Proc. Natd. Acad. Sci. USA Vol. 89, pp. 6580-6584, July 1992 Medical Sciences Active nuclear import of human immunodeficiency virus type 1 preintegration complexes (retrovirus infection/virus entry/cel cyde) MICHAEL I. BUKRINSKY*, NATALIA SHAROVAt, MICHAEL P. DEMPSEY*, TREVOR L. STANWICK*, ALICE G. BUKRINSKAYAt, SHERYL HAGGERTY*, AND MARIO STEVENSON*t§ *Department of Pathology and Microbiology, University of Nebraska Medical Center and tEppley Institute for Cancer Research, Omaha, NE 68198; and tD. I. Ivanovsky Institute of Virology, Academy of Medical Science, Moscow, Russia Communicated by Duard L. Walker, March 16, 1992 (received for review January 23, 1992)

ABSTRACT After cell infection by the human immuno MATERIALS AND METHODS virus 1 nascent viral DNA in the form deficiency type (HIV-1), Cell and Virus Stocks. Preparation of cloned virus stocks, of a high molecular weight nucleoprotein preintegration comin standardization of virus titers, and conditions for virus in- plex must be transported to the nucleus of the host cell prior to fection are as described elsewhere (4). Before cell infection, integration ofviral DNA with the host genome. The mechm virus preparations were treated with DNase to remove con- used by retroviruses for nuclear targeting of preintegration taminating proviral DNA (5). complexes and dependence on cell division has not been estab- Isolation and Analysis of HIV-1 Preintegration Complexes. lished. Our studies show that, after infection, the preintegra- HIV-1-infected cells were lysed in hypotonic medium (6) tion complex of HIV-1 was rapidly transported to the nucleus using multiple strokes ofa Dounce homogenizer, and nuclear of the host cell by a process that required ATP but was integrity during cell lysis was monitored by phase-contrast independent of cell division. Functional HIV-1 integrase, an microscopy. Nuclei were extracted with a hypertonic buffer essential component of the preintegration complex, was not (6) and both nuclear and cytoplasmic extracts were fraction- required for nuclear import of these complexes. The ability of ated on nonionic density gradients as described (2). Integra- iHV-1 to use host cell active transport processes for nuclear tion activity in each gradient fraction was analyzed in vitro by import of the viral preintegration complex obviates the re- a modification of a previous protocol (7). Briefly, 100 ul of quireenlt for host cell division in establishment of the inte- each gradient fraction was mixed with 1 pug of ir AN7 target grated provires. These findgs are pertinent to our under- DNA (8) in a reaction volume of 150 Al and was incubated 60 ding of early events in the life cycle of HIV-1 and to the min at 220C. Samples were treated with DNA polymerase I mode of HIVE1 replication in terminally differentiated nondi- and deproteinated; in vitro integration products were identi- viding cells such as macrophages (monocytes, tissue macro- fied by two rounds of PCR with nested HIV-1 U5 and R long phages, follicular dendritic cells, and microglial cells). terminal repeat (LTR) primers. PCR products were visual- ized by Southern blot hybridization with 32P-end-labeled Integration of the retroviral genome with cellular DNA and oligonucleotide probes as described elsewhere (4). establishment ofthe provirus is an essential step in retrovirus PCR Analysis of HIV-1 DNA in Nuclear and Cytoplasmic replication (1). The integration reaction is catalyzed by a Cell Extracts. Cells were washed once in ice-cold phosphate- virus-encoded integrase, which is derived from the virus buffered saline (pH 7.2) and resuspended in lysis buffer (0.1 particle and which, after reverse transcription of genomic M NaCl/10 mM Tris HCI, pH 7.9/0.5% Nonidet P-40/1.5 viral RNA, remains associated with the viral cDNA in a high mM MgCl2) at 1 x 107 cells per mi. After 10 min at 40C, nuclei molecular weight nucleoprotein preintegration complex (2). were pelleted (5000 rpm1JA20 rotor). Supernatant (cytoplas- Targeting of the viral preintegration complex to host cell mic) and nuclear fracti6ns were extracted as described (9). DNA is therefore dependent on transport of this complex to The pelleted nuclei were deproteinated with proteinase K in the nucleus of the host cell. The process that directs nuclear the presence of 0.1% SDS, extracted with phenol and then localization of retroviral preintegration complexes after in- chloroform, and precipitated with ethanol. on cell division are DNA samples were subjected to repeated rounds of am- fection and the dependence ofthis process plification (25-35 cycles) exactly as detailed (4). For two unknown. Oncogenic retroviruses require a dividing host cell rounds of PCR with nested primers, 1 Al of first-round PCR for completion of the virus life cycle. This refractiveness to products was reamplified with internal primers in a reaction productive infection in stationary cells appears to be manifest vol of 50 ul. at a stage in the virus life cycle preceding establishment ofthe Locations of PCR Primrs and Oligonacleotde Probes Used integrated provirus and may be attributable to inefficient in This Study. For details ofthe HIV-1 pol and mitochondrial virus infection, reverse transcription, nuclear targeting of amplification primers, see Stevenson et al. (4); for tubulin viral DNA, or a combination ofthese factors (reviewed in ref. primers, see Bukrinsky et al. (10). 1). The pattern of oncovirus replication in stationary cells In vitro integration products were amplified as follows: first contrasts that oflentivirus replication, in particular the ability round, + primer (9650-9669), - primer (9126-9143); second ofhuman immunodeficiency virus type 1 (HIV-1) to replicate round, + primer (9668-9688), - primer (9099-9117); probe in nondividing, terminally differentiated cells (3). In the (996-9713); one-LTR circle primers, env + primer (9008- experiments presented here, we demonstrate that active 9027), gag - primer (794-815), probe (9427-9446); two-LTR uptake processes are used by HIV-1 to target its genome to + R - the host cell nucleus, obviating the requirement for a dividing circle primers, U5 primer (9650-9669), primer (9591- host cell in establishment of the provirus. Abbreviations: HIV-1, human immunodeficiency virus type 1; LTR, long terminal repeat; AZT, 3'-azido-3'-deoxythymidine. The publication costs of this article were defrayed in part by page charge §To whom reprint requests should be addressed at: Department of payment. This article must therefore be hereby marked "advertisement" Pathology and Microbiology, University of Nebraska Medical Cen- in accordance with 18 U.S.C. §1734 solely to indicate this fact. ter, 600 South 42nd Street, Omaha, NE 68198-5120. 6580 Downloaded by guest on October 6, 2021 Medical Sciences: Bukrinsky et al. Proc. Natl. Acad. Sci. USA 89 (1992) 6581

9610), probe (9427-9446). Primers are numbered according to activity was associated with the nuclear cell extract in the sequence of Ratner et al. (11). Only 3' LTR map coor- gradient fractions with a density of 1.20 g/ml (Fig. 1C). This dinates are given. Plus-strand (minus-strand complementary) was consistent with the density at which integration activity and minus-strand (plus-strand complementary) primers are was observed in gradient fractionated nuclear extracts from denoted by + and -, respectively. control HIV-1-infected MT-4 cells (data not shown). The gradient fractions containing the greatest integration activity and viral DNA did not coincide with the distribution of RESULTS genomic DNA (tubulin primers) (Fig. 1C), indicating that Nuclear Import of HIV-1 Preintegration Complexes in HIV-1 DNA did not represent integrated viral sequences. Growth-Arrested Cells. Cells were arrested in the G1-S Contamination of nuclear extracts by cytoplasmic compo- phases of the cell cycle by using aphidicolin, an inhibitor of nents (as indicated by the presence of mitochondrial DNA in eukaryotic DNA polymerase a (12). Treatment of CD4+ each fraction; Fig. 1C) could not account for the nuclear MT-4 cells with aphidicolin (5 pg/ml) for 24 hr prior to HIV-1 association of HIV-1 DNA since integration activity, when infection was sufficient to dramatically reduce the level of normalized to the amount of viral DNA, was greatest in the DNA synthesis (Fig. 1A) and cell cycling in the culture (Fig. nuclear fractions. The distribution of in vitro integration 1B). Growth-arrested MT-4 cells were infected with the activity was indistinguishable from that observed in nuclear cloned virus isolate HIV-1 Mf as described (4) and, after 24 and cytoplasmic extracts prepared after acute HIV-1 infec- hr, nuclear and cytoplasmic cell extracts were prepared and tion of untreated (non-growth arrested) cells (data not fractionated by density equilibrium centrifugation in nonionic shown). Taken together, the data indicate that cell division gradients (2). Gradient fractions were removed and assessed was not required for nuclear localization ofthe preintegration for density, for the presence of viral DNA by PCR using complex of HIV-1. HIV-1 pol-specific primers (4), and for integration activity (as In analysis of the nuclear import of HIV-1 preintegration diagrammed in Fig. 1D). The highest level of integration complexes in growth-arrested cells, the contribution of com-

A B C 120 CYTOPLASMIC NUCLEAR FRACTION FRACTION G1 = 44% G1 = 88% 100 S =47% S =11% a = = 1% r=- qt W -_ Rco G2+M 9%/6 G2+M "~~cJ\!\!7 ""-Density (g/ml)

- 60 976 b.p. in vitro integration E 40 products

20

MT4 MT4 MT4 MT4 MT4 Aphidicolin 4=C Aphidicolin

D Gradient fractionated HIV-1 pre-integration i|L + n 479 b.p.- complex I,3 885 b~p 0 Pol

0

integration in vitro 288 b.p.- Tubulin

PCR amplification of 426 b.p. - Mitochondrial n AN7 sequences using HIV-1 LTR specific primers (976 b.p. product)

FIG. 1. Nuclear localization ofthe HIV-1 preintegration complex in growth-arrested cells. (A and B) DNA synthesis and cell cycle parameters in aphidicolin-treated MT-4 cells. MT-4 cells were incubated for 24 hr in medium with or without aphidicolin (5 ,ug-ml-1) and then analyzed for incorporation of [3H]thymidine (A) (presented as mean, n = 12; cells incubated at 4°C for 24 hr provided basal [3H]thymidine uptake levels) and DNA content (B) by flow cytometry of propidium iodide-stained cell DNA. (C) Characterization of HIV-1 preintegration complexes in gradient fractionated nuclear and cytoplasmic extracts after infection of growth-arrested MT4 cells. (D) Schematic representation of assay for HIV-1 integration activity in gradient fractions. Integration of HIV-1 within substrate DNA (ir AN7) in vitro is identified by two rounds of PCR with nested primers directed to U5 and R sequences of the HIV-1 LTR. The PCR product size of 976 base pairs (bp) represents the size of AN7 (8) together with incorporated HIV-1 U5 and R sequences from the 5' and 3' LTRs. Downloaded by guest on October 6, 2021 6582 Medical Sciences: Bukrinsky et al. Proc. Nad. Acad Sci. USA 89 (1992) plexes that were associated with the nuclear membrane, b observed in HIV-1-infected cultures containing the reverse that had not entered the nucleus, could not be excluded. I transcriptase inhibitor 3'-azido-3'-deoxythymidine (AZT) at address this issue, the kinetics of HIV-1 circle genon a concentration of 0.3 FLM (Fig. 2B). formation was monitored after infection of growth-arrest 1HV-1 Integrase Function Is Not Required for Preintegration cells. Circular forms ofthe retroviral genome containing oi Complex Transport. Components of the integration complex and two LTRs (Fig. 2A) are produced only after synthesis of HIV-1 include viral DNA and integrase protein (14). To linear viral DNA and its transport to the nucleus (1, 1 determine whether integrase function was necessary for presumably because the nucleus provides the conditio nuclear localization of HIV-1 DNA, growth-arrested MT4 necessary for correct circularization of retroviral DN. cells were infected with a mutant of HIV-1 deleted in the Consistent with this model, we were unable to detect circul C-terminal half of the integrase coding region (15) and the forms of the viral genome in cytoplasmic extracts of HIV- kinetics of virus circle formation was monitored. The inte- infected cells by PCR. Thus, although circle forms ofthe vii grase mutant was able to direct the formation of two-LTR genome do not appear to be preintegration precursors (1: circle forms of the viral DNA in growth-arrested cells with they represent a convenient marker with which to monit kinetics similar to that ofwild-type virus (Fig. 2C), suggesting nuclear localization of viral DNA. The PCR-based strate that the presence of a functional integrase protein is not used for identification of one- and two-LTR circle forms necessary for nuclear import of HIV-1 DNA. the viral genome is shown in Fig. 2A. After infection, t 1HV-1 Preintegration Complex Import Is ATP Dependent. earliest time for detection of viral DNA synthesis preced Intracellular ATP was depleted in MT4 cells by treatment circular genome formation by ;3 hr (Fig. 2B) and indicat with the metabolic inhibitors sodium azide and 2-deoxy-D- a rapid translocation of viral DNA to the nucleus a glucose in a glucose-free RPMI medium containing dialyzed consequent DNA circularization. Aphidicolin treatmenti fetal calf serum (16). Initial experiments indicated that syn- tarded neither cell infection by HIV-1 (determined by P( thesis of HIV-1 DNA was less efficient in ATP-depleted cell using primers to pol gene products) nor accumulation cultures; therefore, viral DNA synthesis was allowed to circular viral genomes (Fig. 2B). All samples contain proceed in the absence of metabolic inhibitors for the first 3 equivalent amounts of cellular DNA as analyzed by PCR a hr after HIV-1 infection. At 3 hr postinfection, culture tubulin primers (data not shown). Using the cloned vii medium was replaced with glucose-free medium containing isolate and culture conditions described in this study, no vi metabolic inhibitors. A dramatic, yet reversible, reduction in DNA synthesis or accumulation of circle DNA forms w intracellular ATP levels (from 2 x 10-5 to 3 x 10-10 M) was A B Mo-T/HIV Mo-T/ HIV -Aphidkoxin + Aphidicolir'

Time post-infection (h) 12 3 4 6 8 24 3 4 6 8 24 Linear Genorme R-U3RUU R- U5+

gag po/ env Pol X AM*...... of....t*_

1 -LTR circle .4

2-LTR circle V

C MT4/HIV MIN MT4/HIV MT4/ HIV MT4. HIV + Aphidicolin -Aphidicolin + Aphidicolin A27

Time post-infection (h) 0.5 3 5 8 24 Time post-infection (h) 3 5 8 24 3 5 8 4 3

POI Pol . ..

2 LTR cirde ij 2-LTR circle .::: 111'...': 4 4.

FIG. 2. Kinetics of nuclear HIV-1 DNA import in dividing and in growth-arrested cells. (A) PCR strategy for identification of circular HIV-1 DNA forms. U5 and R primers span the LTR-LTRjunction of the two-LTR circle and produce a 595-base-pair (bp) amplification product. gag- and env+ primers span both one- and two-LTR circle forms, resulting in amplification of 891-bp and -1.7-kilobase products, respectively. (B) Kinetics of viral DNA synthesis and circle genome formation in dividing and nondividing CD4+ T cells. After HIV-1 infection of aphidicolin-arrested MT4 and Mo-T cultures, cells were harvested at the indicated intervals and analyzed for the presence of viral DNA. (C) Integrase (IN)-negative mutant of HIV-1 directs formation ofcircular viral DNA. Aphidicolin-treated MT-4 cells were infected with IN-negative mutant virions and kinetics of two-LTR circle formation was monitored as outlined in B. Downloaded by guest on October 6, 2021 Medical Sciences: Bukrinsky et al. Proc. Natl. Acad. Sci. USA 89 (1992) 6583 observed within 30 min of initiation of the metabolic block the metabolic block 8 hr postinfection resulted in nuclear (Fig. 3A). At 8 hr postinfection, metabolic inhibitors were accumulation of viral DNA to levels approximating those in removed and replaced with fresh medium containing AZT control cultures 24 hr postinfection as well as the appearance (0.3 AIM). The synthesis of all viral DNA was monitored by of circle forms of the viral genome (Fig. 3D). These results PCR with pol-specific primers and nuclear localization of suggest an energy-dependent mechanism for the transport of HIV-1 DNA was followed by PCR with primers directed to HIV-1 DNA to the nucleus of the host cell. two-LTR circle products. Consistent with the results pre- Lack of HIV-1 DNA in Nuclei of Quiescent T Cells in Vivo. sented in Fig. 2, circular genome forms appeared several We (4) and others (5) have described restricted HIV-1 infec- hours after the synthesis of viral DNA in control cultures tion of metabolically inactive primary quiescent T lympho- (Fig. 3B). By contrast, there was no detectable two-LTR cytes (Go), which are phenotypically distinct from metabol- circle formation in cultures that had been depleted of ATP ically active T cells arrested in S phase by the action of (Fig. 3C). Upon removal ofthe metabolic block, intracellular aphidicolin. We have also reported that quiescent T lympho- ATP rapidly returned to control levels (10-5 M; Fig. 3A) and cytes ofHIV-i-infected individuals harbor full-length, extra- was accompanied by renewed two-LTR circle formation -3 chromosomal HIV-1 DNA, but not integrated viral se- hr later (Fig. 3C). The presence of high concentrations of quences (10). We therefore extended our observations to AZT ensured that renewed circle formation was due not to determine whether inefficient nuclear localization of HIV-1 the resumption of new rounds of HIV-1 infection and DNA DNA could account for the refractiveness ofquiescent T cells synthesis after removal of the metabolic inhibitors but rather to HIV-1 integration in vivo. The distribution of linear and to the nuclear import and circularization of presynthesized circular viral DNA in nuclear and cytoplasmic extracts of HIV-1 DNA that existed prior to removal of the metabolic enriched quiescent and activated lymphocytes from a HIV- block. 1-seropositive asymptomatic individual was analyzed. In the To ensure that the lack oftwo-LTR circle formation in low activated (HLA Dr+) cell fraction, total viral DNA (identified ATP conditions was a consequence of the inhibition of by HIV-1 pol-specific primers) was detected in both cyto- nuclear import of HIV-1 DNA and not interference with the plasmic and nuclear compartments of the cell. Consistent circularization process of viral DNA in the nucleus, the with the nucleus-specific location of circle forms of the presence oftotal viral DNA and oftwo-LTR circles in nuclear genome of oncogenic retroviruses (1, 13), two-LTR circle fractions prepared from ATP-depleted and control MT-4 cell forms of the HIV-1 genome were detected specifically in the cultures was analyzed (Fig. 3D). In the presence ofmetabolic nuclear fraction (Fig. 4). By contrast, viral DNA was found inhibitors, the nuclear accumulation of total viral DNA was predominantly in the cytoplasmic compartment of quiescent markedly reduced up to 9 hr postinfection as compared with (HLA Dr-) T lymphocytes while two-LTR circle forms ofthe normal cell controls (Fig. 3D Top). This was consistent with viral genome were not detectable (Fig. 4). Identical results the lack of detectible two-LTR circle formation, while the were obtained with fractionated lymphocyte populations metabolic block was in effect (Fig. 3D Middle). Removal of from two additional HIV-1-seropositive asymptomatic indi-

A Release of D metabolic block Sodium Azide/d-D-glucose4 AZT Time post-infection (h) 0.5 5.5 9 24 Cell Viability .- (% of control) Metabolic block + -+ + -+ .5 C .0 _ Ak .0 Pol _41_ c 4- CLa. u

1 2 3 4 5 6 7 8 9 10 11 12 13 2-LTR Circle Time post-infection (h)

B C

Time post-infection (h) 0.5 2.5 5.5 8 9 11 12 13 0.5 2.5 5.5 8 9 11 12 13 Tubulin A.AMLAIMLAMLS&ss

Pol

2-LTR Circle 4-1 -_

FIG. 3. ATP-dependent nuclear import of HIV-1 DNA. (A) Cell viability and ATP levels in metabolically arrested cells. At the indicated times postinfection, samples of control (B) and metabolically arrested (C) cells were removed for measurement of intracellular ATP levels by a bioluminescence assay (ATP levels in metabolically arrested cultures are expressed as percentage of ATP levels in HIV-1-infected control MT-4 cultures) and HIV-1 DNA accumulation. (D) Nuclear accumulation of HIV-1 DNA in metabolically arrested (lanes +) and control (lanes -) MT-4 cells. Downloaded by guest on October 6, 2021 6584 Medical Sciences: Bukrinsky et al. Proc. Nad. Acad. Sci. USA 89 (1992) B33 B33 Our studies provide evidence for an energy-dependent HLA Dr-- HLA Dr+ mode of transport of HIV-1 preintegration complexes to the nucleus of the host cell. The active import of nucleophilic z) z Q Z eukaryotic and viral proteins and of large viral ribonucleo- protein complexes is mediated by the presence of a nuclear localization signal, usually located at the N terminus of the protein, and requires ATP (reviewed in ref. 20). Additional studies are required to elucidate the viral and cellular com- w** HIV pol ponents involved in active nuclear import of HIV-1 preinte- gration complexes. We thank S. Rhode III for discussion, B. Cullen for communicat- ing results before publication, C. Kuszynski and G. Perry for assistance in cell cycle analysis, J. Edwards for artwork, and K. Hansen for manuscript preparation. M.S. was supported by Grants 2 - LTR circle A124481 and A130386 from the National Institutes of Health and by funds from the Nebraska Research Initiative. S.H. is a scholar ofthe American Foundation for AIDS Research (AmFAR). 1. Varmus, H. E. & Swanstrom, R. (1984) inRNA Tumor Viruses, eds. Weiss, R. A., Teich, N., Varmus, H. & Coffin, J. (Cold Spring Harbor Lab., Cold Spring Harbor, NY), pp. 369-512. 2. Bowerman, B., Brown, P. O., Bishop, J. M. & Varmus, H. E. _w w ~~~Tubulrn (1989) Genes Dev. 3, 469-478. 3. Gendelman, H. E., Narayan, O., Kennedy-Stoskopf, S., Ken- nedy, P. G. E., Ghotbi, Z., Clements, J. E., Stanley, J. & Pezeshkpour, G. (1986) J. Virol. 58, 67-74. 4. Stevenson, M., Stanwick, T. L., Dempsey, M. P. & Lamonica, FIG. 4. Subcellular location of HIV-1 DNA in T cells from a C. A. (1990) EMBO J. 9,1551-1560. HIV-1-infected individual. Nuclear and cytoplasmic extracts from 5. Zack, J. A., Arrigo, S. J., Weitsman, S. R., Go, A. S., Haislip, enriched quiescent (HLA Dr-) and activated (HLA Dr+) lympho- A. & Chen, I. S. Y. (1990) Cell 61, 213-222. cytes were analyzed for the presence of total viral DNA (pol) and 6. Sugasawa, K., Murakami, Y., Miyamoto, N., Hanaoka, F. & two-LTR circle forms ofthe viral genome. The level ofHLA Dr+ cell Ui, M. (1990) J. Virol. 64, 4820-4829. contamination (determined by fluorescence-activated cell sorting 7. Ellison, V., Abrams, H., Roe, T., Lifson, J. & Brown, P. 0. with interleukin 2 receptor antibodies) of HLA Dr- enriched cells (1990) J. Virol. 64, 2711-2715. was <1%. 8. Lutz, C. T., Hollifleld, W. C., Seed, B., Davie, J. M. & Huang, H. V. (1987) Proc. Nati. Acad. Sci. USA 84, 4379-4383. viduals (data not shown). Presumably, quiescent primary T 9. Panganiban, A. T. & Temin, H. M. (1983) Nature (London) 306, 155-160. lymphocytes, which are highly differentiated but metaboli- 10. Bukrinsky, M. I., Stanwick, T. L., Dempsey, M. P. & Steven- cally inactive (17), do not provide the energy requirements son, M. (1991) Science 254, 423-427. for active nuclear import ofHIV-1 preintegration complexes. 11. Ratner, L., Haseltine, W. A., Patarca, R., Livak, K. J., Star- cich, B., Josephs, S. J., Doran, E. R., Rafalski, J. A., White- horn, E. A., Baumeister, K., Ivanoff, L., Petteway, S. R., DISCUSSION Pearson, M. L., Lautenberger, J. A., Papas, T. S., Ghrayeb, J., Chang, N. T., Gallo, R. C. & Wong-Staal, F. (1985) Nature The studies outlined in this report are pertinent to our (London) 313, 277-284. understanding of the relationship between cell cycling and 12. Pedrali-Noy, G., Spadari, S., Miller-Faures, A., Miller, nuclear import of HIV-1 preintegration complexes. These A. 0. A., Kruppa, J. & Koch, G. (1980) Nucleic Acids Res. 8, studies also illustrate that, in contrast to oncoviruses, the 377-387. completion of early events in the lentivirus life cycle (fol- 13. Brown, P. O., Bowerman, B., Varmus, H. E. & Bishop, J. M. lowing virus infection and preceding provirus establishment) (1987) Cell 49, 347-356. 14. Farnet, C. M. & Haseltine, W. A. (1991) J. Virol. 65, 1990- does not require a dividing host cell. As demonstrated here, 1915. growth-arrested T cells (G1-S) allow efficient HIV-1 infec- 15. Stevenson, M., Haggerty, S., Lamonica, C. A., Meier, C. M., tion, reverse transcription of viral DNA, and nuclear import Welch, S. & Wasiak, A. (1990) J. Virol. 64, 2421-2425. of viral preintegration complexes. Our results are consistent 16. Richardson, W. D., Mills, A. D., Dilworth, S. M., Laskey, with recent studies demonstrating provirus establishment R. A. & Dingwall, C. (1988) Cell 52, 655-664. and virus production after HIV-1 infection of irradiated 17. Maul, G. G. (1977) Int. Rev. Cytol. (Suppl.) 6, 75-186. primary monocytes completely lacking cellular DNA synthe- 18. Weinberg, J. B., Mathews, T. J., Cullen, B. R. & Malim, M. H. (1991) J. Exp. Med. 174, 1477-1482. sis (18). These observations explain in part the ability of 19. Stevenson, M., Bukrinsky, M. I. & Haggerty, S. (1992) AIDS HIV-1 to replicate in metabolically active nondividing cells Res. Hum. Retroviruses 8, 107-117. such as monocyte-macrophages, dendritic cells, and micro- 20. Garcia-Bustos, J., Heitman, J. & Hall, M. N. (1991) Biophys. glial cells (reviewed in ref. 19). Acta 1071, 83-101. Downloaded by guest on October 6, 2021