Proc. Nati. Acad. Sci. USA Vol. 88, pp. 45%-4600, June 1991 Biochemistry activities in four different forms of human immunodeficiency 1 poi products (/RNase H/crossover linker mutagenesis/baculoirus expression system) YU-WEN HU AND C. YONG KANG Department of Microbiology and Immunology, University of Ottawa, Faculty of Medicine, Ottawa, ON, Canada K1H 8M5 Communicated by Max D. Summers, February 19, 1991 (received for review December 3, 1990)

ABSTRACT Five cassettes of the pol gene of human im- activity (10, 11) but no RNase H activity (3, 7, 12, 13). The munodeficiency virus 1 were constructed and inserted under reverse transcriptase isolated from purified HIV-1 has a the control of the polyhedrin gene promoter of Autographa molecular mass of 95 (14) to 110 (15) kDa, as determined by calfornica nuclear polyhedrosis virus by homologous recom- gel filtration and glycerol gradient centrifugation, respec- bination. The first cassette polF contains the full-length pol tively. However, the 98-kDa nonprocessed polyprotein ex- open reading frame; the second cassette pollOO starts with the pressed in Escherichia coli displayed little or no reverse first AUG codon of the pol gene and deletes 103 amino acids transcriptase activity (16). The p66/pSi heterodimer ex- from the amino terminus of the pol gene product; the third pressed in E. coli has an apparent molecular mass of 120-130 cassette po197 deletes the entire coding sequence; the kDa and was active (5, 6). In addition, there is a 165-kDa fourth cassette po166 deletes both the protease and endonucle- putative gag-pol precursor polyprotein that also possesses ase/ coding sequences; and the fifth cassette polSl reverse transcriptase activity (10). contains the reverse transcriptase coding sequences plus 39 The enzymatically active forms ofHIV-1 reverse transcrip- 3'-terminal nucleotides of the RNase H coding sequences. We tase have been the subject of much debate. An important have expressed these five forms of the pol gene in Spodoptera question related to the pol gene products is whether only frugiperda SF9 cells and have analyzed for both reverse tran- certain form(s) possesses reverse transcriptase and/or scriptase and RNase H activities. The polF construct expressed RNase H activity or whether many different forms, including several processed forms, 66 kDa, 51 kDa, and 34 kDa , processing intermediates, are active. To answer this ques- that were detected only by Western blot. In contrast, pollW, tion, we constructed and expressed several forms of the pol po197, po166, and pol51 products were expressed at high levels gene product in SF9 cells and analyzed them for reverse and were readily detectable in gels by staining. The levels of transcriptase and RNase H activities by using activity gel expression of these four products were estimated to be >150 analysis. We show that four forms of the pol gene product, mg/liter ofculture (5 x 10" cells). Activity gel analyses showed ranging from the unprocessed 100-kDa polyprotein to the that the pol10, po197, po166, and pol5 products possess shortest 51-kDa , are independently active for reverse reverse transcriptase activity; however, only po197 and po166 transcriptase; however, only two proteins, p97 and p66, show have RNase H activity. Our results demonstrate that many RNase H activity. forms, including partially cleaved forms of human immuno- deficiency virus 1 pol gene products, possess reverse transcrip- MATERIALS AND METHODS tase activity but only certain forms have RNase H activity. Construction of Recombinant Autographa calafornica Nu- clear Polyhedrosis Virus (AcNPV) Containg Different Forms Conversion of the genetic information in human immunode- of the HIV-1 poi Gene. We first constructed the po1100 ficiency virus (HIV) genomic RNA to proviral DNA by cassette that deletes 273 base pairs (bp) from the 5' terminus reverse transcriptase is an essential step in replication of ofthe pol open reading frame [nucleotides (nt) 23%-51311 by HIV. The polymerase (pot) gene of HIV-1 contains within it using the crossover linker mutagenesis method (17-19). The a viral-specific protease, a reverse transcriptase, an RNase Bgl II-Sal I fragment ofplasmid pHXB-2D (1) containing the H, and an endonuclease/integrase in the amino- to carboxyl- HIV-1 pol coding region was isolated and inserted into terminal direction (1). The poi gene is expressed as a poly- BamHI and Sal I sites of pUC18. The resulting recombinant protein precursor that is subsequently cleaved to yield the plasmid (pUC18-Dpol 1) was cut with Sst I and dephosphor- following gene products: the 15-kDa viral protease that in ylated. A synthetic double-strand crossover linker containing turn is responsible for processing the polyprotein precursor; an Sst I cohesive end, a BamHI site, the putative ribosome a 66-kDa protein that contains reverse transcriptase and binding site, CCTATAAAT that was derived from the nt -9 RNase H activities; a 51-kDa protein that represents a to -1 ofthe polyhedrin gene (20, 21), and 15 nt of "homology truncated form of p66 and is one of the subunits of a p66/pSl searching sequences" that overlap with the 5' terminus ofthe heterodimer; and a 34-kDa endonuclease/integrase that is pollO0 gene was ligated to the Sst I site and the resulting required for integration of the proviral DNA into host DNA. construct was used to transform E. coli. Recombinant plas- p66 contains several catalytic functions including RNA- mid (pUC18-Dpol2) was isolated, digested with Sph I, de- directed DNA polymerase, DNA-directed DNA polymerase, phosphorylated, and ligated with another crossover linker and RNase H activities (2-6). p51 is a cleaved product DNA containing Sph I cohesive end, a BamHI site, and 15 nt representing the amino terminus of p66 (7, 8). Several labo- of "homology searching sequences" that recognized the 3' ratories have reported that pSl contains neither reverse terminus of the pol gene. The resulting recombinant plasmid transcriptase nor RNase H activities (2, 7, 9). However, (pUC18-Dpol3) contains the putative ribosome binding site others have reported that p51 possesses reverse transcriptase followed by the pol open reading frame starting with the first

The publication costs of this article were defrayed in part by page charge Abbreviations: AcNPV, Autographa californica nuclear polyhedro- payment. This article must therefore be hereby marked "advertisement" sis virus; HIV, human immunodeficiency virus; DTT, dithiothreitol; in accordance with 18 U.S.C. ยง1734 solely to indicate this fact. AMV, avian myeloblastosis virus; nt, nucleotide(s). 4596 Downloaded by guest on September 24, 2021 Biochemistry: Hu and Kang Proc. Natl. Acad. Sci. USA 88 (1991) 4597 ATG (nt 2396-2398) in the pol gene and ending with the The infected cell lysate was prepared in denaturing buffer [60 translation termination codon TAG (nt 5132-5134). This mM Tris HCI, pH 7.6/1% SDS/65 mM 2-mercaptoethanol/ pollOO cassette was flanked with BamHI sites. The BamHI 10% (vol/vol) glycerol/0.01% bromophenol blue], incubated fragment was isolated, inserted into the BamHI site of the at 370C for 5 min, and then electrophoresed at 40 mA for 2 hr pAcYM1 baculovirus transfer vector (21, 22), and used to in a Mini-Protean II electrophoresis cell (Bio-Rad). The gel cotransfect SF9 cells along with wild-type AcNPV DNA to was removed and washed by shaking gently in 1 liter of produce recombinant baculovirus as described (19, 22). The renaturing buffer [50 mM Tris HCI, pH 7.5/10 mM MgCI2/1 full-lengthpolgene (polF) was constructed as follows; the Bgl mM dithiothreitol (DDT)/20 mM KCI] at room temperature II-Sal I fragment of plasmid pHXB-2D was isolated and for 1 hr with several changes. The gel was gently washed in ligated with a synthetic double-stranded DNA linker that renaturing buffer at 40C for another 16 hr with three changes provides 12 nt missing immediately upstream from the Bgl II of the buffer. The washed gel was incubated in 20 ml of site plus a translation initiation codon, a putative ribosome reverse transcriptase buffer [50 mM Tris HCI, pH 7.9/6 mM binding site, a BamHI site, and an Sst I cohesive end. The MgCl2/10% glycerol/80 mM KCI/bovine serum albumin (0.4 DNA was inserted into the Sst I and Sal I sites ofpUC18. The mg/ml)/0.01 mM EDTA/1 mM DDT] containing 150,uCi of resulting recombinant plasmid (pUC18-Fpoll) was cut with [a-32P]dTTP (800 Ci/mmol; 1 Ci = 37 GBq; Amersham) and Sst I and EcoRV and a 900-bp fragment was isolated. The (dT)12.18 (20,g/ml) at 370C for 2 hr. The gel was then washed 900-bp Sst I-EcoRV fragment and a 5.4-kbp Sst I-EcoRV with ice-cold 5% (wt/vol) trichloroacetic acid containing fragment ofpUC18-Dpol3 were ligated and used to transform 0.25% sodium pyrophosphate for 1 hr at room temperature E. coli. The resultant plasmid (pUC18-Fpol2) contains the with two changes ofsolution followed with 5% trichloroacetic putative ribosome binding site followed by a newly intro- acid washing at 40C for 20 hr with four changes ofthe washing duced translation initiation codon, the full-length pol open solution. The gel was wrapped and exposed to x-ray film. reading frame (nt 2087-5131), and the translation termination In Situ Gel Assay for RNase H Activity. In situ gel assays for codon and flanked by BamHI sites at either end. This cassette RNase H activity was carried out as modified (23, 24). (polF) was isolated and inserted into the BamHI site of the Briefly, a hybrid of32P-labeled RNA and unlabeled DNA was pAcYMi baculovirus transfer vector and was used to prepared by transcription of single-stranded M13 DNA as a cotransfect SF9 cells along with the wild-type AcNPV to template with [a-32P]CTP and the three remaining ribonucle- produce recombinant baculovirus. We have used the same oside triphosphates using E. coli RNA polymerase. The crossover linker mutagenesis methods to construct pol97 (nt [32PJRNA*DNA hybrid was purified by Sephadex G-50 spun 2588-5131), pol66 (nt 2588-4273) and polSi (nt 2588-3864 column chromatography. SDS/polyacrylamide gels were plus 4193-4232) (Fig. 1) cassettes. These were introduced prepared in the presence of -1 ,ug of [32P]RNA*DNA hybrid into recombinant baculoviruses to allow expression of dif- (4 x 106 cpm). After electrophoresis of the infected cell ferent forms of pol gene products. lysates, the gel was soaked at room temperature for 2 days Expression of pol Gene Products in SF9 Cells by Using with several changes of buffer (50 mM Tris HCI, pH 8.0/50 Recombinant Baculovirus. SF9 cells infected with recombi- mM NaCl/2 mM DTT/10 mM MgCl2/2 mM MnCl2) to permit nant baculoviruses were harvested 72 or 96 hr after infection. renaturation of the proteins, digestion of [32P]RNA from Lysates of infected or uninfected cells were electrophoresed [32P]RNA-DNA hybrid substrate by RNase H, and elimina- in 12% polyacrylamide Laemmli gels and proteins were tion of degraded radiolabeled oligonucleotides. identified by Coomassie blue staining and/or Western blot analyses using the standard National Institutes of Health RESULTS HIV-positive human sera as described (19). Construction and Expression of Different Forms of the pol Activity Gel Analysis for Reverse Transcriptase. Activity gel Gene Product. We have constructed five cassettes of the pol analysis for reverse transcriptase was performed according to open reading frame by using the crossover linker mutagenesis published protocols (2, 10). Briefly, poly(rA) (Pharmacia; 120 method (17-19), which allowed for deletion or addition of ,ug/ml) was incorporated into a 10o polyacrylamide gel precise numbers ofnucleotides at the 5' and 3' ends ofthe pol containing SDS to serve as a template for cDNA synthesis. open reading frame. The first cassette polF contains the

p 2 7 7 pol F 5134 Bamrn TI TTr Oi-qE-fATffTATA- A TL UCTATAAATATUTTTTTTTATAT&TtlTTTT..-.-- t&~?'8415TTAG GATAT TTATACAAAAAA Int _ ITCATCCTAG-s FIG. 1. Construction of five cassettes of the HIV-1 pol open reading frame. Cassettes of the HIV-1 pol gene were con- 5GTG^TATATT^ATAC:3 RT ^lniIft v CTCvvzv CTAGT5' structed by the crossover linker mutagenesis method using syn- Barn Hi thetic double-stranded oligode- BarnHIBam Hi eI~~~~~~~~~~~~~~~~~~~~TTJ oxyribonucleotides. P denotes GATCCTATAAAATACCTGTTAn KVgCAGGATAG~cAI5 the putative ribosome binding 2588 po197 5134 site and TI and TT are transla- tion initiation and translation GATATTTATACGGGTAA RT vGAGvGATA(v Ban Hi termination codons, respec- tively. Pr, RT, H, and Int desig- nate the viral protease, reverse transcriptase, RNase and P _ 2588 H, poI66 4273 HO endonuclease/integrase , Ba_ -I I LT WAeAhAl IALWKSTAA* I ' respectively. Numbers on top of 1 iiiAATiiiCCTAG-5' each cassette indicate nucleo- Barn N tide positions in the provirus ge- nome. polF, pollOO, pol97, p _ 25b88"po15l 3864 4 7 (900bp) pol66, and pol5l cassettes are Sam HI capable of synthesizing 1016, II5- GATCCTATAAAA AJ8GCCCATT-TTIRT GG^TAWG8A 8GATGAGAAATGAAACT~gTAG- - &AG- GATATTI TAT CGG T AG ~TATCGGCAT TkCATGOTC IFTTCTTAACCTCCTTTA ATC~ CCA _SI 912, 849, 563, and 439 amino TCCaG-5 acids, respectively. Downloaded by guest on September 24, 2021 4598 Biochemistry: Hu and Kang Proc. Natl. Acad. Sci. USA 88 (1991) full-length pol open reading frame starting from nt 2087. This and endonuclease/integrase genes and is able to code for a full-length pol open reading frame contains coding sequences protein of 563 amino acids (Fig. 1). This construct expressed for viral-specific protease, reverse transcriptase, RNase H, a large amount of a 66-kDa protein in SF9 cells that is and endonuclease/integrase. Since the pol open reading recognized by antibody present in HIV-1-positive human frame does not have its own translation initiation site, we sera (Fig. 2, lanes 2). The last cassette pol5i (nt 2588-3864 inserted an ATG in front of the pol open reading frame. In plus 4193-4232) contains only the reverse transcriptase por- addition, we also included the putative ribosome binding site tion ofthepol open readingframe. This cassette has the entire CCTATAAAT in front ofthe translation initiation codon. We protease coding sequence, the endonuclease/integrase cod- have deleted all noncoding sequences at both 5' and 3' termini ing sequence, and a major portion of the RNase H coding ofthe gene and added aBamHI cloning site at either end. The sequence deleted. For the convenience of cloning, we in- resulting polF construct (nt 2087-5131) is capable of synthe- cluded a small portion of the RNase H gene (nt 4193-4232) sizing a product of 1016 amino acids (Fig. 1). When we that adds 13 amino acids to the carboxyl terminus of the expressed this polF gene in SF9 cells using recombinant reverse transcriptase (Fig. 1). The pol5i cassette has a coding baculovirus, we could not detect a precursor polyprotein capacity of439 amino acids. This cassette expresses a 51-kDa band by staining with Coomassie blue (Fig. 2A, lane 5). protein in SF9 cells (Fig. 2A, lane 1) and the 51-kDa protein However, when we performed Western blot analysis using reacted with antibody present in HIV-i-positive human sera the infected cell lysate, we found 66-kDa, 51-kDa, and (Fig. 2B, lane 1). With the exception of polF, the levels of 34-kDa proteins that reacted with antibodies present in expression of pollOO, pol97, pol66, and polSl were >150 HIV-positive human sera (Fig. 2B, lane 5). This result mg/liter of culture (5 x 108 cells). suggests that the HIV protease must be active to cleave the Analysis of poi Gene Products for Reverse Transcriptase precursor polyprotein to yield the final products p66, p5l, Activity. We analyzed reverse transcriptase activity in and p34. We observed that cells infected with recombinant pollOO, pol97, pol66, and polSi by using activity gels (2, 10). baculovirus containing polF cassette lysed by 30 hr of infec- We were unable to detect the po1 gene product in SF9 cells tion (data not shown). We assume that the viral protease infected with recombinant baculovirus containing the polF expressed by this recombinant baculovirus is extremely toxic cassette by Coomassie blue staining (Fig. 3a, lanes 2 and 7) to the cells, and the cells cannot synthesize large quantities or polymerase activity gel assay (Fig. 3b, lanes 2 and 7). In ofthe polyprotein. The second cassette pollOO starts from the contrast, pollOO, pol97, pol66, and pol5i proteins indepen- first AUG codon of the po1 open reading frame, deleting 103 dently possess reverse transcriptase activity (Fig. 3B, lanes amino acids at the amino terminus of the protease (nt 3, 6, 4, and 5, respectively). To determine the location of 2396-5131) but retaining 64 amino acids at the carboxyl these pol gene products in SF9 cells, we separated nuclei terminus of the protease. This cassette contains genetic from the cytoplasm and carried out activity gel analyses on information for 912 amino acids (Fig. 1). When we expressed both fractions. We found enzymatically active forms of the pollOO cassette in SF9 cells, large quantities of unpro- po197, pol66, and polSi in the cytoplasm (Fig. 3, lanes 9-12). cessed 100-kDa protein accumulated in the recombinant In contrast, all pollOO activity was found in the nucleus of baculovirus-infected SF9 cells (Fig. 2A, lane 4). The 100-kDa infected SF9 cells (data not shown) and we could not detect protein is immunoreactive with antibodies present in HIV- any pollOO protein in the cytoplasm ofinfected SF9 cells (Fig. 1-positive human sera (Fig. 2B, lane 4). The third cassette 3, lanes 9). This result suggests that the 64 amino acids at the po197 (nt 2588-5131) has the entire viral protease gene deleted (Fig. 1). This cassette is capable ofproducing a protein of849 amino acids and expressed a 97-kDa protein in SF9 cells (Fig. 2A, lane 3) that also reacted with antibodies present in 97- HIV-1-positive human sera (Fig. 2B, lane 3). The fourth cassette pol66 (nt 2588-4273) has a deletion of both protease 66- A B M 1 2 3 4 5 1 2 3 4 5

- -100 b 97- _rise "-97 66- -*- -66 1-40 -51 43- r>"

31- -34

22 - 14- FIG. 3. Detection of HIV-1 reverse transcriptase activity in pol gene products by activity gel analysis. (a) Coomassie blue stain of a FIG. 2. Expression of pol genes in SF9 cells by recombinant polyacrylamide gel of various recombinant-baculovirus-infected cell baculoviruses. SF9 cells infected with recombinant baculoviruses lysates. The polF-infected cell lysate (lane 2) and cytoplasmic were harvested 72 hr after infection. Lysates of the infected cells fraction (lane 7) show no detectable precursor polyprotein or pro- were denatured and electrophoresed in 12% polyacrylamide Laem- cessed proteins. Lanes 3-6 show 100-kDa (-), 66-kDa (D), 51-kDa mli gels. The proteins in the gel were visualized by Coomassie blue (0), and 97-kDa (*) proteins, respectively, in whole-cell lysates. staining (A) or Western blot analysis (B) using standard National Lane 9 shows the cytoplasmic fraction of pollOO recombinant Institutes of Health HIV-positive human sera. Lanes 5, 4, 3, 2, and virus-infected cells, which has no detectable 100-kDa protein. Lanes 1 represent the SF9 cell lysates infected with recombinant baculo- 10-12 show 66-kDa (D), 51-kDa (K), and 97-kDa (*) proteins in the carrying polF, pollOO, poI97, poI66, and pol5l po1 gene cytoplasm of infected cells. Lanes 1 show molecular mass markers cassettes, respectively. Lane M contains molecular mass markers and lanes 7 show uninfected cell cytoplasmic extracts. The gel (a) shown in kDa. Lanes 1-4 in A and B show 51 kDa, 66 kDa, 97 kDa, was stained with Coomassie blue after activity gel analysis (b). (b) and 100 kDa proteins, respectively. Lane 5 in B shows processed Autoradiogram of the activity gel showing 32P-labeled cDNA prod- proteins of 66 kDa, 51 kDa, and 34 kDa. ucts associated with reverse transcriptase. a and b are the same gel. Downloaded by guest on September 24, 2021 Biochemistry: Hu and Kang Proc. Natl. Acad. Sci. USA 88 (1991) 4599

amino terminus ofpoll0 derived from the carboxyl terminus 2 3 4 5 of the viral protease contains a nuclear localization signal. To confirm that the reverse transcriptase activity associated - 3 J r%'., with pollOO and po197 is not the result of protein processing that may have occurred during the incubation period for reverse transcription, we preincubated the pol97 recombinant virus-infected cell lysate at 37C for 12 hr in the presence or absence of bovine serum albumin. The cell lysate was elec- trophoresed and activity gel analysis was carried out. The DNA products associated with the reverse transcriptase was visualized by autoradiography (Fig. 4A) and the presence of protein was determined by Coomassie blue staining (Fig. 4B). We were unable to detect any processed protein by direct g staining or by reverse transcriptase activity gel analysis. It is F i W clear from this data that pol97 is not cleaved during the e incubation period for reverse transcription and unprocessed |I pol97 protein indeed contains reverse transcriptase activity. | We obtained the same results with the pollOO (data not | shown). We have also analyzed cDNA products transcribed from a globin mRNA template using oligo(dT) as primer to .t determine the quality ofthe cDNA products. Fig. 5 shows the ::6 :xt cDNA products analyzed in a sequencing gel. Lanes 1-4 show .:#e ::p; FIG. 5. cDNA product analysis. Recombi- the cDNA products synthesized by polSl, pol66, pol97, and # .eiiF. pollOO, respectively. Lane 5 shows the products from avian A*. nant-virus-infected cells were harvested 72 hr e after infection and the reverse transcriptase - myeloblastosis virus (AMV) reverse transcriptase as a positive . Wt *-r .s reaction was carried out with crude cell lysates control. It is evident that cDNA synthesized by pol66 and 4 - using globin mRNA as template and (dT)12_18 as pol97 are reasonable length and pollOO can synthesize full- F primer. The cDNA products were analyzed in a length cDNAs of globin mRNA. The reverse transcriptase a: DNA sequencing gel. Lanes: 1-4 represent activity associated with pol5i is relatively weak and much 4',# cDNA products synthesized by polSl, pol66, shorter cDNAs were synthesized (Fig. 5, lane 1). pol97, and pollOO, respectively; 5, cDNA prod- RNase H Activity Associated with pol Gene Products. Ly- ucts synthesized by commercial AMV reverse sates of recombinant-baculovirus-infected SF9 cells were transcriptase (Pharmacia). 940b represents the analyzed for RNase H activity after SDS/PAGE in gels full-length cDNA. containing radiolabeled RNA in an RNADNA hybrid. After virus-infected.jl cells (lanes 6 and 4, respectively) show RNase electrophoresis, the gel was incubated in buffer at room H activity. It is interesting to observe that pollOO shows no temperature to allow renaturation of proteins, digestion of RNase.w...H activity although it contains the complete RNase H radiolabeled RNA in the RNA-DNA hybrid, and release of digestion products. Autoradiography ofthe dried gel revealed 1 2 3 4 5 6 7 8 9 clearing zones against a dark background (Fig. 6). Extracts 200- from control uninfected cells (Fig. 6a, lane 9) and AMV 116- reverse transcriptase (Fig. 6, lane 7) showed no RNase H 97- activity. In addition, extracts from poll0 (lane 3) and pol51 66- 43- (lane 5) recombinant-baculovirus-infected cells show no ,; . RNase H activity. In contrast, pol97 and pol66 recombinant- A B 1 2 3 4 1 2 3 4

i. .". .. 'Z.-

-974 *0ow -A -68

-43 FIG. 6. RNase H activity gel. (a) Coomassie blue-stained SDS/ polyacrylamide gel of crude extracts from various recombinant- virus-infected cell lysates. Position ofmolecular weight mass (lane 1) FIG. 4. Reverse transcriptase activity gel analysis of pol97 pro- are shown to the left in kDa. Lanes: 2, polF recombinant-virus- tein after preincubation. The pol97 recombinant virus-infected cell infected cell lysate; 3-6, pollOO, pol66, polSl, and pol97 recombi- lysate was prepared as described in Fig. 2 and incubated in 10 mM nant-virus-infected cell lysates, respectively; 7, commercial AMV Tris'HCl (pH 7.5) containing 1 mM EDTA, 0.25 M DTT, 0.05% reverse transcriptase (Pharmacia); 8, E. coli RNase H; lane 9 Triton X-100, and 25% glycerol at 370C for 12 hr prior to SDS/PAGE. uninfected cell lysate. (b) Autoradiogram of gel in a demonstrating The gel was removed after electrophoresis and enzyme activity gel RNase H activity (white arrows). A gel was cast with 32P-labeled analysis was carried out as described in Fig. 2. (A) Autoradiogram. RNA in RNADNA hybrid, and recombinant-virus-infected cell (B) Coomassie blue staining of the gel in A. Lanes: 1, pol97 lysates were electrophoresed as shown in a. The gel was removed, recombinant-virus-infected cell lysate; 2, same sample as in lane 1 washed extensively, and incubated to allow proteins to renature and plus bovine serum albumin; 3 and 4, same sample as lanes 1 and 2, to digest [32P]RNA in the RNA-DNA hybrid substrate. The gel was respectively, but treated with Pronase (Calbiochem; 50 units/ml) fixed, dried, and exposed to x-ray film. RNase H activity associated during the 12-hr incubation period. Arrows show the 97-kDa protein with small proteins in the bottom of the gel may be derived from and the letter A with the arrow indicates the position ofbovine serum baculovirus gene products since uninfected cells (lane 9) have no albumin. Molecular masses in kDa are indicated. such activity. Downloaded by guest on September 24, 2021 4600 Biochemistry: Hu and Kang Proc. Natl. Acad. Sci. USA 88 (1991) domain. We conclude that both the fully processed form p66 However, the polF cassette produced very small amounts of and the p97 precursor form of the pol gene products exhibit processed pol gene products. We believe this is due to the RNase H activity. toxic effects of the viral protease that destroys SF9 cells rapidly. This result underscores the obvious inverse relation- DISCUSSION ship between toxicity ofaforeign gene product and production The results presented here demonstrate that both unprocessed of large quantities of the protein. If the cellular function is precursor polyproteins and fully processed forms of the pol destroyed by the expressed protein, it is evident that the cell gene products have reverse transcriptase activity. Our data will be incapable of expressing large quantities ofthe product. confirm the observations (10, 11, 14, 15) that different forms ofthepol gene product including p51 are enzymatically active. We thank Drs. S. Josephs, F. Wong-Staal, and R. C. Gallo for However, it is not clear why the p51 subunit (2, 7, 9), pHXB-2D clone. We also thank J. McCulloch, N. Delcellier, and B. recombinant pol gene products with deletions in the carboxyl Mah for technical assistance and Drs. K. Dimock and H. C. Birn- boim for the critical review of this manuscript. This study was terminus (25, 26), and the 98-kDa protein expressed in E. coli supported by grants from the Medical Research Council of Canada, (16) failed to show reverse transcriptase activity in other Oxford Virology plc., and the Ontario Ministry of Colleges and studies. It is possible that cellular copurified with Universities. these proteins degrade the pol gene products rapidly (27, 28). In contrast, pol gene products expressed in SF9 cells appear 1. Ratner, L., Haseltine, W., Patarca, R., Livak, K. J., Starcich, very stable. This suggests that a bacterial protease(s) can act B., Joseph, S. F., Doran, E. R., Rafalski, J. A., Whitehorn, on these pol gene products, whereas SF9 cell proteases are E. A., Baumeister, K., Ivanoff, L., Petteway, S. R., Jr., Pear- son, M. L., Lautenberger, J. S., Papas, T. K., Ghrayeb, J., less effective. It is also possible that some pol gene products Chang, N. T., Gallo, R. C. & Wong-Staal, F. (1985) Nature fail to exhibit enzymatic activity because they fail to fold (London) 313, 277-284. properly. Although the specific activity of each protein may 2. Starnes, M. C., Gao, W., Ting, R. C. & Cheng, Y. C. (1988) J. vary, multiple forms of the enzyme could contribute to syn- Biol. Chem. 263, 5132-5134. thesis of proviral DNA during virus replication. Our results, 3. Mizrahi, V. (1989) Biochemistry 28, 9088-9094. confirming that reverse transcriptase precursor can be enzy- 4. Tarnese, N., Roth, M. R. & Goff, S. P. (1986) J. Virol. 59, matically active, could explain the presence of intracellular 328-340. reverse transcription before the formation of the virions. 5. Muller, B., Restle, T., Weiss, S., Gautel, M., Sczakiel, G. & In contrast to reverse transcriptase activity, we observed Goody, R. S. (1989) J. Biol. Chem. 264, 13975-13978. 6. Restle, T., Muller, B. & Goody, R. S. (1990) J. Biol. Chem. 265, that only p97 and p66 proteins were active for RNase H. It 8986-8988. was surprising that the 100-kDa protein expressed by the 7. Hansen, J., Schulze, T., Mellert, W. & Moelling, K. (1988) pollO0 recombinant virus had very little or no RNase H EMBO J. 7, 239-243. activity although it contains the complete RNase H domain 8. Farmerie, W. G., Loeb, D. D., Casavant, N. C., Hutchison, as does the 97-kDa protein. The difference between the C. A., III, Edgell, M. H. & Swanstrom, R. (1987) Science 236, 100-kDa protein and the 97-kDa protein is the presence of 64 305-308. extra amino acids at the amino terminus of the 100-kDa 9. Lightfoote, M. M., Coligan, J. E., Folks, T. M., Fauci, A. S., protein, derived from the carboxyl-terminal sequences of the Martin, M. A. & Venkatesan, S. (1986) J. Virol. 60, 771-775. viral protease. These extra amino acids, in some way, block 10. Lori, F., Scovassi, A. I., Zella, D., Achilli, G., Cattaneo, E., Casoli, C. & Bertazzoni, U. (1988) AIDS Res. Hum. Retrovi- RNase H. It is possible that protein folding during the ruses 4, 393-398. renaturation process is altered or that cleavage of this seg- 11. Prasad, V. R. & Goff, S. P. (1989) Proc. Natl. Acad. Sci. USA ment is required for activity. 86, 3104-3108. We synthesized a full-length globin cDNA with only 12. Lowe, D. M., Aitken, A., Bradley, C., Darby, G. K., Lader, poll00. The combination of both high level of reverse tran- B. A., Powell, K. L., Purifoy, D. J. M., Tisdale, M. & Stam- scriptase activity and absence of RNase H activity in the mers, D. K. (1988) Biochemistry 27, 8884-8889. 100-kDa protein may have made full-length cDNA synthesis 13. Tisdale, M., Ertl, P., Larder, B. A., Purifoy, D. J. M., Darby possible (Fig. 5). Our result is consistent with the observation G. & Powell, K. L. (1988) J. Virol. 62, 3662-3667. 14. Chandra, A., Gerber, T. & Chandra, P. (1986) FEBS Lett. 197, that recombinant reverse transcriptase that lacks RNase H 84-88. activity synthesizes much longer cDNA products. 15. Starnes, M. C. & Cheng, Y. (1989) J. Biol. Chem. 264, 7073- We observed that only the 100-lcDa protein is found in the 7077. nucleus of infected SF9 cells (data not shown). This result 16. Leuthardt, A. & Le Grice, S. F. J. (1988) Gene 68, 35-42. suggests that the 64 amino acids in the carboxyl terminus of 17. Sung, W. L., Zahab, D. M., MacDonold, C. A. & Tam, C. S. the protease contain a nuclear localization signal. However, (1986) Gene 47, 261-267. we were unable to locate a typical nuclear localization signal, 18. Garson, K., Percival, H. & Kang, C. Y. (1990) Virology 177, 4 or 5 residues of arginine and/or lycine flanked by proline or 106-115. glycine, in the 64 amino acids. Both deletion and/or site- 19. Luo, L., Li, Y. & Kang, C. Y. (1990) Virology 179, 874-880. 20. Smith, G. E., Fraser, M. J. & Summers, M. D. (1983) J. Virol. directed mutagenesis of this 64-amino acid region may pro- 46, 584-593. vide information on sequences responsible for inhibition of 21. Kang, C. Y. (1988) Adv. Virus Res. 35, 177-192. RNase H activity and for nuclear localization signal. 22. Matsuura, Y., Possee, R. D., Overton, H. A. & Bishop, The RNase H domain has been mapped to the carboxyl D. H. L. (1987) J. Gen. Virol. 68, 1233-1250. terminus of p66 and the enzyme activity is also present in 23. Rucheton, M., Lelay, M. N. & Jeanteur, P. H. (1979) Virology cleaved p15 (7). The pol5i cassette contains 13 amino acids 97, 221-223. from the carboxyl terminus of the RNase H gene but this does 24. Tanese, N. & Goff, S. P. (1988) Proc. Natl. Acad. Sci. USA 85, not provide RNase H activity (Fig. 6). 1777-1781. The baculovirus AcNPV is an extremely useful helper- 25. Larder, B., Purifoy, D., Powell, K. & Darby, G. (1987) Nature independent eukaryotic (London) 327, 716-717. expression vector that is easily engi- 26. Hizi, A., McGill, C. & Hughes, S. H. (1988) Proc. Natl. Acad. neered (20). When insect cells are infected with recombinant Sci. USA 85, 1218-1222. baculovirus carrying a foreign gene, they synthesize high 27. Tanese, N., Sodroski, J., Haseltine, W. A. & Goff, S. P. (1986) levels of recombinant products under the control of the J. Virol. 59, 743-745. polyhedrin gene promoter (19-22). We have expressed high 28. Flexner, C., Broyles, S. S., Earl, P., Chakrabarti, S. & Moss, levels of four cassettes of the HIV pol gene in SF9 cells. B. (1988) Virology 166, 339-349. Downloaded by guest on September 24, 2021