Antimicrobial properties of the medicinal plant Cardiospermum halicacabum L: new evidence and future perspectives.

The emergence and rapid spread of multidrug-resistance in human pathogenic microorganisms urgently require the development of novel therapeutic strategies for the treatment of infectious diseases. From this perspective, the antimicrobial properties of the natural plant-derived products may represent an important alternative therapeutic option to synthetic drugs. Among medicinal plants, the Cardiospermum halicacabum L. (C. halicacabum), belonging to Sapindaceae family, could be a very promising candidate for its antimicrobial activity against a wide range of microorganisms, including both Gram-positive and Gram-negative bacteria, as well as fungal pathogens. Although the antimicrobial properties of C. halicacabum have been intensively studied, the mechanism/s by which it exerts the inhibitory activity towards the pathogenic microbes have not yet been completely understood. This review focuses on the main antimicrobial activities displayed in vitro by the plant extract, with particular attention on our recent advances. We demonstrated that C. halicacabum is able to exert in vitro a dose-dependent fungistatic effect against Trychophyton rubrum (T. rubrum) through molecular interaction with the fungal heat shock protein (Hsp)-90 chaperone. These findings are supported by a growing body of research indicating the crucial role played by the Hsp90 in the virulence of the pathogenic microorganisms, including fungal pathogens. The possible future use of C. halicacabum for treating a wide range of infectious diseases is also discussed.

Effect of maraviroc on non-R5 tropic HIV-1: refined analysis of subjects from the phase IIb study A4001029.

We characterized maraviroc susceptibility of dual/mixed tropic viruses from subjects enrolled onto phase IIb study A4001029. Maraviroc baseline plasma samples from 13 multidrug-experienced subjects were sequenced and the HIV-1-env gene cloned into pNL4.3Δenv to obtain recombinant viruses. The V3 region was sequenced by the Sanger method and ultradeep sequencing. By analysing subjects having a weighted optimized background therapy susceptibility (wOBT) score of <1, 3/7 subjects were characterized by good in vivo and in vitro response to maraviroc therapy. Molecular docking simulations allowed us to rationalize the maraviroc susceptibility of dual/mixed tropic viruses. A subset of subjects with dual/mixed tropic viruses responded to maraviroc. Further investigations are warranted of CCR5 antagonists in subjects carrying dual/mixed tropic virus that explore the feasible use of maraviroc in subjects that is potentially larger than those infected with a pure R5 virus.

Natural polymorphisms of HIV-1 subtype-C integrase coding region in a large group of ARV-naïve infected individuals.

PURPOSE: Integrase (IN) is an enzyme produced by human immunodeficiency virus (HIV)-1 that enables its genetic material to be integrated into the DNA of the infected cell. Still now, few data are available with detailed analysis of the natural IN polymorphisms of HIV-1 subtype-C in datasets retrieved from antiretroviral-naïve patients; this study focuses on these polymorphisms. METHODS: The analysis included 335 HIV-1 subtype-C IN sequences (one per patient). Multi-alignment of IN sequences was performed, and for the definition of a polymorphism, only amino acid changes with prevalence ≥3 % among treatment-naïve patients were considered. RESULTS: Seventy IN amino acid positions were fully conserved. Differently, forty-six IN amino acid polymorphic positions were observed, 12 within the N-terminal domain and 13 within the C-terminal domain. In the DDE-catalytic motif, only one mutation per site (D64G/D116G/E152K) was found, while a low variability (<1 %) was observed for IN positions interacting with LEDGF/p75. A major drug resistance mutation for raltegravir (RAL) and elvitegravir (EVG), Q148H, was retrieved from one patient and another RAL primary resistance mutation, Y143H, was also retrieved from another patient. CONCLUSIONS: The results from the IN sequences analyzed underlined that some unexpected baseline substitutions affecting the susceptibility to RAL/EVG could be detected in drug-naïve individuals, and, therefore, it should be genotyped before the consideration of HIV-1 IN inhibitors (INIs). The impact of these mutations on the baseline drug susceptibility of HIV-1 subtype-C to INIs may need to be addressed prior to the introduction of these drugs in some Asiatic and African countries.

NeuroAIDS: virological aspects of HIV infection.

NeuroAIDS is one of the main complications of chronic HIV-infection. The Central Nervous System is an immunologic sanctuary for HIV and allows the persistence of the virus despite an efficient antiretroviral therapy. HIV-1 could promote the neurodegeneration through the induction of inflammation by the release of neurotoxins from infected cells. In addition, several viral proteins can directly contribute to the neuronal damages, activate cell-signaling involved in the control of cellular survival and apoptosis, favoring functional alterations in the target cells. Macrophages play a key role in the pathogenesis of NeuroAIDS, they are the main reservoirs of the infection in brain, promoting the inflammatory escalation, astrogliosis and degeneration process. This review aims to highlight the virological aspects associated with NeuroAIDS including pathogenesis, and treatment of HIV-1 in the CNS sanctuaries.

Inhibition of human immunodeficiency virus (HIV-1) infection in human peripheral blood leucocytes-SCID reconstituted mice by rapamycin.

The capacity of the immunomodulatory drug rapamycin (RAPA) to inhibit replication of the CCR5 strain of human immunodeficiency virus (HIV) in vitro prompted us to test its effects in a murine preclinical model of HIV infection. RAPA (0.6 or 6 mg/kg body weight) or its vehicle were administered daily, per os, to SCID mice reconstituted with human peripheral blood leucocytes (hu-PBL) starting 2 days before the intraperitoneal challenge with the R5 tropic SF162 strain of HIV-1 (1000 50% tissue culture infective dose/ml). Relative to hu-PBL-SCID mice that received no treatment, HIV-infected hu-PBL-SCID mice treated with the vehicle control for 3 weeks exhibited a severe depletion of CD4(+) cells (90%), an increase in CD8(+) cells and an inversion of the CD4(+)/CD8(+) cell ratio. In contrast, treatment of HIV-infected mice with RAPA prevented a decrease in CD4(+) cells and the increase of CD8(+) cells, thereby preserving the original CD4(+):CD8(+) cell ratio. Viral infection also resulted in the detection of HIV-DNA within peritoneal cells and spleen, and lymph node tissues of the vehicle-treated mice within 3 weeks of the viral challenge. In contrast, treatment with RAPA decreased cellular provirus integration and reduced HIV-RNA levels in the blood. Furthermore, in co-cultivation assays, spleens from RAPA-treated mice exhibited a reduced capacity for infecting allogeneic T cells which was dose-dependent. These data show that RAPA possesses powerful anti-viral activity against R5 strains of HIV in vivo and support the use of additional studies to evaluate the potential application of this drug in the management of HIV patients.

Carbohydrate-binding agents (CBAs) inhibit HIV-1 infection in human primary monocyte-derived macrophages (MDMs) and efficiently prevent MDM-directed viral capture and subsequent transmission to CD4+ T lymphocytes.

Carbohydrate-binding agents (CBAs) have been proposed as innovative anti-HIV compounds selectively targeting the glycans of the HIV-1 envelope glycoprotein gp120 and preventing DC-SIGN-directed HIV capture by dendritic cells (DCs) and transmission to CD4(+) T-lymphocytes. We now show that CBAs efficiently prevent R5 HIV-1 infection of human primary monocyte-derived macrophage (MDM) cell cultures in the nanomolar range. Both R5 and X4 HIV-1 strains were efficiently captured by the macrophage mannose-binding receptor (MMR) present on MDM. HIV-1 capture by MMR-expressing MDM was inhibited by soluble mannose-binding lectin and MMR antibody. Short pre-exposure of these HIV-1 strains to CBAs is able to prevent virus capture by MDM and subsequent syncytia formation in cocultures of the CBA-exposed HIV-1-captured MDM and uninfected CD4(+) T-lymphocytes. The potential of CBAs to impair MDM in their capacity to capture and to transmit HIV to T-lymphocytes might be an important property to be taken into consideration in the eventual choice to select microbicide candidate drugs for clinical investigation.

Antiviral profile of HIV inhibitors in macrophages: implications for therapy.

Macrophages (M/M) are identified as the second cellular target of HIV and a crucial virus reservoir. M/M are persistently infected cells and not susceptible to the HIV cytophatic effects typical of infected CD4+ T-lymphocytes. HIV replication in M/M is a crucial pathogenetic event during the whole course of the disease. Moreover, the dynamics of HIV-1 replication and cumulative virus production is quite different in M/M and CD4+ T-lymphocytes in the presence or in the absence of antiviral drugs. Thus, for their unique cellular characteristics, the activity of anti-HIV compounds could be different in M/M than in CD4+ T-lymphocytes. Indeed, nucleoside analogues inhibitors of HIV-reverse transcriptase (NRTIs) show potent antiviral activity in macrophages, although the limited penetration of these compounds in sequestered body compartments and the scarce phosphorylation ability of macrophages, suggest that a phosphate group linked to NRTIs may confer a greater anti-HIV activity in such cells. The antiviral activity of non-nucleoside reverse transcriptase inhibitors (NNRTIs) in macrophages is similar to that found in CD4-lymphocytes. Interestingly, protease inhibitors (PIs), acting at post-integrational stages of virus replication, are the only drugs able to interfere with virus production and release from macrophages with established and persistent HIV infection. For these reasons, a careful analysis of the distribution of antiviral drugs, and the assessment of their activity in cells of macrophage lineage, represent key factors in the development of therapeutic strategies aimed to the treatment of the HIV-infected patients.

HIV/HCV co-infection: the magnitude of the problem.

Hepatitis C virus (HCV) and human immunodeficiency virus (HIV) share routes of transmission, therefore their coinfection is relatively common. Nevertheless, the clinical relevance of this event has been minimal until few years ago when, due to the increased survival of HIV-infected individuals (favoured by highly active antiretroviral therapy) morbility and mortality caused by pathologies not strictly related to HIV (such as HCV infection) raised sharply. Despite differences in their general characteristics (including lifecycle, target cells, and type of persistence in the infected host) a remarkable level of interaction exists between HCV and HIV; this makes the progression of both liver disease and immunological damage easier and more rapid. A therapeutic approach to HIV/HCV coinfection thus requires the utilization of drugs and strategies effective against both viruses, yet, timing, drug types, and effective combinations still remain poorly defined. New and innovative studies specifically focused on HIV/HCV coinfection are thus warranted to increase the knowledge about their interaction, and define therapeutic strategies aimed to the best management of the infection by both viruses during coinfection.

Antiretrovirus activity of a novel class of acyclic pyrimidine nucleoside phosphonates.

A novel class of acyclic nucleoside phosphonates has been discovered in which the base consists of a pyrimidine preferably containing an amino group at C-2 and C-4 and a 2-(phosphonomethoxy)ethoxy (PMEO) or a 2-(phosphonomethoxy)propoxy (PMPO) group at C-6. The 6-PMEO 2,4-diaminopyrimidine (compound 1) and 6-PMPO 2,4-diaminopyrimidine (compound 11) derivatives showed potent activity against human immunodeficiency virus (HIV) in the laboratory (i.e., CEM and MT-4 cells) and in primary (i.e., peripheral blood lymphocyte and monocyte/macrophage) cell cultures and pronounced activity against Moloney murine sarcoma virus in newborn NMRI mice. Their in vitro and in vivo antiretroviral activity was comparable to that of reference compounds 9-[(2-phosphonomethoxy)ethyl]adenine (adefovir) and (R)-9-[(2-phosphonomethoxy)-propyl]adenine (tenofovir), and the enantiospecificity of (R)- and (S)-PMPO pyrimidine derivatives as regards their antiretroviral activity was identical to that of the classical (R)- and (S)-9-(2-phosphonomethoxy)propyl purine derivatives. The prototype PMEO and PMPO pyrimidine analogues were relatively nontoxic in cell culture and did not markedly interfere with host cell macromolecular (i.e., DNA, RNA, or protein) synthesis. Compounds 1 and 11 should be considered attractive novel pyrimidine nucleotide phosphonate analogues to be further pursued for their potential as antiretroviral agents in the clinical setting.

Macrophages: a crucial reservoir for human immunodeficiency virus in the body.

The replication of Human Immunodeficiency Virus (HIV) in cells of macrophage lineage represents a key pathogenetic event of the neurological damages typically found during the course of this disease. Macrophages are persistently infected cells and thus not susceptible to the cytophatic effect typical of infected activated CD4-lymphocytes. The resistance of macrophages to HIV infection is at least in part mediated by the autocrine production of the nerve growth factor (NGF), a neurokine able to sustain the survival of some cells of bone marrow origin, including monocyte-derived macrophages. This anti-apoptotic effect of NGF in HIV-infected macrophages can be even more relevant at the central nervous system level, where many cells are able to physiologically produce NGF, thus further increasing the survival of macrophages infected by HIV, and enhancing the damages that these cells may induce upon bystander neurons. The proapoptotic effect of soluble factors released by HIV-infected macrophages may heavily affect the survival and functions also of astrocytes, that in turn become unable to sustain neuronal homeostasis. Taken together, this information supports the importance of therapeutic attempts aimed at attacking virus replication in infected macrophages and/or to selectively eliminate these chronically infected and persistently virus-producing cells.

Productive infection of primary macrophages with human herpesvirus 7.

Here we demonstrate replication of human herpesvirus 7 (HHV-7), a T-lymphotropic virus, in macrophages. Productive replication was lost after 2 weeks, but HHV-7 DNA was detected up to 1 month after infection. Thus, macrophages become infected by HHV-7 and might play an important role as a viral reservoir, as has been demonstrated for human immunodeficiency virus type 1.

The LD78beta isoform of MIP-1alpha is the most potent CC-chemokine in inhibiting CCR5-dependent human immunodeficiency virus type 1 replication in human macrophages.

The CC-chemokines RANTES, macrophage inflammatory protein 1alpha (MIP-1alpha), and MIP-1beta are natural ligands for the CC-chemokine receptor CCR5. MIP-1alpha, also known as LD78alpha, has an isoform, LD78beta, which was identified as the product of a nonallelic gene. The two isoforms differ in only 3 amino acids. LD78beta was recently reported to be a much more potent CCR5 agonist than LD78alpha and RANTES in inducing intracellular Ca2+ signaling and chemotaxis. CCR5 is expressed by human monocytes/macrophages (M/M) and represents an important coreceptor for macrophage-tropic, CCR5-using (R5) human immunodeficiency virus type 1 (HIV-1) strains to infect the cells. We compared the antiviral activities of LD78beta and the other CC-chemokines in M/M. LD78beta at 100 ng/ml almost completely blocked HIV-1 replication, while at the same concentration LD78alpha had only weak antiviral activity. Moreover, when HIV-1 infection in M/M was monitored by a flow cytometric analysis using p24 antigen intracellular staining, LD78beta proved to be the most antivirally active of the chemokines. RANTES, once described as the most potent chemokine in inhibiting R5 HIV-1 infection, was found to be considerably less active than LD78beta. LD78beta strongly downregulated CCR5 expression in M/M, thereby explaining its potent antiviral activity.

Cyclosaligenyl-2',3'-didehydro-2',3'-dideoxythymidine monophosphate: efficient intracellular delivery of d4TMP.

Cyclosaligenyl-2',3'-didehydro-2', 3'-dideoxythymidine-5'-monophosphate (cycloSal-d4TMP) is a potent and selective inhibitor of human immunodeficiency virus replication in cell culture and differs from other nucleotide prodrug approaches in that it is designed to selectively deliver the nucleotide 5'-monophosphate by a controlled, chemically induced hydrolysis. Its antiviral efficacy in cell culture is at least as good as, if not superior to, that of d4T. CycloSal-d4TMP was found to lead to the efficient intracellular release of d4TMP in a variety of cell lines, including both wild-type CEM and thymidine kinase-deficient CEM/TK(-) cells. Under similar experimental conditions, exposure of CEM/TK(-) cells to d4T failed to result in significant d4TTP levels. The intracellular conversion of cycloSal-d4TMP proved to be both time and dose dependent. The half-life of d4TTP generated intracellularly from d4T- or cycloSal-d4TMP-treated CEM cells was approximately 3.5 h, and the intracellular ratios of d4TTP/d4TMP in cells exposed to cycloSal-d4TMP gradually increased from 1 to 3.4 upon prolonged incubation. Radiolabeled cycloSal-d4TMP could be separated as its two R(p) and S(p) diastereomers on high-performance liquid chromatography. The R(p) diastereomer of cycloSal-d4TMP was 3- to 7-fold more efficient in releasing d4TMP and generating d4TTP than the S(p) cycloSal-d4TMP diastereomer. This correlated well with the 5-fold more pronounced antiviral activity of the R(p) diastereomer versus the S(p) diastereomer. d4TMP is a poor substrate for the cytosolic 5'(3')-deoxyribonucleotidase (V(max)/K(m) for d4TMP: 0.08 of V(max)/K(m) for dTMP) and is only slowly hydrolyzed to d4T. This contributes to the efficient conversion of the prodrug of d4TTP.

Primary macrophages infected by human immunodeficiency virus trigger CD95-mediated apoptosis of uninfected astrocytes.

Infection of macrophages (M/M) by human immunodeficiency virus (HIV) is a main pathogenetic event leading to neuronal dysfunction and death in patients with AIDS dementia complex. Alteration of viability of neurons and astrocytes occurs in vivo even without their infection, thus it is conceivable that HIV-infected M/M may affect viability of such cells even without direct infection. To assess this hypothesis, we studied the effects of HIV-infected M/M on an astrocytic cell-line lacking CD4-receptor expression. Exposure to supernatants of HIV-infected M/M triggers complete disruption and apoptotic death of astrocytic cells. This effect is not related to HIV transmission from infected M/M, because HIV-DNA and p24 production in astrocytic cells remained negative. Apoptotic death of astrocytes is mainly mediated by Fas ligand released in supernatants of HIV-infected M/M (as demonstrated by complete reversal of such phenomenon by adding neutralizing antibodies against CD95 receptor). Treatment of astrocytic cells with recombinant (biologically active) Tat induces < 10% apoptosis, and gp120 was totally ineffective. Treatment of HIV-infected M/M with AZT completely reverses the proapoptotic effect of their supernatants on astrocytes, thus demonstrating that productive virus replication within M/M is required for the induction of astrocytic cell death. Taken together, data suggest that homeostasis of astrocytes may be affected by HIV-infected M/M in the absence of productive infection of target cells. This phenomenon may help to explain the cellular damage found in HIV-infected patients also in areas of the brain not strictly adjacent to HIV-infected M/M.

Presence of 2',5'-Bis-O-(tert-butyldimethylsilyl)-3'-spiro-5"-(4"-amino-1",2"-oxath iole-2",2"-dioxide) (TSAO)-resistant virus strains in TSAO-inexperienced HIV patients.

HIV-1 samples from six patients undergoing diverse anti-HIV therapies possessed the E138A mutation in their reverse transcriptase (RT) genome. Patients were receiving the following therapies: TIBO monotherapy (one patient); zidovudine plus didanosine combination therapy (one); zidovudine monotherapy (one); sequential therapy with zidovudine, then stavudine and finally zalcitabine plus didanosine (one); and two were drug naive. E138K, not E138A, is a known TSAO-specific resistance mutation, emerging under selective pressure in vitro. Our phenotypic data on the patient isolates, confirmed by data on an E138A mutant acquired through in vitro mutagenesis, indicated that an alanine substitution for glutamate at codon 138 of the HIV-1 RT renders the virus TSAO resistant, confirming the importance of this amino acid residue in the activity of TSAO derivatives. In addition, we have demonstrated through phenotypic analysis of the E138A and A98S mutants (after in vitro mutagenesis) that the mutation A98S, found in one of these patients, could be partially responsible for the phenotypic reversal of TSAO resistance. This reversal could be explained by the restoration of a hydrogen bond between 98S and the main-chain residue L349, which compensates for the loss of the E138-G99 main-chain hydrogen bond. As TSAO derivatives have not been used in the clinical setting, the presence of the E138A mutation at a frequency of 6.7% in our study of 90 TSAO-inexperienced HIV-seropositive individuals implies that 138A of the RT must be a natural variant and that the mutant virus is replication competent. Our observations suggest that the E138A mutation may likely arise in patients under the selective pressure of TSAO or related compounds that show a decreased antiviral potency toward the E138A variant.

Viral dynamics and mutations in the pol gene during primary HIV-1 infection.

The understanding of viral dynamics and appearance of mutations during primary infection could be useful for the design of an efficient therapy. For this reason a cohort of samples from naive primary patients was examined. The results pointed out that only a few secondary mutations in protease gene (having no effect on resistance) were found, while a single mutation conferring resistance to non-nucleosides inhibitors of reverse transcriptase was found both in plasma and cerebrospinal fluid of a patient. As both the protease secondary mutations and the single non nucleoside reverse transcriptase mutation map far from the catalytical sites of the enzymes, neither one is able to impair viral fitness. Overall data suggest that treated donors carrying resistant strains may be in part unable to transfer them to the recipient, and/or virus in the recipient tends to revert to wild type. These results should be taken into account in the planning of early HAART treatment of HIV infection.

Activities of masked 2',3'-dideoxynucleoside monophosphate derivatives against human immunodeficiency virus in resting macrophages.

The anti-human immunodeficiency virus (HIV) activity of aryloxyphosphoramidate protides of a number of anti-HIV nucleoside analogues was assessed in resting primary monocyte-macrophages (M/M). While 2',3'-dideoxythymidine (d4T), 2',3'-dideoxyadenosine (ddA), and 2',3'-dideoxy-2',3'-didehydroadenosine (d4A) protides showed an anti-HIV activity that was 25- to 625-fold greater than the parent nucleotides d4T, ddA, and d4A, respectively, other aryloxyphosphoramidate protides showed similar or even lower anti-HIV activities than their parent compounds. This variable anti-HIV effect is most likely related to the different dynamics of intracellular nucleoside monophosphate release from the protides. Our results indicate the potential advantage of therapeutic use of this approach for some nucleotide analogues to affect HIV replication in M/M, one of the major reservoirs of HIV in vivo.

Intracellular metabolism of CycloSaligenyl 3'-azido-2', 3'-dideoxythymidine monophosphate, a prodrug of 3'-azido-2', 3'-dideoxythymidine (zidovudine).

The administration of CycloSaligenyl 3'-azido-2',3'-dideoxythymidine monophosphate (CycloSal-AZTMP) to CEM cells resulted in a concentration- and time-dependent conversion to the 5'-monophosphate (AZTMP), 5'-diphosphate (AZTDP), and 5'-triphosphate (AZTTP) derivatives. High ratios of AZTMP/AZTTP were found in the CEM cell cultures treated with CycloSal-AZTMP. The intracellular T(1/2) of AZTTP in CEM cell cultures treated with either AZT and CycloSal-AZTMP was approximately 3 h. A variety of human T- and B-lymphocyte cell lines efficiently converted the prodrug to the AZT metabolites, whereas peripheral blood lymphocytes and primary monocyte/macrophages showed at least 10-fold lower metabolic conversion of the prodrug. CycloSal-AZTMP failed to generate marked levels of AZT metabolites in thymidine kinase-deficient CEM/TK(-) cells, an observation that is in agreement with the substantial loss of antiviral activity of CycloSal-AZTMP in CEM/TK(-) cells. The inability of CycloSal-AZTMP to generate AZTMP in CEM/TK(-) cells is presumably due to a relatively high hydrolysis rate of AZTMP to the parent nucleoside AZT, combined with the inability of CEM/TK(-) cells to phosphorylate AZT to AZTMP through the cytosolic salvage enzyme thymidine kinase.

Nerve growth factor is an autocrine factor essential for the survival of macrophages infected with HIV.

Nerve growth factor (NGF) is a neurotrophin with the ability to exert specific effects on cells of the immune system. Human monocytes/macrophages (M/M) infected in vitro with HIV type 1 (HIV-1) are able to produce substantial levels of NGF that are associated with enhanced expression of the high-affinity NGF receptor (p140 trkA) on the M/M surface. Treatment of HIV-infected human M/M with anti-NGF Ab blocking the biological activity of NGF leads to a marked decrease of the expression of p140 trkA high-affinity receptor, a concomitant increased expression of p75(NTR) low-affinity receptor for NGF, and the occurrence of apoptotic death of M/M. Taken together, these findings suggest a role for NGF as an autocrine survival factor that rescues human M/M from the cytopathic effect caused by HIV infection.

Relative potency of protease inhibitors in monocytes/macrophages acutely and chronically infected with human immunodeficiency virus.

The activity of three human immunodeficiency virus (HIV) protease inhibitors was investigated in human primary monocytes/macrophages (M/M) chronically infected by HIV-1. Saquinavir, KNI-272, and ritonavir inhibited the replication of HIV-1 in vitro, with EC50s of approximately 0.5-3.3 microM. However, only partial inhibition was achievable, even at the highest concentrations tested. Also, the activity of these drugs in chronically infected M/M was approximately 7- to 26-fold lower than in acutely infected M/M and approximately 2- to 10-fold lower than in chronically infected H9 lymphocytes. When protease inhibitors were removed from cultures of chronically infected M/M, production of virus rapidly returned to the levels found in untreated M/M. Therefore, relatively high concentrations of protease inhibitors are required to suppress HIV-1 production in chronically infected macrophages, and such cells may be a vulnerable point for the escape of virus in patients taking these drugs.