Identification of Sequences Downstream of the ... - Journal of Virology

JOURNAL OF VIROLOGY, Aug. 1997, p. 6003–6010 0022-538X/97/$04.0010 Copyright © 1997, American Society for Microbiology

Vol. 71, No. 8

Identification of Sequences Downstream of the Primer Binding Site That Are Important for Efficient Replication of Human Immunodeficiency Virus Type 1 XUGUANG LI,1,2 CHEN LIANG,1 YUDONG QUAN,1 RAVI CHANDOK,2 MICHAEL LAUGHREA,1,2 MICHAEL A. PARNIAK,1,2 LAWRENCE KLEIMAN,1,2,3 AND MARK A. WAINBERG1,2,3* McGill University AIDS Centre, Jewish General Hospital,1 and Departments of Medicine2 and Microbiology,3 McGill University, Montreal, Quebec, Canada Received 16 December 1996/Accepted 26 March 1997

Reverse transcription of retroviruses is initiated from an 18-nucleotide (nt) primer binding site (PBS), located within the 5* region of viral genomic RNA, to which the host cell-derived tRNA primer is annealed and also involves viral genomic sequences outside the PBS. We constructed proviral DNA clones of human immunodeficiency virus (HIV) that had selective deletions of either a 7-nt segment found immediately downstream of the PBS or an extended nontranslated 54-nt stretch located immediately downstream of the PBS and containing the aforementioned 7-nt segment. Synthesis of minus-strand strong-stop DNA was assessed with MT-4 cells infected with viruses derived from COS-7 cells that had been transfected with these various constructs. We found that similar levels of minus-strand strong-stop DNA as well as DNA produced after template switching were expressed in MT-4 cells infected with COS-7-derived wild-type viruses or with viruses that had the 7-nt segment deleted. In contrast, significantly lower levels of viral DNA were detected in MT-4 cells after infection with viruses that had deletions of the 54-nt stretch. Furthermore, the molecular clone containing the 7-nt deletion was able to replicate with wild-type kinetics, while that containing the 54-nt deletion displayed a significantly diminished capacity in this regard. Further deletion analysis showed that a 16-nt segment at the 3* end of this 54-nt segment was largely responsible for these effects. We also conducted studies to determine levels of viral mRNA in COS-7 cells that had been transfected with equivalent amounts of DNA derived from either a wild-type HIV construct or our various deletion mutants. In the case of transfections performed with the 7-nt deletion mutant and wild-type HIV DNA, high levels of viral mRNA transcripts were detected, which was not the case for the 54 nt-deletion mutant. However, these various mRNAs possessed similar stabilities, as shown through studies in which transcript formation was arrested by treatment of cells with actinomycin D. Thus, the 54-nt segment of 5* nontranslated RNA, located downstream of the PBS, is involved in efficient expression of each of viral DNA, mRNA, and infectious virus. Formation of the reverse transcription initiation complex involves base pairing between the PBS and a complementary 18-nt region at the 39 end of tRNA, as well as additional interactions between sequences that neighbor the PBS and the remainder of the tRNA primer. In avian retroviruses, the efficiency of a tRNATrp-PBS complex in initiation of reverse transcription was enhanced by inclusion of viral genomic sequences upstream of the PBS and the TCC loop of tRNATrp (1, 34). Furthermore, disruption of a stem-loop structure, i.e., the U-IR stem near the PBS, caused diminished reverse transcription in both avian and murine retroviruses (12, 13, 44, 48). We have studied the role in viral replication of noncoding sequences that lie downstream of the PBS by introducing a deletion of 7 nt immediately downstream of this region (33), designated pHIV/del-7. Alternatively, we generated a 54-nt deletion of the 59 portion of the noncoding region, designated pHIV/del-LD, located immediately downstream of the PBS and containing the aforementioned 7-nt sequence. Previous studies by our group have shown that the first of these deletions is not required for cell-free synthesis of minus-strand strong-stop DNA in reactions performed with recombinant reverse transcriptase (RT), NCp, tRNALys and viral RNA tem3 plate (37). We now show that deletion of the 7-nt segment (pHIV/del-7) had relatively minor effects on in vivo reverse transcription of the viral DNA product in MT-4 cells, in contrast to results obtained with deletion of the 54-nt segment, i.e., pHIV/del-LD. The latter sequence was also independently in-

Reverse transcription begins at the primer binding site (PBS) of unspliced retroviral RNA, to which a tRNA primer is bound (38). The PBS of human immunodeficiency virus type 1 (HIV-1) is located approximately 180 nucleotides (nt) from the 59 terminus of genomic RNA and is flanked at its 59 end by a region referred to as R/U5 (49). This R/U5 region possesses a number of functional activities, including a role in packaging of viral RNA, a role in binding of the Tat transactivator protein, and involvement in reverse transcription and integration of proviral DNA (1, 7, 12, 13, 14, 21, 24, 25, 28, 29, 34, 43, 44, 52, 56, 57). A 133-nt noncoding and untranslated region is located downstream of the PBS and upstream of the gag initiation codon (49). The function of this sequence, especially its 59 portion, is not well understood, even though its 39 end is thought to be involved in packaging, splicing, and dimerization of genomic RNA and in translation of viral proteins (2, 9, 11, 15, 32, 35, 41, 42, 45, 51). The PBS region of HIV-1 RNA and surrounding sequences appear to be highly structured as determined by computer modelling and chemical analysis (6, 8, 22). The unfolding of the tRNA primer and of the RNA template is thought to be mediated by the viral nucleocapsid protein (NCp) (30, 31, 37). * Corresponding author. Mailing address: McGill University AIDS Centre, Lady Davis Institute-Jewish General Hospital, 3755 Cote-SteCatherine Rd., Montreal, Quebec H3T 1E2, Canada. Phone: (514) 340 8260. Fax: (514) 340 7537. E-mail: [email protected]. 6003

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volved in efficient expression of viral mRNA. The pHIV/del-7 virus displayed replication kinetics similar to those of wild-type viruses, while the pHIV/del-LD virus, as well as viruses containing other deletions in this region, were significantly impaired in this regard. (The research performed by Xuguang Li was in partial fulfillment of the requirements for a Ph.D. from the Faculty of Graduate Studies and Research, McGill University, Montreal, Canada.) MATERIALS AND METHODS Molecular clones with deletion mutations in sequences surrounding the PBS. The HxB2D recombinant clone of infectious DNA, obtained from the National Institutes of Health reagent repository, was used as a starting material for further genetic alteration. We modified a previously described PCR-based megaprimer mutagenesis procedure to generate deletions in the vicinity of the PBS (47). The primer selected for the 7-nt deletion (pHIV/del-7), immediately downstream of the PBS (nt 654 to 660), was 59-TGGCGCCCGAACAGGGACCTGAAAGGG AAACCAGAG-39. The primer for deletion of the 54-nt segment (pHIV/delLD), also downstream of the PBS (nt 654 to 707), was 59-TGGCGCCCG AAC AGGGACCGCGCACGGCAAGAGGCG-39. These primers were used as forward primers in conjunction with a backward primer (termed Pst 1) (nt 1405 to 1422; 59-CCATTCTGCAGCTTCCTC-39) to specifically amplify sequences in regard to each of these deletions. The resulting amplified products were used as megaprimers with an additional primer, termed UPBS (upstream of PBS), located at the 59 terminus of the R region (59-AGACCAGATCTGAGCCTGGG AG-39). Amplified fragments were then digested with BglII and PstI and were inserted into a pSVK3 vector (Pharmacia Biotech, Montreal, Quebec, Canada). The cloned fragments were sequenced to verify that correct modifications of viral gene sequences had been made and were inserted into the HXB2D clone of infectious DNA as described previously (36). To further define minimal sequences in the 54-nt deletion (pHIV/del-LD), three additional constructs that had deletions at nt 654 to 671, 672 to 691, and 692-707, respectively, were generated. These were constructed by using the primers 59-GAGAGAGCTCTGGGTCCCTGTTCGGCG-39, 59-CCGTGCGC GCTTCAGCAAGCCGAGTCTTTCCCTTTCGCTTTC-39, and 59-CCGTGCG CGCCTGCGTCGAGAGAGC-39 in conjunction with the primer UPBS (see above). Figure 1 shows a graphic description of the mutant viruses generated. Wild-type HXB2D viral DNA was designated pHIV/WT. Replication potential of viral constructs. Molecular constructs containing the above-described mutations in leader regions surrounding the PBS were purified twice by CsCl2 gradient ultracentrifugation. These plasmids were transfected into COS-7 cells by using a standard calcium coprecipitation procedure (40). Viruscontaining culture fluids were harvested approximately 72 h after transfection and were clarified by centrifugation for 30 min at 4°C at 3,000 rpm in a Beckman GS-6R centrifuge, prior to filtration with a 0.2 mm-pore-size sterile membrane. Viral preparations were stored at 270°C until use. For purposes of infection, the viral stock was thawed and treated with 100 U of DNase I in the presence of 10 mM MgCl2 at 37°C for 1 h to ensure that any contaminating plasmids had been eliminated from the transfection inocula (36). Infection of MT-4 cells was performed by incubating cells at 37°C for 2 h with virus (50 ng of p24), following which the cells were washed three times with phosphate-buffered saline and incubated at 37°C with fresh medium. In some experiments, HIV-IIIB, kindly provided by R. C. Gallo, National Institutes of Health, Bethesda, Md., was used as a positive control. Culture fluids were monitored for virus production by RT assay (10) and by p24 (capsid protein [CA]) antigen detection enzyme-linked immunosorption assay (Abbott Laboratories, Abbott Park, Ill.). Detection of viral DNA. At various times after infection (4 to 8 h), MT-4 cells were collected and washed extensively with serum-free medium. To ensure that no contaminating plasmids were present, fluids from each wash were routinely checked by PCR with HIV-specific primers (36). Total cellular DNA was then isolated from these cells (40) and analyzed by PCR with specific primer pairs to amplify minus-strand strong-stop DNA (20, 60). Cellular DNA isolated from cells inoculated with heat-inactivated wild-type viruses served as a negative control to ensure that potentially contaminating plasmids had been eliminated. For minus-strand strong-stop DNA, UPBS, located at the 59 terminus of the R region (nt 468 to 489) (49), was employed as a forward primer, while the backward primer was AA559 (nt 621 to 604), which was modified from a previously published procedure (60). The expected product of this primer pair (i.e., UPBS-AA559) is 153 bp in length. To amplify viral DNA generated after the first template switch, we employed U3 (nt 1 to 21) as a forward primer and AR (nt 532 to 511) as a backward primer. To amplify viral DNA made after the second template switch, we used UPBS as a forward primer and PST, in the gag gene (nt 1422 to 1398), as a backward primer. As a negative control, we also employed cells that had been pretreated with 2 mM zidovudine (AZT) for 3 h prior to viral inoculation and maintained these cells in the presence of the drug for an additional 4 to 8 h prior to extraction of total DNA. PCR assays were performed with 50 mg of sample DNA,

FIG. 1. Schematic depiction of deletion mutations surrounding the PBS of HIV-1 proviral DNA. pHIV/del-7 represents a 7-nt deletion immediately downstream of the PBS; pHIV/del-LD represents a 54-nt deletion also immediately downstream of the PBS and containing the aforementioned 7-nt sequence. The initiation codon of the gag gene is indicated, along with relevant nucleotide positions. 50 mM Tris-Cl (pH 8.0), 50 mM KCl, 2.5 mM MgCl2, 2.5 U of Taq polymerase, 0.2 mM deoxynucleoside triphosphates, 10 pmol of 32P-end-labelled forward primer, and 20 pmol of unlabelled backward primer. Reactions were standardized by simultaneous amplification of b-globin sequences as an internal control (36, 60) and involved 30 cycles in which samples were subjected to 94°C (1 min), 60°C (1 min), and 72°C (1 min). Analysis of viral RNA by Northern and slot blotting. Analysis of viral mRNA expression in COS-7 cells, transfected with various DNA constructs, was performed by slot and Northern blotting procedures as described previously (10). The efficiency of transfection was routinely monitored by detection of viral CA, using monoclonal anti-p24 antibodies in an immunofluorescence assay (10). For Northern blots, total cellular RNA extracted from COS-7 cells was purified with a commercial RNA extraction kit (Biotecs, Houston, Tex.). The extracted RNA was treated with 100 U of DNase I, followed by phenol-chloroform extraction and ethanol precipitation, to ensure removal of any contaminating plasmids and cellular DNA. The RNA pellets were resuspended in diethylpyrocarbonatetreated double-distilled water. RNA samples (up to 20 mg) were fractionated on 1% agarose gels containing formaldehyde as a denaturant (10). RNA molecules were transferred to a Hybond-N nylon membrane (Amersham, Toronto, Canada) and hybridized with pBH10 viral DNA as a radiolabelled probe (nick translation system; Life Technologies, Toronto, Canada) as described previously (10). To quantify viral RNA transcripts derived from COS-7 cells, total cellular RNA (harvested at various times after transfection) was immobilized on nylon membranes, using a slot blot apparatus, followed by UV irradiation (Amersham). Hybridization reactions were performed as described for Northern blots (10). The quantity of viral RNA was determined by counting the radioactivity on the relevant filter pads by liquid scintillation. In some cases, viral RNA that had been packaged into virions (purified by sucrose gradient centrifugation) was also quantified by the slot blot protocol. To rule out the possibility that the samples tested also contained residual DNA, which might have been hybridized by the radiolabelled DNA probe, RNase digestion of RNA extracted from virions was performed with RNase A (Boehringer-Mannheim, Montreal, Canada) at a final concentration of 10 mg/ml at 37°C for 30 min, following which phenol-chloroform extraction was performed. RNA stability assay. Thirty-six hours after transfection, actinomycin D was added to the culture medium to block the transcriptional activity of RNA polymerase II (19). At different times, e.g., 0, 1, 3, and 6 h after addition of the drug, total cellular RNA was extracted by using an ultraspecTM-II RNA isolation system (Biotecs) and was treated with 100 U of RNase-free DNase I which was

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FIG. 2. Viral replication capacities of various constructs. Cell-free viruses harvested from COS-7 cells transfected with various molecular constructs (72 h posttransfection) were used to infect MT-4 cells. Culture fluids were collected and monitored for RT activity. The decreased viral production in MT-4 cultures after 1 week in the case of cells infected by pHIV/WT (h) and pHIV/del-7 ({) was due to viral cytopathology; fresh cells were not added to these cultures. E, cells infected by pHIV/del-LD; Ç, mock-infected cells. then removed by phenol-chloroform extraction. Two micrograms of RNA was used in reverse transcription reactions, with 59-TTTATTGAGGCTTAAGCAG TGGG-39 (nt 56 to 78) as an antisense primer in a total volume of 20 ml. One microliter of product was then amplified in a 15 cycle-PCR with 59-AGACCA GATCTGAGCCTGGGAG-39 (nt 14 to 35) as a sense primer and the same antisense primer mentioned above to yield a 65-bp DNA fragment. Products were analyzed on 5% polyacrylamide gels and further quantified by molecular imaging analysis. Detection of viral proteins produced by transfected COS-7 cells. Expression of viral proteins in transfected COS-7 cells was determined by using a commercial kit for detection of p24 CA antigen and by RT assay as described previously (10). Both intracellular and extracellular CA levels were determined in order to shed light on the efficiency of viral assembly. Viral proteins were also analyzed by Western blotting as described previously (10). For this purpose, protein samples (standardized on the basis of viral p24) were fractionated on sodium dodecyl sulfate–12% polyacrylamide gels and transferred to nitrocellulose filters (10). The filters were then blocked with 5% skim milk–0.05% Tween 20–phosphate-buffered saline at 37°C for 2 h, followed by exposure to sera obtained from HIV-1-seropositive individuals (10). After extensive washing with 0.05% Tween 20–phosphate-buffered saline, 125I-labelled goat anti-human immunoglobulin G (ICN, Mississauga, Canada) was added and left for 1 h at 37°C. The filters were then washed three times, dried, and exposed to Kodak X-Omat film at 270°C.

RESULTS Replication of virus deletion mutants. The mutations introduced into proviral DNA constructs (Fig. 1) include a deletion of the conserved 7-nt stretch located immediately downstream of the PBS (pHIV/del-7) and an extensive 54-nt deletion downstream of the PBS containing the aforementioned 7-nt segment (pHIV/del-LD) (Fig. 1). In addition, the 54-nt deletion region was subdivided by smaller deletions termed pHIV/delLD1, pHIV/del-LD2, and pHIV/del-LD3 (Fig. 1). To investigate the replication potentials of these constructs, viruses (containing 50 ng of p24) derived from COS-7 cells that had been appropriately transfected were used to infect MT-4 cells. Figure 2 shows that wild-type virus (pHIV/WT) and one of the deletion mutants (pHIV/del-7) replicated efficiently, as determined by levels of RT activity in culture fluids after 3 and 7 days. In contrast, the pHIV/del-LD mutant was significantly impaired in the ability to produce viral progeny (Fig. 2). Further analysis revealed that the pHIV/del-LD3 mutant was most severely diminished in its ability to replicate. We also studied the ability of viruses derived from transfec-

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tions of COS-7 cells to infect MT-4 cells, using a p24 antigen capture assay. In this instance, we also examined viruses that had been subjected to more extensive deletion mutagenesis than that for pHIV/del-LD. Two different concentrations of viral inoculum were used in each case. The results in Table 1 show that the pHIV/del-7 construct yielded levels of p24 similar to those from wild-type virus over 13 days, while both pHIV/del-LD and the pHIV/del-LD3 construct, with a deletion of 16 nt at the 39 end of the 54-nt LD deletion, were severely impaired and produced only low levels of p24 for at least 90 days in culture. In contrast, little or no effect was observed when pHIV/del-LD1, with a deletion of 18 nt at the start of this 54-nt segment, was employed. Moderate inhibition of p24 synthesis was noted when pHIV/del-LD2 was studied; this construct lacks a stretch of 20 nt at the center of this 54-nt region. These findings are consistent with the data in Fig. 2 and with the work presented below on synthesis of viral DNA in infected cells. Production of minus-strand strong-stop DNA in infected cells. We found that similar levels of viral RNA were packaged into viruses derived from COS-7 cells that had been transfected 72 h earlier with our various constructs (Fig. 3) (results, based on RNA/p24 ratios, are shown for pHIV/WT and pHIV/ del-LD). When these RNA preparations were digested with RNase as a negative control, little or no hybridizable material remained, indicating that contaminating viral DNA was not present in these preparations. Since previous work had shown that certain sequences surrounding the PBS were involved in reverse transcription in cell-free systems (24, 25, 33), we asked whether the modifications introduced into our constructs would result in impaired generation of viral DNA. Toward this end, total cellular DNA was isolated at 4 and 8 h after infection of MT-4 cells with viruses derived from COS-7 cells and was analyzed by PCR with primer pairs that specifically amplify minus-strand strongstop DNA as well as viral DNA that is generated after each of the first and second template switch events. We found that similar levels of minus-strand strong-stop DNA were present in MT-4 cells infected by pHIV/del-7 (Fig. 4A, lanes 6 and 12) and by wild-type virus (Fig. 4A, lanes 4 and 10) after 4 to 8 h. In contrast, MT-4 cells infected with the pHIV/del-LD mutant contained significantly decreased levels of minus-strand strong-stop DNA (Fig. 4A, lanes 8 and 14) (i.e., about 10 times TABLE 1. Levels of p24 antigen expression in infected MT-4 cellsa Viral construct

pHIV/WT pHIV/del-7 pHIV/del-LD pHIV/del-LD1 pHIV/del-LD2 pHIV/del-LD3

Inoculum (ng of p24)

50 10 50 10 50 10 50 10 50 10 50 10

p24 concn (ng/ml) on days 7

23.5 18.9 15.6 12.7 1.5 0.4 18.7 18.5 10.3 8.6 0.9 0.6

10

27.3 16.8 19.4 21.9 2.0 1.1 26.2 20.8 14.5 11.4 1.2 1.6

13

30

90

2.0 1.2

1.8 1.3

1.2 1.6

1.5 1.9

b

19.7 13.9b 16.5b 17.9b 2.4 1.3 17.4b 16.8b 15.7b 13.2b 2.1 1.9

a MT-4 cells were infected with various viral constructs, and p24 levels in culture fluids were measured. b After day 13, cytotoxicity resulted in the death of cultures that produced relatively high levels of p24. In contrast, cultures infected by the pHIV/del-LD and pHIV/del-LD3 viruses continued to generate low levels of p24 activity over extensive periods.

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FIG. 3. Relative quantities of viral RNA packaged into viral structures. ) or pHIV/WT COS-7 cells were transfected with either pHIV/del-LD ( (u). After 60 h, viruses in culture fluids were purified by sucrose gradient ultracentrifugation. RNA was extracted from equal amounts (on the basis of p24 content) of viruses and quantified by slot blot and liquid scintillation analysis (see Materials and Methods). Experiments were performed with three replicate samples; error bars represent standard deviations. In some cases, viral RNA was digested with RNase, and all hybridizable material was eliminated. Results are standardized to 100 for pHIV/WT (7 ng).

less than with wild-type virus as quantified by densitometry). As a control, we performed mock infections with culture fluids of COS-7 cells that had been transfected with DNA from cells inoculated with heat-inactivated viruses and were unable to detect a DNA signal (Fig. 3A, lanes 1, 2, and 3 for wild-type, pHIV/ del-7, and pHIV/del-LD, respectively). Consistent results were obtained with total cellular DNA by using primer pairs that amplify DNA that is present after the first template switch (Fig. 4B) as well as full-length reverse-transcribed DNA (Fig. 4C) (60). As an additional important control, we treated cells with 2 mM AZT in order to prevent synthesis of the viral DNA product generated after the first template switch. Indeed, we found, as expected, that such treatment did not affect levels of minusstrand strong-stop DNA in the case of either wild-type virus (Fig. 4A, lanes 5 and 11) or pHIV/del-7 (Fig. 4A, lanes 7 and 13), nor, in fact, did the presence of AZT affect the already diminished levels of minus-strand strong-stop DNA found in cells infected by pHIV/del-LD (Fig. 4A, lanes 9 and 15). In contrast, treatment with 2 mM AZT significantly impaired synthesis of DNA products generated after both the first and template switch events for each of the viruses tested (Fig. 4B and C) (compare lanes 4 and 5, lanes 6 and 7, lanes 8 and 9, lanes 10 and 11, lanes 12 and 13, and lanes 14 and 15). In the case of the full-length product (Fig. 4C), it should be noted that pHIV/del-LD, as expected, yielded a DNA product (lanes 8 and 14) smaller than that obtained with wild-type virus. In this case, treatment with AZT prevented the appearance of any detectable DNA product (Fig. 4C, lanes 9 and 15). Thus, the 54-nt untranslated sequence, located immediately downstream of the PBS, is necessary for both efficient reverse transcription and efficient infectivity. As expected from the results shown in Fig. 4, far less proviral DNA became integrated into MT-4 cells after infection with pHIV/del-LD than after infection with pHIV/WT or pHIV/del-7, but the pHIV/ del-LD DNA persisted for up to 3 months (data not shown). No evidence of revertant virus was observed as determined by

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sequencing during extensive cultivation, although p24 antigen could be detected at low levels for as long as 3 months. To further define the minimal necessary sequences within this 54-nt region, the pHIV/del-LD1, pHIV/del-LD2, and pHIV/del-LD3 viruses were used to infect MT-4 cells, and levels of reverse-transcribed DNA were determined (Fig. 5). Molecular imaging analysis showed that, in comparison with wild-type virus (Fig. 5, lanes 4 and 8), pHIV/del-LD3 was severely (i.e., .90%) impaired in synthesis of minus-strand strong-stop DNA (Fig. 5, lanes 3 and 7). Only a modest diminution ('50%) in generation of such material occurred when pHIV/del-LD2 was studied (Fig. 5, lanes 2 and 6), while no effect whatever was seen in the case of pHIV/del-LD1 (Fig. 5, lanes 1 and 5). These findings are consistent with the results on viral replication described above. Role of the untranslated region downstream of the PBS in viral gene expression. The data described above indicate that the 54-nt region is involved in generation of viral DNA, consistent with observations for cell-free systems (37). However, the observed reductions ('10-fold) might not have led directly to the near lethality of pHIV/del-LD, since postintegrational effects, e.g., generation of viral mRNA and proteins, might also have played a role. We therefore assessed what role the untranslated region downstream of the PBS might play in expression of viral mRNA. Figure 6 depicts the results of Northern blot analysis of viral RNA extracted from COS-7 cells transfected with either mutant or wild-type constructs. Levels of viral RNA transcripts in cells transfected with pHIV/del-LD were much lower than those in cells transfected with pHIV/

FIG. 4. Detection of viral DNA. Viruses harvested from culture fluids of COS-7 cells that had been transfected with various molecular constructs were standardized on the basis of p24 content and used to infect MT-4 cells. Total cellular DNA (approximately 50 mg) was isolated from infected cells at 4 and 8 h after infection and subjected to PCR analysis with primers that specifically amplify minus-strand strong-stop DNA (59). Primers amplifying b-globin were used as an internal control to monitor the input of sample DNA (59). Mock infections involved culture fluids derived from COS-7 cells that had been transfected with DNA from cells inoculated with heat-inactivated viruses. Lanes 1 to 3, cells exposed to heat-inactivated viruses HIV/WT, HIV/del-7, and HIV/delLD, respectively; lanes 4, 6, and 8, cells infected with HIV/WT, HIV/del-7, and HIV/del-LD, respectively; lanes 5, 7, and 9, cells infected with HIV/WT, HIV/ del-7, and HIV/del-LD in the presence of 2 mM AZT, respectively. Lanes 1 to 9, cells were maintained for 4 h after exposure to virus prior to extraction of DNA; lanes 10 to 15, same order of experiments as in lanes 4 to 9 except that DNA was extracted after 8 h, lanes 16 to 19, several dilutions of HxB2D plasmid as a positive control (i.e., 10-fold dilutions of plasmids in terms of copy numbers [5 3 102, 5 3 103, 5 3 104, and 5 3 105, respectively]). (A) Detection of minus-strand strong-stop DNA; (B) Detection of viral DNA generated after the first template switch. (C) Detection of viral DNA generated after the second template switch. (D) PCR amplification of b-globin DNA as an internal control.

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FIG. 5. Detection of minus-strand strong-stop DNA [(2)ssDNA]. Lanes: 1 and 5, infection by pHIV/del-LD1; 2 and 6, infection by pHIV/del-LD2; 3 and 7, infection by pHIV/del-LD3; 4 and 8, infection by pHIV/WT; 9, inoculation of cells with heat-inactivated wild-type virus. Positive controls of serially diluted HXB2D plasmids are as shown in Fig. 4.

WT, although the major three bands representing unspliced, singly spliced, and multiply spliced RNA were present in each case (Fig. 6A). Similar viral RNA transcript patterns were observed in COS-7 cells transfected with pHIV/del-7 and wildtype virus (data not shown). These results were further confirmed by quantitative slot blot analysis. Figure 6B shows that dramatically reduced levels of RNA transcript were present in cells transfected with pHIV/ del-LD compared with those transfected with wild-type virus (pHIV/WT) or pHIV/del-7. The differences were most pronounced at early time points after transfection (16 h). To exclude the possibility that the reduced levels of viral mRNA in pHIV/del-LD-transfected cells were due to instability, we determined the half-lives of the viral mRNA molecules produced following transfection of COS-7 cells by wild-type and mutated constructs. Toward this end, cells were treated with actinomycin D at 36 h after transfection, as described in Materials and Methods, and total RNA was extracted at 0, 1, 3, and 6 h thereafter and reverse transcribed to yield DNA. The results of specific PCR amplifications revealed an expected disappearance of relevant amplified genetic material over time (Fig. 7, top). Consistent with the results shown in Fig. 6, cells transfected with the HIV/del-LD construct produced much lower overall levels of mRNA than did those transfected by wild-type material. However, the rates of disappearance of viral RNA in both cases were nearly identical as shown by molecular imaging analysis (Fig. 7, bottom). Indeed, no differences in regard to stability were observed among mRNA molecules derived from the wild-type, pHIV/del-LD, pHIV/del-LD1, pHIV/del-LD2, or pHIV/del-LD3 construct (data not shown). Effects on viral protein synthesis. We next wished to investigate protein expression and viral assembly in COS-7 cells that had been transfected with wild-type DNA and the pHIV/ del-LD construct. Toward this end, p24 detection and Western blot analyses were performed with culture fluids and cell lysates. As expected on the basis of the RNA transcript results described above, COS-7 cells transfected with pHIV/del-LD produced lower levels of both intracellular and extracellular p24 after 16 h than cells transfected with pHIV/WT (Table 2). Interestingly, transfection with pHIV/del-LD did not result in an excess accumulation of intracellular p24 relative to that with other transfections, suggesting that viral protein assembly had proceeded normally. It was also important to determine whether the diminished synthesis of viral proteins associated with pHIV/del-LD would affect the profiles of the viral proteins produced by transfected COS-7 cells, in a system in which the same total amount of


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protein was analyzed in each case by gel electrophoresis. Figure 8 shows a Western blot analysis of proteins produced by COS-7 cells that had been transfected by pHIV/del-LD (lane 1) or pHIV/WT (lane 2) or mock transfected (lane 4). Lane 3 of Fig. 8 represents MT-4 cells infected by wild-type HIV. The contents of lanes 1 to 3 were equalized on the basis of the amount of p24 as determined by enzyme-linked immunosorbent assay. We found that viral protein profiles were essentially nondistinguishable among COS-7 cells transfected by the mutant (Fig. 8, lane 1) or the wild-type construct (lane 2) or MT-4 cells infected by wild-type virus (lane 3), nor were differences observed in regard to transfections by the pHIV/del-LD1, pHIV/del-LD2, or pHIV/del-LD3 construct (data not shown). Thus, deletion of the 54-nt stretch downstream of the PBS did not affect patterns of viral protein synthesis but rather led to a

FIG. 6. (A) Northern blots for detection of viral RNA. Total cellular RNA was purified from COS-7 cells 16 h after transfection with either pHIV/del-LD or pHIV/WT. Lane 1, RNA (20 mg) from cells transfected with pHIV/del-LD; lane 2, RNA (20 mg) from cells transfected with pHIV/WT; lane 3, RNA (10 mg) from cells transfected with pHIV/del-LD; lane 4, RNA (10 mg) from cells transfected with pHIV/WT; lane 5, RNA (20 mg) from mock-transfected COS cells. Molecular size markers are indicated. (B) Quantitative determination of viral RNA transcripts by slot blot analysis. Total cellular RNA was harvested from COS-7 cells and purified at 16, 24, 48, and 72 h, respectively, after transfection with various molecular constructs. Relative intensities were calculated by comparison with levels of radioactivity obtained with wild-type transfections after 72 h, which were defined as 100 (i.e., 2,478 cpm). Standard deviations (for four separate experiments) are indicated by error bars.

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FIG. 7. RNA stability assay. Actinomycin D was added to culture medium at 36 h after transfection of COS-7 cells, and total cellular RNA was extracted at 0, 1, 3, and 6 h thereafter. Levels of viral RNA were determined by RT-PCR (top) and analyzed by molecular imaging (bottom). Mock, RT-PCR performed with wild-type HIV RNA in the absence of RT; HIV/del-LD and HIV/WT, infections performed with HIV/del-LD and wild-type constructs, respectively.

marked decrease in levels of all viral proteins produced. This is because the untranslated sequences downstream of the PBS can influence the production of infectious progeny virus by affecting both reverse transcription and the expression of viral mRNA. DISCUSSION Reverse transcription is initiated at the PBS to which the tRNA primer is bound. The HIV-1 PBS is located about 180 nt TABLE 2. Intracellular and extracellular p24 levels in COS-7 cells transfected with pHIV/del-LD or pHIV/WT p24 level (ng/ml)a h after transfection

16 24 48 72

Intracellular

Extracellular

pHIV/del-LD transfection

pHIV/WT transfection

pHIV/del-LD transfection

pHIV/WT transfection

5.22 6 0.48 17.9 6 1.55 64.8 6 6.02 62.9 6 5.89

170 6 15.2 241 6 22.2 233 6 24.5 245 6 23.3

4.49 6 0.32 18.7 6 16.2 97.0 6 10.2 145 6 13.5

187 6 16.8 258 6 25.3 304 6 29.8 300 6 31.2

a At various times after transfection, both intracellular and extracellular viral p24 levels were determined. Data are means 6 standard deviations from four separate experiments.

FIG. 8. Viral protein analysis by Western blotting. Proteins isolated from COS-7 cells were analyzed by Western blotting as described in Materials and Methods. Lanes: 1, proteins from COS cells transfected with pHIV/del-LD; 2, proteins from COS cells transfected with pHIV/WT; 3, positive control, using proteins derived from MT-4 cells infected by HIV-IIIB; 4, proteins from mocktransfected COS-7 cells.

from the 59 terminus of unspliced RNA (23). The PBS is flanked by the R/U5 region at its 59 border and by a 133-nt untranslated sequence at its 39 end (23). Increasing evidence suggests that the untranslated sequences that flank the PBS are involved in several steps of viral replication, including reverse transcription, integration, expression of the proviral genome, and packaging. Two lines of data prompted us to initiate an identification of regions downstream of the PBS that are essential for viral replication. First, efficient retroviral reverse transcription requires interaction between primer tRNA and the RNA at multiple sites (1, 4, 5, 12, 13, 24–26, 51). Second, HIV-1 has evolved to choose tRNALys as a primer for optimal 3 growth (16, 27, 36, 58). As an additional control, we also employed AZT, as described in Materials and Methods, to block synthesis of viral DNA products. Consistent with previous observations, we found that AZT had no effect on the presence of minus-strand strong-stop DNA, which is carried into cells by the virions in which it is made (39, 54). However, the use of AZT efficiently interfered with production of DNA products that are generated after each of the first and second template switch events, consistent with previous observations (3). Nonessentiality of a small, restricted region downstream of the PBS. A short, 6-nt sequence, located immediately downstream of the PBS, was previously shown to be important in specifying utilization of the tRNA primer (33). We have now shown that this sequence is apparently unnecessary either for synthesis of minus-strand strong-stop DNA in infected cells (pHIV/del-7) after transfection (Fig. 2) or for viral replication,

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consistent with in vitro studies that employed the nucleocapsid protein (37). Others have reported that nucleotide substitutions downstream of the PBS may be observed in HIV-1 revertants that have deletions within the PBS; these revertants appeared to have wild-type replication capacity (50). Thus, this small, 6- or 7-nt sequence may not be required in either maintenance of viral secondary structure or interaction with tRNALys 3 . Likewise, an 18-nt segment encompassing the abovementioned 7 nt downstream of the PBS is not essential for synthesis of viral DNA and viral replication, despite being DNase sensitive (17). It should be noted that our experiments were performed with a transient-transfection system, while others employed “integrated templates” (17). While studies on integrated templates would have provided additional information, the results in Fig. 2 show that virus infectivity was not severely affected by deletion of a 7-nt stretch downstream of the PBS. Identification of sequences important for viral replication downstream of the PBS. An extended 54-nt deletion, which does not compromise packaging or dimerization signals or the splice donor (2, 11, 15, 35, 41, 52), was found to significantly diminish virus replication (pHIV/del-LD) (Fig. 2). The proximity of this 54-nt region to the PBS might play a role in limiting viral replication, due to a defect in reverse transcription (37). Measurement of reverse-transcribed DNA, shortly after viral entry, revealed only low levels of product (Fig. 4). This was most pronounced when an 18-nt segment at the 39 end of the 54-nt region was removed (Fig. 5). This deficit in reverse transcription could be due to poor NCp-mediated interactions between the tRNA primer and the RNA template (37). In the case of minus-strand strong-stop DNA, it can be argued that the above-described experiments do not distinguish between synthesis of material that is present in virions versus that produced in cells after infection (39, 54). For this reason, we employed AZT to influence the synthesis of viral DNA products that are generated after the first template switch. As previously demonstrated, we found that the use of AZT in our protocols did not result in chain termination until after the first template switch event (3, 60). The potential role of contaminating DNA plasmids was also ruled out through mock infection protocols performed with DNA from cells inoculated with heat-inactivated viruses (see Materials and Methods). We also found that deletion of the 54-nt region resulted in a significant decrease in accumulation of viral RNA transcripts in transfected cells (Fig. 6). The relationship between defects in reverse transcription and mRNA production is unknown. The 54-nt segment could conceivably have affected both of these activities. Of course, the observed differences could be explained if pHIV/del-LD mRNA was less stable than wildtype viral RNA. However, we did not observe differences in stability between the various wild-type and mutated RNA transcripts studied. It is also unlikely that this region would influence efficiency of integration, since the latter process is primarily affected by the termini of reverse-transcribed viral DNA (46, 56). Indeed, MT-4 cells infected with the pHIV-1/del-LD virus contained integrated proviral DNA. Our results show that the 54-nt stretch downstream of the PBS is important for virus replication and that a 16-nt sequence located at the 39 end of this segment is principally involved. Further studies on nucleosome structure and/or identification of potential transcription factor binding sites, through the use of reporter genes, will yield additional functional information, as will further deletion analysis of the 16-nt segment itself (17, 18, 53, 55). Most binding elements for inducible or constitutive cellu-


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lar transcripts are located upstream of the PBS (for a review, see reference 13). However, untranslated sequences downstream of the PBS can also affect viral transcription activity, as documented here. It is also interesting that the 54-nt deletion had little effect on packaging of viral RNA, despite overlap at its 39 end with a substitution mutation that extends further downstream and that can affect RNA encapsidation (32). Differences between these constructs could have resulted in RNA structures that may have been differentially recognized by viral proteins involved in selection and encapsidation. The use of different viral strains and cell lines might also have contributed to differences between the studies. In summary, we have shown that a 54-nt segment in the noncoding region of the HIV-1 genome, downstream of the PBS, is important for viral replication in two principal ways. First, this region clearly is necessary for efficient reverse transcription of the viral DNA product, including that which is generated both prior to and after each of the two template switch events. In addition, this region is important for the efficient generation of viral mRNA and consequently for the synthesis of viral protein and infectivity. Each of these effects, i.e., on reverse transcription and on synthesis of viral transcripts, seems to be independent of the other. This was shown through (i) studies in which transfection of cells with deleted DNA constructs failed to generate significant levels of viral mRNA and (ii) independent experiments in which infection by viruses containing relevant deletions in viral RNA yielded extremely low levels of viral DNA products generated both before and after template switching. ACKNOWLEDGMENTS Xuguang Li and Chen Liang contributed equally to this project. This research was supported by grants from the Medical Research Council of Canada and by Health Canada. Xuguang Li was supported by a postdoctoral fellowship from the National Health Research Development Programme of Health Canada. Mark A. Wainberg is a National AIDS Scientist of Health Canada. REFERENCES 1. Aiyar, A., D. Cobrinik, Z. Ge, H. J. Kung, and J. Leis. 1992. Interaction between U5 viral RNA and the TCC loop of the tRNATrp primer is required for efficient initiation of reverse transcription. J. Virol. 66:2464–2472. 2. Aldovini, A., and R. Young. 1990. Mutations of RNA and protein sequences involved in human immunodeficiency virus type 1 packaging result in production of noninfectious virus. J. Virol. 64:1920–1926. 3. Arts, E. J., and M. A. Wainberg. 1994. Preferential incorporation of nucleoside analogs after template switching during human immunodeficiency virus reverse transcription. Antimicrob. Agents Chemother. 38:1008–1016. 4. Arts, E. J., X. Li, Z. Gu, L. Kleiman, M. A. Parniak, and M. A. Wainberg. 1994. Comparison of deoxy-oligonucleotide and tRNALys.3 as primers in an endogenous HIV-1 in vitro reverse transcription/template switching reaction. J. Biol. Chem. 269:14672–14680. 5. Arts, E. J., S. R. Strtor, X. Li, J. W. Rausch, K. J. Howard, B. Ehresmann, T. W. North, B. M. Wohrl, R. Goody, M. A. Wainberg, and S. F. J. LeGrice. 1996. Initiation of (2) strand DNA synthesis from tRNALys.3 on lentiviral RNAs: implications of specific HIV-1 RNA-tRNALys.3 interactions inhibiting primer utilization by retroviral reverse transcriptions. Proc. Natl. Acad. Sci. USA 93:10063–10068. 6. Baudin, F., R. Marquet, C. Isel, J.-L. Darlix, B. Ehresmann, and C. Ehresmann. 1993. Functional sites in the 59 region of HIV-1 RNA form defined structural domains. J. Mol. Biol. 229:382–397. 7. Berkhout, B., R. H. Silverman, and K. T. Jeang. 1989. Tat transactivates the human immunodeficiency virus through a nascent RNA target. Cell 59:273– 282. 8. Berkhout, B., and L. Schoneveld. 1993. Secondary structure of the HIV-2 leader RNA comprising the tRNA-primer binding site. Nucleic Acids Res. 21:1171–1178. 9. Berlioz, C., and J.-L. Darlix. 1995. An internal ribosome entry mechanism promotes translation of murine leukemia virus gag polyprotein procursors. J. Virol. 69:2214–2222. 10. Boulerice, F., S. Bour, R. Geleziunas, A. Lvovich, and M. A. Wainberg. 1990.

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