1 Department of Experimental Medicine and Biochemical Sciences, University of Rome Tor Vergata, Via Montpellier 1, 00133 Rome; 2 Department of Histology, Microbiology and Medical Biotechnologies, University of Padua, Padua, Italy; 3 Rega Institute for Medical Research, Katholieke Universiteit Leuven, B-3000 Leuven, Belgium
Received 22 December 2004; returned 2 February 2005; revised 21 February 2005; accepted 22 February 2005
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Methods: Cellular passages in the presence of lamivudine were performed every 2 weeks by transferring supernatants of infected M/M to fresh M/M. A fitness assay using wild-type virus and a lamivudine-resistant HIV-1 virus (harbouring the M184V RT mutation) was performed in peripheral blood mononuclear cells. Culture supernatants were tested for p24 antigen production and RT activity. The M184V RT mutant virus was obtained by site-directed mutagenesis on a CCR5-using HIV-1 backbone.
Results: The mutagenized M184V RT virus showed full resistance to lamivudine in M/M. However, no detectable phenotypic and genotypic resistance (neither virus breakthrough, nor RT resistance-related mutations) developed in M/M infected by HIV-1 and cultured for up to seven passages in vitro (i.e. 105 days). This inefficiency of M/M to develop M184V RT mutated virus is tightly related to the low 2'-deoxynucleotide (dNTP) pool in such cells, which in turn decreases the kinetics of HIV-1-RT. Despite this, the M184V RT mutant virus replicates in M/M, although with a 30% decreased efficiency compared with the wild-type.
Conclusions: Our results show that the chances of development of resistance are far lower in M/M than in lymphocytes. This underlines the importance and the peculiar role of M/M as reservoirs of either wild-type or resistant strains in human organs.
Keywords: reservoirs , fitness , M184V
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
HIV-1 infection in M/M is characterized by viral dynamics substantially different from those of lymphocytes. In fact, activated lymphocytes can sustain a rapid and exponential viral production followed by massive cell death.18 In contrast, M/M are resistant to the cytopathic effect of HIV-119,20 and produce virus over a prolonged period, with dynamics that increase linearly during the first 12 weeks of infection, followed by a plateau of high-level replication ( > 108 copies of unspliced/spliced RNA produced) lasting at least 60 days after infection.21 Moreover, M/M can survive HIV-1 infection for long periods of time owing to the autocrine secretion of the nerve growth factor (NGF) associated with enhanced expression of the high-affinity NGF receptor p140 trkA on their surface, thus continuously representing a powerful viral reservoir.22 The long-term dynamics of HIV-1 replication in M/M closely reflects the cellular characteristics of these cells. In fact, M/M are resting cells, characterized by a poor deoxynucleotide metabolism and low endogenous nucleotide pools that in turn slow down the activity of the HIV-1 reverse transcriptase (RT) enzyme.23,24 As a consequence, the activity of antiretroviral drugs in M/M is substantially different from that in actively replicating lymphocytes.2527 In fact, nucleoside analogue inhibitors of RT (NRTIs) have been shown to be 10- to 100-fold more active in acutely infected M/M than in lymphocytes.27,28
It is thus conceivable that the development of resistance to antiretroviral drugs in M/M may also be different from that observed in lymphocytes. Mechanisms that regulate the development of resistance in M/M are poorly understood. In an attempt to clarify these mechanisms, we focused our attention on the development and progression of resistance to lamivudine (2',3'-dideoxy-3'-thiacytidine, 3TC), a prototype of NRTIs, in HIV-1-infected M/M. A single mutation at position 184 in the highly conserved YMDD motif of HIV-1-RT (methionine to valine) (transiently preceded by M184I) appears rapidly under lamivudine pressure, and confers complete resistance to lamivudine, both in vitro and in up to 100% of patients failing an antiretroviral therapy containing lamivudine.2932 Our study aims to understand whether the selective pressure of lamivudine also determines the emergence of the M184V RT mutation in HIV-1-infected M/M in vitro, and to characterize a monocytotropic strain harbouring the M184V RT mutation in M/M by comparing the mutant's replicative capacity with that observed in lymphocytes.
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Primary M/M were prepared and purified as described elsewhere.23,33 Briefly, peripheral blood mononuclear cells (PBMCs) were obtained from healthy HIV-1-seronegative donors, separated over a Ficoll gradient, and seeded in 48-well plates at 1.5 x 106 cells/well in 1 mL of RPMI 1640 (Gibco, Paisley, UK) containing 20% heat-inactivated fetal calf serum (Hyclone Laboratories, Logan, UT, USA), 50 U/mL penicillin, 50 µg/mL streptomycin (Euroclone) and 2 mM L-glutamine (Euroclone) (hereafter referred to as complete medium). Complete medium was used in every experiment. Five days after plating and culturing the PBMCs at 37°C in a humidified atmosphere enriched with 5% CO2, non-adherent cells were carefully removed with repeated washings with warmed RPMI 1640 as previously described,23,33 leaving a monolayer of adherent cells, which were finally incubated in complete medium. Cells treated under these conditions have been shown to be > 95% M/M, as determined by non-specific esterase staining and morphology.34 M/M were then challenged with the virus (see below) without detachment or further manipulation.
PBMCs were separated from peripheral blood as mentioned above, and seeded in 75 cm2 flasks at 106 cells/mL in 30 mL of complete medium added with phytohaemagglutinin. After 3 days, interleukin-2 (10 U/mL) was added to the culture medium, and then cells were seeded in 96-well plates at 3 x 105 cells/well.
HeLa cells were maintained in Dulbecco's modified Eagle's medium containing 10% heat-inactivated fetal calf serum (Hyclone Laboratories), 50 U/mL penicillin and 50 µg/mL streptomycin (Euroclone).
CEM cells were obtained from the ATCC and cultured as described previously.35
Virus
Two CCR5-using strains of HIV-1, HIV-1BaL and HIV-1 81A, and one CXCR4-using strain HIV-1IIIB, were used. Details about these HIV-1 strains have been published elsewhere.1,35,36 HIV-1BaL and HIV-1IIIB were expanded in primary M/M and H9 cells, respectively, collected, filtered and stored in liquid nitrogen. HIV-1 81A was obtained from HeLa cells after transfection (Fugene 6 transfection reagent; Roche, Mannheim, Germany) with plasmid p81A,36 collected and stored in liquid nitrogen.
Determination of the nucleotide sequence of HIV-1BaL RT after serial passages in M/M and CEM cells
M/M were exposed to 0.01 µM (concentration was carefully selected, see the Results section) lamivudine and 15 min later infected with 300 TCID50/well (moi of 0.006 infectious dose) of HIV-1BaL. Excess of virus was removed 2 h after infection, then cells were cultured in complete medium added with 0.01 µM lamivudine. M/M were regularly fed every 7 days with complete medium and replenished with lamivudine at the concentration (0.01 µM) known to be able to induce development of lamivudine drug resistance in lymphocytes.35 Culture supernatants were harvested at 14 days after infection and passaged to uninfected M/M obtained from different donors, for a maximum of seven passages. At each passage the virus amounts were quantified by a commercially available kit able to detect HIV-1 p24 protein (Abbott Laboratories, Pomezia, Italy) in the supernatants; at the same time cells were carefully washed and frozen at 80°C. To extract proviral DNA from each passage, cells were washed twice with phosphate buffer, treated with 50 µL of lysis solution containing 0.5% Tween 20 and 0.5% Nonidet P40 for 30 min at room temperature and then transferred in 1.5 mL Eppendorf tubes. The cell suspension was incubated with 2 µL of proteinase K (12.5 mg/mL) at 56°C for 1 h and subsequently heated at 95°C for 10 min. The samples were stored at 20°C until PCR analysis.
Amplification of proviral DNA was performed using 10 µL of cell suspension and 2 µL of each primer (10 mM) in a final volume of 100 µL. The first set of primers (forward 5'-ATTAAAGCCAGGAATGGATGGCCCAAAA and reverse 5'-AAGTGCTTTGGTTCCTCTAAGGAGTTTAC) gave a 900 bp product of the proviral RT gene. One-tenth of the reaction product of the first PCR was then transferred as a template to a new PCR using a second set of primers (forward 5'-CCTGAAAATCCATACAATACTCCAGTATTG and reverse 5'-CTGTATGTCATTGACAGTCCAGCTGTCT) giving a 727 bp RT fragment covering amino acids 50270. The second set of primers primes internally of the first set of oligonucleotides, and thereby amplifies specific products from the first PCR, whereas non-specific products are not further amplified. PCR products were loaded on a 1.5% agarose low melting point gel (Seaplaque agarose; FMC, Philadelphia, PA, USA), purified on synthetic columns (Quiaquick gel extraction kit; Genenco M-Medical, Florence, Italy), directly sequenced with a Taq Dye Deoxy Terminator kit (Applied Biosystems, Foster City, CA, USA) and analysed on a model 373A DNA sequencer (Applied Biosystems).
CEM cells were infected with HIV-1IIIB and kept in culture in the presence of fixed lamivudine concentrations. Briefly, the cell cultures were passaged every 34 days by transferring 50100 µL of the drug-exposed HIV-1-infected cell cultures to fresh uninfected CEM cell cultures in 1 mL wells of a 48-well microtitre plate. Virus breakthrough (in the presence of drug) was monitored prior to each subcultivation by microscopical inspection of the appearance of virus-induced syncytia. The nucleotide sequence of HIV-1IIIB RT of the virus isolates was determined as described previously.35
Site-directed mutagenesis
To construct a convenient vector for site-directed mutagenesis of HIV-1-RT, the fragment in the plasmid p81A encompassing the ApaI restriction site and the EcoRI restriction site was cloned into pBlue Script-SKII() (Stratagene). The QuickChange Site-Directed Mutagenesis kit (Stratagene) was used to introduce the mutation M184V in the subclone mentioned, using a positive and a negative strand oligonucleotide, according to the manufacturer's instructions. The mutagenized fragment was then reintroduced in p81A. The positive-strand oligonucleotide used in the mutagenesis procedure was: 5'-CAGATCCTACGTACAAATCATCCACGTATTGATAG. The negative strand oligonucleotide sequence was antiparallel to that of the positive-strand oligonucleotide.
Virus fitness and resistance test
M/M were exposed to different concentrations of lamivudine (0.1, 1 or 10 µM) and 30 min later infected with 10 000 pg/mL of p24 equivalent of wild-type HIV-1 81A virus or its lamivudine-resistant congener carrying M184V in its RT. Excess of virus was removed 2 h after infection, and cells were cultured with complete medium supplemented with the appropriate concentration of lamivudine. M/M were regularly fed every 7 days with complete medium and replenished with drugs. Aliquots of the culture supernatants were collected at 14 days after infection to detect produced virus using the commercial kit to determine p24 antigen concentrations in the supernatants, mentioned above. Student's t-test was used to assess statistically significant differences in p24 antigen production.
To evaluate the titre of wild-type HIV-1 81A virus and M184V RT lamivudine-resistant virus, peripheral blood lymphocytes (PBLs) and M/M were infected with serial dilutions of each virus beginning from 90 000 pg/mL of p24 antigen equivalent. Excess of virus was removed 2 h after infection, and cells were cultured with complete medium. Virus titre was determined by measuring the p24 antigen produced in each dilution after 7 days for PBLs and 14 days for M/M.
RT activity assay
The RT activity assay was carried out according to the manufacturer's protocol (high sensitivity RT activity assay; Cavidi Tech, Uppsala, Sweden). Briefly, 15 µL of culture supernatant was serially diluted (1/10, 1/100) and added to a 96-well plate with poly(rA) (enzyme template) bound to the bottom of wells and 160 µL of reaction solution containing bromodeoxyuridine triphosphate (BrdUTP) as the enzyme substrate. Polymerization was allowed to proceed at 33°C for 3 h, and for the kinetic test for 30 min, 1 h, 2 h, 4 h and overnight. The incorporated BrdUMP detection with alkaline phosphatase-conjugated anti-BrdUTP antibody was carried out at 33°C for 90 min. The level of bound antibody, which is proportional to the RT activity in the sample, was determined colorimetrically with an alkaline phosphatase substrate, para-nitrophenyl phosphate, in a standard microtitre plate reader (405 nm) at 15 min, 30 min, 2 h and overnight.
RT activity was determined at the time of replicative peak in the wild-type and mutant M184V RT HIV-1-infected M/M cultures, at two different amounts of virus, 5.8 and 50 pg of p24 antigen.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Human primary M/M were infected with the CCR5-using strain HIV-1BaL in both the absence and presence of 0.01 µM lamivudine. Every 2 weeks viral passages were performed by transferring supernatants of infected M/M to fresh M/M from a different donor.
As previously reported, HIV-1BaL infects 100% of M/M cultures in drug-free medium; only a slight decrease in the percentage of viable HIV-1-infected M/M cultures was detected starting from the fourth passage (Figure 1). In contrast, and starting from the third passage, a complete loss of virus was observed in the presence of lamivudine in HIV-1-infected M/M cultures (Figure 1). Indeed, the cell cultures became p24-negative at the lamivudine concentration tested. As a control, HIV-1IIIB-infected CEM cell cultures were cultured in the presence of lamivudine at 0.22, 0.44, 0.9 and 1.7 µM. The virus breakthrough was monitored by scoring the appearance of virus-induced cytopathicity (syncytium formation), and the drug-treated, virus-infected cell cultures were subcultivated every 3 or 4 days in the presence of a fixed concentration of lamivudine. Virus breakthrough in the cell cultures was already microscopically visible after 1, 2, 4 and 5 subcultivations, respectively. Abundant production of mutant virus was evident at passage 4, 5, 6 and 7, respectively (data not shown).
|
Progression of lamivudine resistance in M/M: comparison of fitness between wild-type and lamivudine-resistant variant M184V
The CCR5-using molecular clone HIV-1 81A,36 which carries the entire genome of HIV-1, with the backbone of NL4-3 and the env of BaL, was used to generate the lamivudine-resistant M184V mutation in the RT by site-directed mutagenesis.
More than 99% inhibition of wild-type HIV-1 81A replication was observed in M/M using lamivudine at 1 and 10 µM (P=0.003), while inhibition at 0.1 µM was 80% (P=0.004) compared with control (Figure 2). Interestingly, in the absence of lamivudine, the M184V RT virus showed a 30% decrease in viral replication compared with the wild-type HIV-1 81A (P=0.02) (Figure 2). As expected, lamivudine showed remarkable antiviral efficacy in M/M infected with wild-type HIV-1 81A, while its effect was completely lost in M184V RT mutated HIV-1 81A, even at 10 µM, that is, a concentration 100200-fold greater than its 50% inhibitory concentration for wild-type virus (Figure 2). Thus, M184V does not appear in M/M exposed in vitro to HIV-1 in the presence of lamivudine; however, and similarly to lymphocytes, M184V generated by site-directed mutagenesis confers full resistance to lamivudine also in M/M.
|
|
|
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The potent efficacy of lamivudine, and in general of all NRTIs, in M/M may be explained by the fact that M/M are resting cells characterized by a poor DNA metabolism and consequently by low amounts of nucleotide pools that result in a lower competition by 2'-deoxynucleotide (dNTP).27
A previous study by our group showed that the level of all 2'-deoxynucleosides-5'-triphosphates (with the partial exception of 2'-deoxycytidine-5'-triphosphate) were 10-fold lower in M/M compared with T-lymphocytes.37
In particular 2'-deoxythymidine-5'-triphosphate showed a remarkable 16-fold difference in lymphocytes and M/M (
320.51 and
19.49 pmol/106 cells, respectively).37
It is worth stressing that the remarkable antiviral efficacy of lamivudine, acting on HIV-1 RT and thus at a pre-integrational step of HIV-1 replication, is also due to the fact that our experimental model was represented by acutely infected M/M. Previous studies by our and other groups demonstrated that all NRTIs are completely ineffective on chronically infected M/M (a reservoir poorly sensitive to the cytopathic effect of HIV-1, and therefore able to maintain a constant rate of virus replication). Indeed, chronically infected M/M carry the proviral DNA already integrated within the host genome and thus the virus does not utilize its RT anymore for the replicative cycle.27
For this reason, the assessment of the experimental model based on M/M with established and persistent HIV-1 infection is not reliable for the development of viral resistance induced by NRTIs.38
In fact, both in vitro and in vivo, only protease inhibitors, which act at a post-integrational step of HIV-1 replication, are able to interfere with virus production and release from chronically infected M/M, although complete viral suppression is not achievable in these cells.27
Another important issue raised by our study is that mutations conferring resistance to lamivudine do not emerge during treatment with lamivudine in HIV-1-infected M/M, even at times far longer than those sufficient to induce full-blown resistance in lymphocytes. Also, the low/no ability of HIV-1 to develop resistance to lamivudine in M/M may be explained by the poor DNA metabolism in M/M. In fact, the low level of nucleotide pools, acting as limiting factors for RT activity, decreases the reverse transcription rate,24 and in turn the mutation rates occurring along the HIV-1 genome. This may have important in vivo implications; it is in fact conceivable that the poor dNTP metabolism of resting M/M may reduce the outgrowth of strains resistant to lamivudine in human organs, such as brain and testis, where M/M represent the majority of cells infected by HIV-1. In agreement with this hypothesis, resistance to zidovudine and didanosine develops at a lower rate and at a lower degree in the central nervous system, where wild-type virus strains can be isolated even when highly resistant viral strains are concurrently present in plasma.39 Similarly, recent data from several authors show that the number of mutations present in HIV-1 from cerebrospinal fluid of patients treated with antiviral drugs is lower than that detected in the corresponding plasma samples.4044 Thus, the development of resistance to antiviral drugs in M/M, may occur at a significantly lower rate. Similarly, M/M present in lymphoid organs may carry wild-type strains still susceptible to antiviral drugs even when HIV-1 particles in plasma are fully resistant to the currently used antiviral drugs. Under these conditions, recruitment of newly infected cells, transfer of the virus to lymphocytes, induction of apoptosis of bystander lymphocytes, and production of factors triggering both virus replication and cell death (all phenomena attributed to M/M in the pathogenesis of HIV-1 infection)11,12,45 may be slowed down under antiviral treatments that are apparently no longer effective. This can contribute in part to the discordant results often seen in patients where CD4 counts continue to increase and general conditions improve, including enhancement of responses to opportunistic antigens, all despite virological failure. Thus genotypic and phenotypic changes associated with drug resistance in plasma may not always be synonymous with drug failure.46 Clinical studies are required to provide confirmation of this hypothesis, which has obvious and important clinical implications.
The M184V mutation in HIV-1 RT alters the methionine residue of the conserved YMDD region, which is part of the catalytic site of HIV-1 RT. Several studies have demonstrated that the M184V mutation causes a reduced RT processivity in vitro compared with wild-type enzyme in both primary cells and cell-free virions.4749 Defects in RT processivity, probably owing to an altered interaction of the enzyme with the primer/template duplex,50 are often correlated with reduced replication capacity,48,30 and are somewhat more pronounced in primary cells containing low dNTP levels.48 Thus, the lower replication efficiency of M184V RT-generated virus that we observed in M/M compared with primary lymphocytes may be related mainly to the cell type, and more definitely to the metabolic characteristics of the cells in which the virus replicates, rather than to the intrinsic characteristics of the virus.
These results, although confirmed only for lamivudine, can theoretically be applied also to other nucleoside analogues characterized by a high genetic barrier (i.e. drugs against which the virus must accumulate a relevant number of mutations to achieve resistance). Indeed, the development of resistance against these drugs may be even more difficult for HIV-1. Preliminary experiments conducted with other nucleoside analogues, such as AZT and PMEA (phosphonylmethoxyethyladenine), a prototype of the currently used PMPA (tenofovir) showed no development of resistance in M/M even under different experimental conditions (S. Aquaro, A. Cenci, C. F. Perno and R. Caliò, unpublished data). Whether this also applies to other classes of drugs, such as non-nucleoside reverse transcriptase inhibitors and protease inhibitors, requires further studies addressing this point.
Taken together, our data suggest that tissue M/M harbour HIV-1 strains different from those present at the same time in actively replicating lymphocytes. The inherent properties of HIV-1 infection of M/M should therefore be taken into account in designing therapeutic strategies aimed at achieving an optimal therapeutic effect in all tissue compartments where the virus hides and replicates.
![]() |
Acknowledgements |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
2 . Meltzer, M. S. & Gendelman, H. E. (1992). Mononuclear phagocytes as targets, tissue reservoirs, and immunoregulatory cells in human immunodeficiency virus disease. Current Topics in Microbiology and Immunology 181, 23963.
3 . Lane, J. H., Sasseville, V. G., Smith, M. O. et al. (1996). Neuroinvasion by simian immunodeficiency virus coincides with increased numbers of perivascular macrophages/microglia and intrathecal immune activation. Journal of Neurovirology 2, 42332.[ISI][Medline]
4 . Herbein, G., Coaquette, A., Perez-Bercoff, D. et al. (2002). Macrophage activation and HIV infection: can the Trojan horse turn into a fortress? Current Molecular Medicine 2, 72338.[CrossRef][Medline]
5 . Koenig, S., Gendelman, H. E., Orenstein, J. M. et al. (1986). Detection of AIDS virus in macrophages in brain tissue from AIDS patients with encephalopathy. Science 233, 108993.[ISI][Medline]
6 . Tschachler, E., Groh, V., Popovic, M. et al. (1987). Epidermal Langerhans cells a target for HTLV-III/LAV infection. Journal of Investigative Dermatology 88, 2337.[CrossRef][ISI][Medline]
7
.
McElrath, M. J., Pruett, J. E. & Cohn, Z. A. (1989). Mononuclear phagocytes of blood and bone marrow: comparative roles as viral reservoirs in human immunodeficiency virus type 1 infections. Proceedings of the National Academy of Sciences, USA 86, 6759.
8 . Gabuzda, D. H., Ho, D. D., de la Monte, S. M. et al. (1986). Immunohistochemical identification of HTLV-III antigen in brains of patients with AIDS. Annals of Neurology 20, 28995.[CrossRef][ISI][Medline]
9 . Giulian, D., Vaca, K. & Noonan, C. A. (1990). Secretion of neurotoxins by mononuclear phagocytes infected with HIV-1. Science 250, 15936.[ISI][Medline]
10
.
Tyor, W. R., Power, C., Gendelman, H. E. et al. (1993). A model of human immunodeficiency virus encephalitis in scid mice. Proceedings of the National Academy of Sciences, USA 90, 865862.
11 . Crowe, S. M., Mills, J., Kirihara, J. et al. (1990). Full-length recombinant CD4 and recombinant gp120 inhibit fusion between HIV infected macrophages and uninfected CD4-expressing T-lymphoblastoid cells. AIDS Research and Human Retroviruses 6, 10317.[ISI][Medline]
12
.
Badley, A. D., Dockrell, D., Simpson, M. et al. (1997). Macrophage-dependent apoptosis of CD4+ T lymphocytes from HIV-infected individuals is mediated by FasL and tumor necrosis factor. Journal of Experimental Medicine 185, 5564.
13 . Herbein, G., Mahlknecht, U., Batliwalla, F. et al. (1998). Apoptosis of CD8+ T cells is mediated by macrophages through interaction of HIV gp120 with chemokine receptor CXCR4. Nature 395, 18994.[CrossRef][ISI][Medline]
14
.
Garaci, E., Aquaro, S., Lapenta, C. et al. (2003). Anti-nerve growth factor Ab abrogates macrophage-mediated HIV-1 infection and depletion of CD4+ T lymphocytes in hu-SCID mice. Proceedings of the National Academy of Sciences, USA 100, 892732.
15
.
Shi, B., De Girolami, U., He, J. et al. (1996). Apoptosis induced by HIV-1 infection of the central nervous system. Journal of Clinical Investigation 98, 197990.
16
.
Aquaro, S., Panti, S., Caroleo, M. C. et al. (2000). Primary macrophages infected by human immunodeficiency virus trigger CD95-mediated apoptosis of uninfected astrocytes. Journal of Leukocyte Biology 68, 42935.
17
.
Mollace, V., Salvemini, D., Riley, D. P. et al. (2002). The contribution of oxidative stress in apoptosis of human-cultured astroglial cells induced by supernatants of HIV-1-infected macrophages. Journal of Leukocyte Biology 71, 6572.
18 . Bagnarelli, P., Valenza, A., Menzo, S. et al. (1996). Dynamics and modulation of human immunodeficiency virus type 1 transcripts in vitro and in vivo. Journal of Virology 70, 760313.[Abstract]
19
.
Gendelman, H. E., Orenstein, J. M., Martin, M. A. et al. (1988). Efficient isolation and propagation of human immunodeficiency virus on recombinant colony-stimulating factor 1-treated monocytes. Journal of Experimental Medicine 167, 142841.
20 . Orenstein, J. M., Meltzer, M. S., Phipps, T. et al. (1988). Cytoplasmic assembly and accumulation of human immunodeficiency virus types 1 and 2 in recombinant human colony-stimulating factor-1-treated human monocytes: an ultrastructural study. Journal of Virology 62, 257886.[ISI][Medline]
21 . Aquaro, S., Bagnarelli, P., Guenci, T. et al. (2002). Long-term survival and virus production in human primary macrophages infected by human immunodeficiency virus. Journal of Medical Virology 68, 47988.[CrossRef][ISI][Medline]
22
.
Garaci, E., Caroleo, M. C., Aloe, L. et al. (1999). Nerve growth factor is an autocrine factor essential for the survival of macrophages infected with HIV. Proceedings of the National Academy of Sciences, USA 96, 140138.
23
.
Perno, C. F., Yarchoan, R., Cooney, D. A. et al. (1988). Inhibition of human immunodeficiency virus (HIV-1/HTLV-IIIBa-L) replication in fresh and cultured human peripheral blood monocytes/macrophages by azidothymidine and related 2',3'-dideoxynucleosides. Journal of Experimental Medicine 168, 111125.
24 . O'Brien, W. A., Namazi, A., Kalhor, H. et al. (1994). Kinetics of human immunodeficiency virus type 1 reverse transcription in blood mononuclear phagocytes are slowed by limitations of nucleotide precursors. Journal of Virology 68, 125863.[Abstract]
25
.
Hao, Z., Cooney, D. A., Hartman, N. R. et al. (1988). Factors determining the activity of 2',3'-dideoxynucleosides in suppressing human immunodeficiency virus in vitro. Molecular Pharmacology 34, 4315.
26 . Perno, C. F., Yarchoan, R., Balzarini, J. et al. (1992). Different pattern of activity of inhibitors of the human immunodeficiency virus in lymphocytes and monocyte/macrophages. Antiviral Research 17, 289304.[CrossRef][ISI][Medline]
27 . Aquaro, S., Calio, R., Balzarini, J. et al. (2002). Macrophages and HIV infection: therapeutical approaches toward this strategic virus reservoir. Antiviral Research 55, 20925.[CrossRef][ISI][Medline]
28 . Aquaro, S., Perno, C. F., Balestra, E. et al. (1997). Inhibition of replication of HIV in primary monocyte/macrophages by different antiviral drugs and comparative efficacy in lymphocytes. Journal of Leukocyte Biology 62, 13843.[Abstract]
29 . Wainberg, M. A., Salomon, H., Gu, Z. et al. (1995). Development of HIV-1 resistance to (-)2'-deoxy-3'-thiacytidine in patients with AIDS or advanced AIDS-related complex. AIDS 9, 3517.[ISI][Medline]
30 . Miller, M. D., Anton, K. E., Mulato, A. S. et al. (1999). Human immunodeficiency virus type 1 expressing the lamivudine-associated M184V mutation in reverse transcriptase shows increased susceptibility to adefovir and decreased replication capability in vitro. Journal of Infectious Diseases 179, 92100.[CrossRef][ISI][Medline]
31 . Schuurman, R., Nijhuis, M., van Leeuwen, R. et al. (1995). Rapid changes in human immunodeficiency virus type 1 RNA load and appearance of drug-resistant virus populations in persons treated with lamivudine (3TC). Journal of Infectious Diseases 171, 14119.[ISI][Medline]
32 . Kuritzkes, D. R., Quinn, J. B., Benoit, S. L. et al. (1996). Drug resistance and virologic response in NUCA 3001, a randomized trial of lamivudine (3TC) versus zidovudine (ZDV) versus ZDV plus 3TC in previously untreated patients. AIDS 10, 97581.[ISI][Medline]
33 . Perno, C. F. & Yarchoan, R. (1993). Culture of HIV in monocytes and macrophages. In Current Protocols in Immunology, vol. 3 (Coligan, J. E., Kruisbeek, A. M., Margulies, D. H. et al., Eds), pp. 12.4. 111. John Wiley & Sons, New York, NY, USA.
34
.
Perno, C. F., Yarchoan, R., Cooney, D. A. et al. (1989). Replication of human immunodeficiency virus in monocytes. Granulocyte/macrophage colony-stimulating factor (GM-CSF) potentiates viral production yet enhances the antiviral effect mediated by 3'-azido-2'3'-dideoxythymidine (AZT) and other dideoxynucleoside congeners of thymidine. Journal of Experimental Medicine 169, 93351.
35
.
Balzarini, J., Pelemans, H., Karlsson, A. et al. (1996). Concomitant combination therapy for HIV infection preferable over sequential therapy with 3TC and non-nucleoside reverse transcriptase inhibitors. Proceedings of the National Academy of Sciences, USA 93, 131527.
36 . Toohey, K., Wehrly, K., Nishio, J. et al. (1995). Human immunodeficiency virus envelope V1 and V2 regions influence replication efficiency in macrophages by affecting virus spread. Virology 213, 709.[CrossRef][ISI][Medline]
37 . Aquaro, S., Calio, R., Balestra, E. et al. (1998). Clinical implications of HIV dynamics and drug resistance in macrophages. Journal of Biological Regulators and Homeostatic Agents 12, 237.[ISI][Medline]
38
.
Richman, D. D., Kornbluth, R. S. & Carson, D. C. (1987). Failure of dideoxynucleosides to inhibit human immunodeficiency virus replication in cultured human macrophages. Journal of Experimental Medicine 166, 11449.
39 . Sei, S., Stewart, S. K., Farley, M. et al. (1996). Evaluation of human immunodeficiency virus (HIV) type 1 RNA levels in cerebrospinal fluid and viral resistance to zidovudine in children with HIV encephalopathy. Journal of Infectious Diseases 174, 12006.[ISI][Medline]
40 . Cunningham, P. H., Smith, D. G., Satchell, C. et al. (2000). Evidence for independent development of resistance to HIV-1 reverse transcriptase inhibitors in the cerebrospinal fluid. AIDS 14, 194954.[CrossRef][ISI][Medline]
41
.
Tang, Y. W., Huong, J. T., Lloyd, R. M. et al. (2000). Comparison of human immunodeficiency virus type 1 RNA sequence heterogeneity in cerebrospinal fluid and plasma. Journal of Clinical Microbiology 38, 46379.
42 . Venturi, G., Catucci, M., Romano, L. et al. (2000). Antiretroviral resistance mutations in human immunodeficiency virus type 1 reverse transcriptase and protease from paired cerebrospinal fluid and plasma samples. Journal of Infectious Diseases 181, 7405.[CrossRef][ISI][Medline]
43 . Lanier, E. R., Sturge, G., McClernon, D. et al. (2001). HIV-1 reverse transcriptase sequence in plasma and cerebrospinal fluid of patients with AIDS dementia complex treated with Abacavir. AIDS 15, 74751.[CrossRef][ISI][Medline]
44 . Bestetti, A., Presi, S., Pierotti, C. et al. (2004). Long-term virological effect of highly active antiretroviral therapy on cerebrospinal fluid and relationship with genotypic resistance. Journal of Neurovirology 10, Suppl. 1, 527.
45 . Kaul, M., Garden, G. A. & Lipton, S. A. (2001). Pathways to neuronal injury and apoptosis in HIV-associated dementia. Nature 410, 98894.[CrossRef][ISI][Medline]
46 . Kuritzkes, D. R. (1996). Clinical significance of drug resistance in HIV-1 infection. AIDS 10, Suppl. 5, 2731.
47 . Boyer, P. L. & Hughes, S. H. (1995). Analysis of mutations at position 184 in reverse transcriptase of human immunodeficiency virus type 1. Antimicrobial Agents and Chemotherapy 39, 16248.[Abstract]
48 . Back, N. K., Nijhuis, M., Keulen, W. et al. (1996). Reduced replication of 3TC-resistant HIV-1 variants in primary cells due to a processivity defect of the reverse transcriptase enzyme. EMBO Journal 15, 40409.[Abstract]
49
.
Sharma, P. L. & Crumpacker, C. S. (1999). Decreased processivity of human immunodeficiency virus type 1 reverse transcriptase (RT) containing didanosine-selected mutation Leu74Val: a comparative analysis of RT variants Leu74Val and lamivudine-selected Met184Val. Journal of Virology 73, 844856.
50
.
Huang, H., Chopra, R., Verdine, G. L. et al. (1998). Structure of a covalently trapped catalytic complex of HIV-1 reverse transcriptase: implications for drug resistance. Science 282, 166975.
|