protein conc. (nM)

B

40

30

~) 20 o

10 - - - - e - - + RT

• + BSA

o i |

0 1000 2000

Protein conc. (nM)

FIG. 5. HIV-1 proteinase activity expressed as % substrate (SQNYPIVQ- NH2, 2 mM) cleaved after 2-hr incubation at 37 ° in the absence or pres- ence of different concentrations of HIV-1 RT and HIV-1 p24 CA, RT 216 and RT 235 (A), or BSA (B). The HIV-1 proteinase concentration was 31 and 34 nM, respectively. Values are the mean + SD of duplicates from one of three independent determinations giving similar results.

sence of RT. All known cleavage sites that could be tested in vitro were found to be more efficiently cleaved in the presence of RT (Fig. 6), indicating that RT interacts with the proteinase itself rather than with the peptide substrate. Figure 6 shows the relative effect of RT on the cleavage of different sites. Experiments are underway to determine whether RT also changes the absolute cleav- age efficiency of different sites and thus influences their cleavage order. The octapeptide RQANFLGK-NH2 repre- senting the cleavage site between the p7 nucleocapsid (NC) protein and pl was not cleaved bythe proteinase in vitro, neither in the absence nor presence of RT. Similarly, cleavage of the octapeptide ARVLAEAM-NH2, represent- ing the cleavage site between p24 CA and the p2 spacer peptide of the Gag polyprotein, could not be detected in

the absence or presence of RT under the conditions used here. Cleavage of the site between RT and integrase was the most enhanced, while the cleavage site between proteinase and RT was the least influenced by the pres- ence of RT (Fig. 6).

Intracellular enhancement of HIV-1 proteinase activity by HIV-1 RT

To test whether RT would exert its effect on proteinase activity intracellularly, we used the vaccinia T7 expres- sion system to measure intracellular proteinase activity.

Proteinase was expressed with or without the RT se- quence in cis and its activity monitored as the processing of p55 Gag precursor expressed in trans. Processing was analyzed by Western blots using anti-p24 CA monoclonal antibody EF7 (Hinkula eta/., 1990). As shown in Fig. 7B, expression of proteinase in trans with p55 Gag resulted in the processing of p55 Gag precursor to a 41- and a 24-kDa band. The degree of p55 Gag precursor pro- cessing was dependent on the amount of proteinase expressing plasmid (pT7-PR) transfected into the cells (Fig. 7B).

The 41-kDa band is probably composed of several cleavage intermediates (Gowda et aL, 1989; Mervis et al., 1988), and the 24-kDa band corresponds to the p24/ p25 CA products. When RT was expressed in cis with the proteinase and in trans with p55 Gag (Fig. 7C), larger amounts of p24/p25 CA were cleaved. Reproducible re- sults were obtained using different plasmid preparations and vaccinia vTF7-3 virus stocks.

RNase H/IN

RT/RNase H

PR/RT

poVPR

p1/p6

p2/p7 NC

p17 MA/p24 CA

I~ ATIMMQRG-NH2 ~ + ~ - I

N SQNYPtVQ-NH2 ~ ~ i

O 20 40 60 80

% Cleavage above control

FIG. 6. The cleavage of peptides corresponding to the different HIV- 1 Gag-Pol polyprotein cleavage sites was measured after 2-hr incuba- tion at 37 ° with 16 nM HIV-1 proteinase in the presence and absence of HIV-1 RT (256 nM). Results are presented as the % increase in substrate cleavage in the presence of RT as compared to controls, in the absence of RT. p17 MA, matrix protein; p24 CA, capsid protein; p7 NC, nucleocapsid protein; PR, proteinase; RT, reverse transcriptase; IN, integrase. Values are mean + SD of duplicates of one of three independent determinations which gave similar results.

392 GOOBAR-LARSSON ET AL.

TT.promo~r

pTT-PR

pT7-PR-RT

pT7-55 Gag I ~ I

PR

PR-RT

pS~ C~g

B

pT7-PR (rig): 0 10 50 100 500

p55 Gag

p41 Gag

p24/p25 CA

C 1 2 3

p55 Gag

p41Gag

p24/p25CA

FIG. 7. (A) Schematic representation of plasmid constructs used for intracellular proteinase activity measurements. (B) Western blot of ly- sates of HeLa cells infected with vaccinia vTF7-3, transfected with pT7- 55 Gag, and cotransfected with increasing amounts of pT7-PR. (C) Western blot of lysates of HeLa cells infected with vaccinia vTF7-3 and transfected with pT7-55 Gag, lanes 1, 2, and 3. Lanes 1 and 2 were cotransfected with pT7-PR-RT (lane 1) or pT7-PR (lane 2) in equimolar amounts. Western blots were developed using an anti-p24 CA mono- clonal antibody. Results are from one of four independent transfections giving similar results.

These results show that RT affects the processing of the p55 Gag precursor in eukaryotic cells. Moreover, these experiments indicate that RT enhancement of pro- teinase activity is not a result of the in vitro conditions used and that the effect is present at intracellular condi- tions of pH, ionic strength, and protein concentration.

DISCUSSION

Our resuks show that HIV-1 RT significantly increased HIV-1 proteinase activity not only in vitro but also intracel- lularly. The degree of enhancement in vitro and the RT/ proteinase ratio for maximal enhancement was depen- dent on both the proteinase and RT concentration, as shown in Fig. 1, suggesting a protein-protein interaction. At a proteinase concentration of 128 nM the proteinase/ RT ratio for maximal enhancement was approximately 1,

assuming 100% sp act for both RT and the proteinase and molecular weights of 117,000 and 21,500, respec- tively. This is in agreement with the intracellular ratio of RT to proteinase expressed during virus infection.

One possible explanation for our in vitro results, is that RT stabilizes the proteinase against denaturation (Grant eta/., 1992) or autoproteolysis (Strickler eta/., 1989; Rose et aL, 1993). However, the reaction rate for the proteinase over the incubation times used was linear both in the presence and absence of RT. Moreover, no proteinase degradat ionwas detectable by silver staining of reaction mixtures during this time in the absence of RT. Also, neither BSA nor p24 CA could mimic the effect of RT. Finally, the possible in vitro instability of the proteinase cannot explain the effect of RT observed in cell culture.

Another possible explanation for the enhancement seen both in vitro and in cell culture is that RT promotes the dimer izat ionof the proteinase (Zhang et al., 1991; Kuzmic, 1993; Darke et al., 1994). This cannot be ruled out, especially in the vaccinia expression system, where proteinase dimerization is a rate limiting step (Luukkonen et al., submitted for publication). Several in vitro results point though against this being the only effect of RT on proteinase activity. First, a shift in the dimerization con- stant for the proteinase would affect the cleavage of all substrates to the same degree. However, as shown in Fig. 6, RT enhanced the cleavage of different substrates to different degrees. Second, the effect of RT was additive to that of high Salt concentration, a condition that most probably affects the dimerization of the proteinase.

The cleavage that was most enhanced in the presence of RT corresponds to the site between the C-terminal end of RT p66 and the integrase in the Gag-Pol polypro- tein. This cleavage, which is not one of the most effi- ciently cleaved by the mature proteinase in vitro (Darke et aL, 1988), could in vivo be more efficiently cleaved in the presence of RT, allowing for the release of the RT p66 peptide from the precursor polyprotein. Interestingly, this Pol site is also the most sensitive to mutagenesis (Loeb et al., 1989). The lack of cleavage of RQANFKGK- NH2 corresponding to the cleavage site between p7 NC and pl, is in agreement with previous reports (TOzs6r et al., 1991; Wondrak et aL, 1993), showing that a larger fragment of the p7 NC protein is needed for proteolytic cleavage. The other substrate peptide that did not give detectable product levels was ARVLAEAM-NHz, repre- senting the cleavage site between p24 CA and p2. This is the last site cleaved of the Gag polyprotein precursor (Tritch et al., 1991 ) and probably the least efficient cleav- age site. Higher proteinase Or substrate concentrations or longer incubation times may be needed to obtain de- tectable product levels with this substrate. Further exper- iments are needed to determine whether the order of cleavage of the gag- and pol -gene products is influenced by the presence of RT.

The effect of RT on proteinase activity in HeLa ceils

EFFECT OF HIV-1 REVERSE TRANSCRIPTASE ON HIV-1 PROTEINASE 393

is modest, probably due to the fact that Gag and protein- ase are expressed in trans and, while most of the Gag precursor is transported to the plasma membrane (Kaplan and SwanstrOm, 1991), the proteinase (or pro- teinase/RT) most probably remains in the cytosol. Hu et al. (1990) reported that a construct that contained the entire proteinase gene, together with Gag, would pro- cess p55 Gag less efficiently than constructs expressing Gag-proteinase-RT or the whole Gag-Pol polyprotein. in their system the entire pol gene, or fragments thereof, were expressed in cis with the Gag polyprotein, and after frameshift, as is the case during virus infection. Indeed, the effect of RT on Gag processing seen with their con- structs is even more pronounced.

Karacostas et al. (1993), showed that a block of virus assembly caused by overexpression of the Gag-Pol polyprotein (i.e., increased intracellular proteinase activ- ity), could be partially overcome by shortening of the Gag-Pol polyprotein by deletion of most of the reverse transcriptase, indirectly implying that RT may have an effect on proteinase activity. These results are in agreement with ours and further corroborate the effect of RT in eukaryotic cells. The effect of RT on proteinase may represent a feed back enhancement of enzyme ac- tivity by its substrate. Alternatively, the effect of RT may reflect the activity of the proteinase within the polyprotein precursor or in a processing intermediate. This would be analogous to polio virus polyprotein processing, where the processing intermediate 3CD containing the se- quences for the proteinase and the viral RNA-dependent RNA polymerase is the form of the proteinase required for complete processing of the capsid protein precursor P1 (Ypma-Wong et aL, 1988). RT may also enhance pro- teinase activity at a specific stage of virus replication and function as a regulatory mechanism for proteinase activity.

Experiments are underway to determine the mecha- nism by which RT enhances proteinase activity and its role during virus replication.

ACKNOWLEDGMENTS

We thank M. Hessam Amiri and L. Wiklund for excellent technical assistance, Dr. J. Hincula for anti p24 CA antibodies, and Dr. B. Moss for recombinant vaccinia-T7 virus. Dr. I. B. Duncan and Dr. W. G. Tarpley are gratefully acknowledged for HIV-1 proteinase inhibitors and Dr. F. Volvovitz and Professor B. Wahren for recombinant HIV-1 p24 CA. This work was supported by grants from the Swedish board for Technical Development and by Medivir AB,

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