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    00001

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    Artigo5

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    JAIDS Journal of Acquired inmane Defiieny Syndromes BELO To99 Lappncon Willams & Wiking, ne. Philadelphia Nitric Oxide Modulates HIV-1 Replication *Joan B. Mannick, + jonathan S. Stamler, *Edna Teng, *Neal Simpson, “John Lawrence, *Jeff Jordan, and *Robert W. Finberg "Department of Adult Oncology, Dana Farber Cancer Institute, and Department of Medicine, Brigham and Women’s Hospital and Harvard Medical School, Boston, Massachusetts: and *Department of Medicine, Divisions of Respiratory and Cardiovascular Medicine and Department of Cell Biology, Duke University Medical Center, Durham, North Carolina, U.S.A. Summary: Although nitric oxide (NO) production is increased in HIV-1-infected patients, and NO is known to inhibit the replication of several viruses, very litte is known about the effects of NO on HIV-1 replication. In the present studies, we find that S-nitrosothiols (RSNOs), a class of NO donor compounds present in the human circulatory system, inhibit HIV-1 replication in acutely infected human peripheral blood mononuclear cells (PBMCs) and have an additive inhibitory effect on HIV-1 replication in combination with 3'-azido-3"-deoxythymidylate (AZT). RSNOs inhibit HIV-1 replication in acutely infected PBMCs at a step in the viral replicative cycle afler reverse transcription, but before or during viral protein expression through cGMP-independent mechanism. In the latently infected U1 cell line, NO donor com- pounds and intracellular NO production stimulate HIV-1 reactivation. These studies suggest that NO both inhibits HIV-1 replication in acutely infected cells and stimulates HIV-1 reactivation in chronically infected cells. Thus, NO may have a physiologic role in HIV-1 replication, and NO donor compounds, which have been used for decades in the treatment of coronary artery disease with limited to treatment of HIV-1 disease by inhibiting acute infection, react ‘Virus replication—Peripheral blood mono- both. Key Words: HIV-I—Nitric oxi nuclear cells—UI cells In vivo, cells are exposed to two potential sources of nitric oxide (NO): NO synthesized intracellularly by the enzyme nitric oxide synthase (NOS) and NO synthesized extracellularly by NOS activity in adjacent cells or by naturally occurring NO donor compounds in the circula- tion such as the S-nitrosothiols (RSNOs) (1,2). Similar to cytokines, NO and its adducts or reaction products, re- ferred to here as NO-related species, exert complex and sometimes opposing effects on biologic responses (3). For instance, NO-telated species can be either neuropro- tective or neurotoxic in the central nervous system (CNS) (4); NO-telated species induce apoptosis in mac- rophages (5) but inhibit apoptosis in B cells (6.7); and Address correspondence and reprint requests to Joan B. Mannick, ana 1440, Dana Farber Cancer Insitute, 44 Binney Stret, Boston, MA 02115, USA; email:Joan_Mannick@dfci harvard. Manuscript received October 26, 1998; accepted June 10, 1999 might be useful in the ting latent virus, or NO-related species activate NF-xB in peripheral blood, ‘mononuclear cells (8) but inhibit NF-xB in endothelial cells (9), NO has been shown to inhibit replication of multiple viruses (6,10-26). It remains unclear, however, whether NO has a physiologic effect on HIV-1 replication, al- though HIV-I has been reported to increase NO produe tion in infected monocytes (27), and several studies have documented that NO production is increased in HIV-1— infected patients (28-31). Further, increased NO produc- tion is believed to contribute to HIV-I-associated de- mentia (32-34). Three studies have directly examined the effects of NO on HIV-I replication. In one study, intracellular NO production was induced in the chroni- cally infected UI cell line by crosslinking CD23 in the presence of interleukin-4 (IL-4). The effect of NO on. HIV-1 replication was variable and dependent on the level of constitutive p24 expression in cells (35). A sec- a J.B, MANNICK ET AL. ‘ond study found no effect of endogenous NO production ‘or of NO donor compounds on HIV-1 replication in acutely infected monocyte-derived macrophages (36). ‘The third study found that NO donor compounds inhibit HIV-| replication in an astrocytoma cell ine possibly by inhibiting HIV-1 long terminal repeat (LTR) transcrip- tional activity (37). A potential inhibitory role for NO in HIV-1 infection is also suggested by the recent finding that the NO donor compound NOR-3 inhibits the HIV-1 protease, perhaps through S-nitrosylation of critical cys- teine residues (38). To further assess the potential physiologic or thera- peutic role of NO in HIV-1 infection, the present study analyzes the effects of intracellular NO production or NO donor compounds on HIV-1 replication in acutely infected PBMCs as well as in chronically infected UL cells. Our results suggest that NO donor compounds in- hibit HIV-1 replication in acutely infected PBMCs. In addition, NO donor compounds as well as constitutive intracellular NO production stimulate HIV-1 reactivation in chronically infected cells. M ETHODS Inhibition of HIV-1 Replication in Acutely Infected Peripheral Blood Mononuclear Cells with S-Nitrosothiols Peripheral blood mononuclear cells isolated from HIV-negative adult donors were stimulated for 48 hours with $ pg/ml of phytohe- magglutnin (PHA), infected with HIV-I strain MN at an mulkiplicity ‘of infection (MOD of 0.1 for It 2 hours at 37°C, washed three times and resuspended at 500,000 cellsiml in complete culture medium (RMPI-1640 containing 10% fetal bovine serum [FBS]) supplemented with 10 Uml of IL-2, with or without various concentrations of S- nitroso-N-acetylpenicillamine (SNAP), N-acetypenicllamine (NAP), (0.025 uM AZT. The cells were then plated in 24-well plates with 1 x 10° cellviwell. After 4 days, the cells were split and refed with 1L-2-containing complete culture medium. After 6 days in culture celHfiee supernatants were collected, and p24 antigen levels deter mined using the HIV-1 p24 enzyme-linked immunosorbent assay (ELISA, DuPont Chemical Co,, Wilmington, DE, U.S.A.) per the ‘manufacturer's instructions ‘Measurement of Cellular Proliferation in Acutely Infected Peripheral Blood Mononuclear Cells PHA-stimulated PBMCs resuspended in IL-2-containing medium as already described were plated in 96-well plates with 100 of cells/well Various concentrations of SNAP, NAP andlor 0.025 M AZT in a volume of 100 of IL-2-contaning medium were added to each wel. On day 4, the cells were spit and refed with IL-2-containing medium. ‘On day 6, 1 mCi of witiated thymidine (NEN, Boston, MA, U.S.A.) was ‘added to each well, the cells were harvested 18 hours ater and thymidine levels measured as described previously (39). Polymerase Chain Reaction Analysis of Proviral Levels in Peripheral Blood Mononuclear Cells PHA-stimulated PBMCs were infected with HIV-1 at an MOI of 0.1 and were then incubated in IL-2-containing medium inthe presence or absence of SNAP of NAP. Alter 24 hours (approximately one vial replicative cycle, 1 x 10% ces were lysed in OX of bur (0.001% [v/v] Triton X-100, 0.00015 [w/v] sodium dodecyt sulfate [SDS}, 10 mM Tris-Cl, pH 80, 1 mM ethylenediaminetetraacetic acid [EDTA)) containing 15 pl of 20 mg/ml proteinase K for 1 hour at 56°C, fllowed by inactivation of proteinase K for 60 minutes at 94°C, Twenty-five jl of each cell Iysate was PCR amplified in a 50-pl reaction using gae-specific primers SK38 (ATA ATC CAC CTA TCC CAG TAG GAG AAA 7) and SK39 (TTT GGT-CCT TOT CTT ATG TCC AGA ATG C) (40,41). PCR conditions were 15 seconds denaturation at 94°C, 30 seconds of annealing at 57°C, and 75 seconds of extension at 72°C for 40 cycles sing a Gene Amp 9600 PCR system (Perkin-Elmer Cetus, Norwalk, CT, U'S.A.. The amplified products were analyzed on 1 2% Nusieve agarose GTG, (FMC Corporation, King of Prussia, PA, U.S.A), 1% Sea Kem ME agarose (FMC Corporation) ge containing 05 gil ethidium bromide Inhil mn of HIV-1 Replication in U1 Cells With 1-NG-Monomethyl-t-Arginine Logarthmically growing UI cells were resuspended a 200,000 cellsiml in RPMI-1640 culture medium (GIBCO, Grand Island, NY. U.S.A.) containing 10% FBS, and plated in 24-well plates with 2 ml of cellvwel. After 4 days, the cells were washed three times, resuspended at 200,000 cellsal in RPMI-1640 10% FBS containing 0.1 mM arg nine, and plated in 24-well plates with 1 ml of cellswell, NG ‘monomethy-L-arginine (L-NMA, 0-00) uM), L-arginine (1 mM), 0- arginine (1 mM), SNAP (10 4M) or NAP (10 uM) were added to the appropriate wells. p24 antigen levels were measured in cell-free super- natants using the HIV-1 p24 ELISA (DuPont) per manufacturer's in- Western Blot Analysis Westem blots were performed as described previously (6). RESULTS ‘S-Nitrosothiols Inhibit HIV-1 Replication in Acutely Infected Peripheral Blood Mononuclear Cells To determine the effects of intracellular NO. produ tion or extracellular NO donor compounds on HIV-1 replication in acutely infected cells, human PBMCs were acutely infected with HIV-1 (MN strain), and then cul- tured in IL-2-containing medium in the presence or ab- sence of various concentrations of the NOS inhibitor NG-monomethyl-.-arginine (L-NMA), RSNOs, a natu- rally occurring class of NO donor compounds present in the human circulatory system, or control parent com- pounds that do not generate NO-related species. Intra- cellular NO production had no consistent effect on HIV- 1 replication in acutely infected cells (data not shown). JAIDS Jounal of Acquired Immune Deficiency Syndromes, Vol. 22, No. I, Seplember 1, 1999 NITRIC OXIDE MODULATES HIV-1 REPLICATION However, the RSNO S-nitroso-N-acetylpenicillamine (SNAP), but not by the control compound N-acetylpeni- cillamine (NAP), inhibited HIV-I replication in acutely infected PBMCS in a dose-dependent manner without associated cytotoxicity (Fig. 1A). Similar inhibitory ef- cts were seen using the NO donor compound S-nitroso- cetyleysteine (SNAC) but not with the control parent ‘compound N-acetyleysteine (NAC; data not shown), S-Nitrosothiols and Azidothymidine Have istic Inhibitory Effects on HIV-1 Replication Because of the strong rationale for combination che- motherapy in the treatment of HIV-I infection, the ef- fects of a combination of NO donors and the reverse transcriptase inhibitor AZT on HIV-1 replication were analyzed. PBMCs infected with HIV-1 as previously de- scribed were grown in the presence or absence of AZT. alone or in combination with various concentrations of SNAP or NAP. SNAP in combination with AZT had an additive or synergistic inhibitory effect on p24 levels (Fig. 1B), S-Nitrosothiols Inhibit HIV-1 Replication Without Inhibiting Cellular Proliferation Given that NO-telated activity inhibits lymphocyte proliferation (4244), and inhibition of cellular prolifera- tion is associated with an inhibition of HIV replication (45-47), the NO-induced inhibition of HIV replication might simply result from an antiproliferative effect of NO-generating compounds. However, concentrations of, SNAP =250 4M, alone or in combination with AZT, had no inhibitory effect on lymphocyte proliferation de- spite inhibiting HIV-I replication. Concentrations of RSNO >250 1M were associated with an inhibition of cellular proliferation. Subsequent experiments were per- formed using concentrations of SNAP <250 4M to ana- lyze the antiviral effects of NO-related species that are independent of their antiproliferative effects. S-Nitrosothiols Inhibit HIV-1 Replication Through a GMP-Independent Mechanism ‘One of the mechanisms by which NO exerts biologic effects is activation of guanylate cyclase leading to in- creased cGMP levels. However, neither the CGMP ana- Jogue 8-bromo-cGMP (100 or 1000 wM) nor the CGMP- dependent phosphodiesterase inhibitor zaprinast (1 or 10 1M) inhibited HIV-1 replication in acutely infected PBMCS (data not shown), which suggests that the anti- viral effect of NO is GMP independent. S-Nitrosothiols Inhibit HIV-1 Replication at a Step in the Viral Replicative Cycle After Reverse Transcription To determine the mechanism by which RSNOs inhibit HIV. replication, the effects of SNAP or NAP on full- length HIV-1 proviral DNA levels in acutely infected PBMCs were measured by semiquantitative PCR using gag-specific primers. Inhibition of full-length HIV-1 proviral DNA levels by RSNOs during a single viral replicative cycle would suggest that NO-related species inhibit HIV-1 replication before or during reverse tran- scription. To quantify the level of gag-specific PCR product in each sample, the relative intensity of each PCR signal was compared with the PCR signal obtained from 10-fold serial dilutions of ACH-2, a T-cell clone containing one proviral copy per cell (Fig. 2B). In addi- tion, the relative intensity of gag-specific PCR signals obtained from fivefold serial dilutions of each DNA sample was analyzed (Fig. 28). Twenty-four hours after HIV-1 infection (approximately one viral replicative cycle), the expected 115-bp gag-specific PCR product was detectable in all cell populations at approximately equal levels and the PCR product was undetectable in all samples at a 1:5 dilution, suggesting that HIV-1 proviral DNA levels are not measurably inhibited by RSNOs. Thus, NO-telated species do not appear to inhibit HIV-1 replication before or during reverse transcription in acutely infected PBMCs, ‘To confirm that the inhibitory effect of RSNOs did not result from an inhibition of reverse transcription, their effect on HIV-1 reverse transcriptase activity was ana- lyzed. Six days after infection with HIV-1, PBMCs were incubated in the presence or absence of SNAP or NAP for 24 hours (approximately one viral replicative cycle), and reverse transcriptase activity in cell-free superna- tants was measured (41). No inhibitory effect of SNAP or NAP on HIV-I reverse transcriptase activity was de- tected (Fig. 20). S-Nitrosothiols Inhibit Viral Protein Expression Finally, the effect of exogenous NO production on a later replicative step, that is, viral protein expression, was analyzed, PHA-stimulated PBMCs were infected with HIV-1 at an MOI of 5.0 and then were incubated in the presence or absence of SNAP or NAP. After 24 hours, viral protein expression was analyzed in whole cell ly- sates by western blot using antisera derived from HIV- [infected patients. SNAP (200 1M) but not the control ‘compound NAP (200 1M) significantly inhibited HIV-1 JANDS Journal of Acquired lnmane Deficiency Sydromes, Vo. 22, No.1, September 1, 1999 J. B, MANNICK ET AL 250 im SNAP 2 coo 4 1 Nap 21504 § 1004 © 504 & f t 9 + + a, + A vy" av " 50 " 100 | 200 " 250 0000 4 snap cued BB snapsazt Foon | & Control g so000 4 o Hemme | E soo004 ¥ 20000 T 10000. od ve ve AZT" 1" 100 "150" 200 " 250 e concentration SNAP (uM) 40000 30000 = = S 20000 10000 ° fe tgoeo soe ce coop eee epec ered BBS TOLPRS TPLPa “MEERA TOKENS ‘SNAP j SNAP NAP i c ay AZT AZT {NDS Jounal of mired immune Defic Syndromes Vol 22, Ne I Septem FIG. 1. S-nitrosothiols (RSNOs) inhibit HIV-1 replication in human peripheral blood mononuclear cells (PBMCs); effects alone and in combination with azidothymi dine. (A) The RSNO S-ntroso-N acetyipeniciliamine (SNAP) but not the control compound. N. acetyipenicilamine (NAP) inhibits HIV-1 replication. Phytohemag lutinin (PHA)-stimulated human PBMCs were infected with HIV-1 and then were cultured in the presence or absence of SNAP or NAP. P24 levels were analyzed in the cell free supematants by en zymelinked immunosorbent as say (ELISA) 6 days ater infection. The data are expressed as the percentage of control p24 levels (generated in the absence of added drug). and represent the mean + standard error of the mean of five separate exper ‘ments, * indicates p = .008. + in dicates p = .0001, paired Fest, n 5. (B) The effects of a combina: tion of SNAP and AZT on HIV-1 replication. The same experiment \was performed as in A inthe pres fence or absence of AZT (0.025 uM), various concentrations of ‘SNAP, or a combination of these. The data are expressed as the mean + standard error of the ‘mean of triplicate cultures and are representative of two separate ex periments. (C) SNAP and azido- thymidine inhibit HIV-1 replication ‘at concentrations that d0 not in hibit cellular proliferation. PHA. stimulated human PBMCs were cultured in interleukin-2 (IL-2) ‘containing medium in the pres fence or absence of SNAP, NAP, AZT, of a combination, After 6 ‘ays in culture, counts per minuto (opm) of PH)-thymidine uptake wore measured, The data repre- sentthe mean of triplicate cultures and are representative of two (AZT) or five (SNAP and NAP) Separate experiments, NITRIC OXIDE MODULATES HIV-1 REPLICATION 5 0. - p24 Vv wv SNAP NAP FIG. 2. S:nitroso-N-acetyipenicilamine (SNAP) inhibits HIV-1 replication after reverse transcription but before or during viral protein ‘expression. (A) SNAP does not inhibit the synthesis of ful length HIV-1 proviral DNA. Phytohemagglutinin (PHA)-stimulated peripheral ‘blood mononuclear cells (PBMCs) were not infected (uninfected) or infected with HIV-1 and cultured in the presence or absence of 200, LM SNAP or N-acetyipenicilamine (NAP). After 24 hours, DNA isolated from an equal number of cells in each sample was amplified with polymerase chain reaction (PCR) using gag-specitic primers that detect full-length or nearly completely synthesized viral DNA (GAG). The PCR products were visualized on an ethidium-stained agarose gel. The markers onthe left indicate the basepair size of IX DNA fragments {generated from a Hadll digest. (B). The same experiment was performed as in A using fivefold dilutions of DNA from each cell population. To quantitate levels of HIV-1 DNA during PCR amplification, HIV-1 gag standards were generated by PCR amplification of DNA extracted from log dilutions of ACHE cells which contain one proviral copy per cell. (C) SNAP does not inhibit HIV-1 reverse transcriptase activi. Uninfected (-V) or HIV-1~infected (+V) PBMCs were incubated in the presence of 200 LM SNAP or NAP. After 24 hours, reverse transcriptase activity was measured in cel-free supematants as described previously (41). The data represent the mean of duplicate cultures. (D) SNAP inhibits HIV-1 protein expression. PHA-stimulated PBMCs were uninfected or infected with HIV-1 and grown in the presence or absence of SNAP or NAP. After 24 hours, HIV-1 protein expression was analyzed by immunoblot of whole cell iysates using antisera from HIV-1—infected patients. Markers of molecular mass in kilodaltans are indicated onthe left. The HIV-1 p55 and p24 antigens. are indicated on the right. JAIDS Journal of Acuied Immune Deficiency Syndromes, Vo No.1 September 1,199 6 J. B. MANNICK ET AL. protein expression (Fig. 2D). Total cellular protein ex- pression (as detected by Ponseau $ staining of cell ly- sates transferred onto nitrocellulose) was not signifi- cantly inhibited by this concentration of SNAP (data not ACH-2 & 58 a 116 - 80 20 p24 Concentration (% of cont 0 100 400 LINMA Concentration (uM) ° 1204 100 | 8 ices Concentration p24 (2% of control) 8 NeS N4NAP NL NeD. shown) suggesting that exposure to RSNOs is associated with a selective inhibition of HIV-1 protein expression. Human Cell Lines Latently Infected With HIV-1 Express Type II Nitric Oxide Synthase (NOS) Having analyzed the effects of intracellular NO pro- duction and extracellular NO donor compounds. on HIV-1 replication in acutely infected cells, we analyzed the effects of NO on latent HIV-1 infection using the latently infected promonocytic UI cell line as a model. To confirm that UI cells were capable of synthesizing NO, expression of type II NOS (the NOS isoform typi- ly implicated in host defense [3}) was examined by Western blot analysis in U1 cells, as well as in several other cell lines which support the replication of HIV-1 Type II NOS expression was detected in the UI cell lin the U937 cell line of which is the uninfected parental cell line from which UI was cloned, and the ACH-2 T-cell line, which is chronically infected with HIV-I (Fig. 34), Very low levels of NOS activity (-100 nM of nitro- sothiols) were detected in U937 cells using a photolysis! FIG. 3._Niic oxide (NO) increases HIV replication in the chron cally infected Ut cell line. (A) Type Il nitric oxide synthase (NOS) 's expressed in several cell ines that support the replication of HIV-1. A western blot was performed to detect type Il NOS pro- tein expression in whole cell lysates made from 0.5 x 10° colls ftom the folowing cell lines: ACH-2, a human T-cell line chron! cally infected with HIV-1, US37 (US), a human promonocytic cell line, or Ut, a clone of U837 chronically infected with HIV-1. Rat ‘macrophages stimulated with LPS (RAT) were used as a positive Control. Type iI NOS was detected using an affnty-purfied rabbit antiserum raised against the N-terminal 20 amino acids ofthe rat isoform that recognizes both human and rat type ll NOS (62). (B) Inhibition of NOS activity decreases HIV-1 replication in U1 cells. Ut cells were incubated with 0, 100 or 400 pM concentrations of the competitive NOS inhibitor NG-monomethyl-t-arginine (- NMA). After 3-5 days, p24 antigen levels were measured in the cell supernatants by enzyme-linked Immunosorbent assay (ELISA). The data are expressed as the porcentage of control p24 levels (generated in the absence of t-NMA), and represent the mean + standard error of the mean of 5 (0 oF 100 pM NMA) or 4 (400 IM NMA) separate experiments. The x axis indicates uM concentration of -NMA in each culture * indicates p = 02, n 5, pairod test (tp < 01, n= 4, paired test) (C). The effects of NMA on HIV-1 replication are reversible by excess (arginine but not o-arginine; or by the S-nitrosothiol SNAP but not the Control compound NAP. The same experiment was performed as in (B) using 400 uM L-NMA alone (N), or in combination with 10 UM S-niteoso-N-aootylpenicilamine (SNAP) (N + S), 10 pM N- fcetyipenicilamine (NAP) (N + NAP), 1 mM L-arginine (N +L), or 1'mM b-arginine (N + D), After 5 days, p24 antigen levels were ‘measured in the cell supernatants by ELISA. The data are ex: pressed as the percentage of control p24 levels (generated in the absence of added drug), and represent the mean = standard fertor of the mean of three separate experiments ("p = 03; tp = (002; "p=.04, paired Hest, n= 3). SNAP (10 uM) or L-arginine (1 mM) alone had no significant effect on HIV-1 replication (data not shown), 1 Sepember 1, 1999 NITRIC OXIDE MODULATES HIV-1 REPLICATION 7 chemoluminescence method as described previously (data not shown) (48). Intracellular NO Production Increases HIV-1 Replication in Latently Infected U1 Cells To determine the effects of intracellular NO produc~ tion on latent HIV-1 infection, latently infected UI cells were grown in the presence or absence of various con- centrations of the NOS inhibitor t-NMA. After 3 to 5 days, virus production was measured in cell-free super- natants by ELISA specific for the p24 gag antigen (14). Inhibition of NOS activity in Ul cells by 1-NMA was associated with a small but statistically significant 42% = 11% (mean + standard error of the mean [SEM}) de- crease in basal HIV-1 replication in UI cells (Fig. 3B: p 02, n = 5, paired t-test). t-Arginine (the NOS sub- strate) but not D-arginine reversed the 1-NMA-induced decrease in HIV-1 replication indicating that the t-NMA cffect was likely due to a specific inhibition of NOS (Fig. 30). In addition, the NO donor SNAP, but not the control parent compound NAP, reversed the inhibitory effects of L-NMA (Fig. 30). Concentrations of RSNO >10 uM were cytotoxic to UI cells and therefore were not asso- ciated with an increase in HIV-I replication. Thus, both intracellular NO. production and low concentrations of extracellular RSNOs increase HIV-I reactivation in la- tently infected UI cells. In contrast, low concentrations (10 UM) of RSNOs had no significant effect on HIV-1 replication in acutely infected PBMCs (data not shown), DISCUSSION These results have several biologic and therapeutic implications. They suggest that low level constitutive NOS activity in human leukocytes is capable not only of inhibiting latent virus reactivation (as is the case with Epstein-Barr virus), but also of stimulating latent virus reactivation (as is the case with HIV). Although the ef- fects of NOS activity on HIV-I reactivation were small in these studies, it is possible that the effects will be more dramatic in latently infected cells in vivo. The mecha- nism by which intracellular NO production reactivates latent HIV-1 has not been investigated. Because NO do- nors activate NF-xB in PBMCs (8), it is possible that intracellular NO production activates NF-KB in latently infected cells and thereby induces HIV-I transcription. Our results suggest that extracellular RSNOs as well as intracellular NO production modulate HIV-1 replica- tion. In vivo, RSNOs are the most abundant NO donor ‘compound in the circulation (48). The concentrations of RSNOs used to inhibit HIV-I replication in these experi- ments (100-250 uM) were higher than those detected in humans (1-10 uM) (48). However, NO donor com- pounds release NO more efficiently in vivo than in vitro, and therefore it is possible that concentrations of RSNOs present in the human circulation regulate HIV-1 replica- tion. ur findings are supported by studies of hydroxyurea and IL-2 in HIV-I infection. Hydroxyurea, a known ri- bonucleotide reductase inhibitor, has also been recently identified as an NO donor compound (49,50). The thera- peutic efficacy of hydroxyurea in sickle cell disease now thought to be secondary to increased NO produ tion, and subsequent vasodilation (50). Hydroxyurea has also been shown to have synergistic inhibitory effects on HIV-I replication in combination with deoxynucleoside analogues (51-54). It is possible that the inhibitory ef- fects of hydroxyurea on HIV-I replication are mediated in part by its NO donor capability. Although hydroxy- urea and NO inhibit ribonucleotide reductase (55), addi- tional mechanisms may be involved in their inhibitory effects on HIV-1 replication since RSNOs do not inhibit reverse transcription, Similarly, IL-2 in combination with other activating stimuli reactivates HIV-1 in latently infected cells (56). Given that IL-2 is known to increase intracellular NO production (57,58), itis possible that the stimulatory effects of IL-2 on HIV-I latency are medi- ated in part by increased intracellular NO levels. The effects of RSNOs may have clinical as well as biologic relevance. Our data suggest that RSNOs, and pethaps other related compounds (such as organic ni- trates, which mediate their effects through biotransfor- mation to RSNO (59), and which have been used for decades for the treatment of angina without significant associated toxicity), may have therapeutic efficacy in HIV-1 infection. NO therapy may be of particular inter- est in light of recent data demonstrating persistent reser voirs of inducible latent HIV-1 during highly active an- tiretroviral therapy (56,60,61). It is possible that NO do- hors in combination with other antiretrovirals will help eliminate latent reservoirs of infection as well as sup- press viral replication, REFERENCES 1, Stamler 18, Jaraki O, Osbome J, etal. Nitric oxide circulates in ‘mammalian plasma primarily as an S-nitroso adduct of serum al bbumin, Proc Nat! Acad Sei USA 1992:89:7674-7. 2. Stamler JS, Jia L, Ew JP, et al, Blood flow regulation by S. nitrosohemoglobin in the physiological oxygen gradient. 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