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The M184V substitution in HIV-1 RT develops rapidly following initiation of therapy with 3TC and confers high-level phenotypic resistance to this drug both in vitro and in vivo. Interestingly, the presence of M184V is also associated with alteration of several mechanisms relating to RT function that include decreased RTprocessivity, reduced nucleotide-dependent primer unblocking, increased fidelity, hypersensitization to other NRTIs, impaired viral fitness, and delayed appearance of mutations in RT that are responsible for resistance to thymidine analogues (i.e. thymidine-associated mutations or TAMs). In addition, M184V may affect viral transmission and immunological response. Collectively, these factors might explain the residual antiviral effect and clinical benefit observed with continued use of 3TC in combination therapy regimens following the emergence of M184V.

The drug resistance profile of treatment-naive HIV-infected individuals living in Buenos Aires, Argentina, was studied. Samples taken from 94 drug-naive individuals with established HIV infection and 13 patients with primary HIV infection were assessed by nucleotide sequencing and LIPA. The prevalence of drug-associated primary mutations in individuals with established infection was very low. In the viral protease region, 1/86 (1.2%) individuals carried the D30N mutation, whereas 1/85 (1.2%) had the M41L mutation in the reverse transcriptase (RT) region. Secondary mutations in both the protease and RT regions were found in almost 90% of the individuals. In individuals with primary infection, primary mutations were detected in 2/13 (15.4%) patients, one of them carrying M461 mutation in the protease while the other patient had a mutation at codon 184 of the RT. In accordance with current drug resistance testing guidelines, the results of this study suggest that susceptibility tests need not be performed at this time prior to initiation of antiretroviral therapy in HIV-1-infected people in Argentina. However, the public health implications of this subject warrant follow-up studies that will examine a larger number of drug-naive patients, not only in Buenos Aires but also in other major Argentinian cities and in rural areas.

The widespread use of antiretroviral agents (ARVs) and the growing occurrence of HIV strains resistant to these drugs have given rise to serious concerns regarding the transmission of resistant viruses to newly infected persons. Plasma viral RNA from 80 individuals newly infected between 1997 and 1999 was genotyped by automated sequencing to analyze the profile of viruses resistant to nucleoside and non-nucleoside reverse transcriptase inhibitors (NRTIs and NNRTIs) and to protease inhibitors (PIs). The prevalence of mutations that conferred primary resistance to PIs (L10I, D30Y, V82A, L90M) was 15% of the cohort. RT genotypic variants, associated with high-level resistance to ARVs, were observed in 21% of individuals, including NRTI, NNRTI and multidrug (MDR) resistance in 6, 5, and 10% of cases, respectively. The phenotypic susceptibility of viral isolates to ARVs was also assayed and showed transmission of high-level resistance to ZDV, 3TC, and PIs in those individuals with MDR. The transmission of drug-resistant HIV genotypic variants is a serious problem that merits further attention by public health officials, virologists, and clinicians.

The selective packaging of the primer tRNA(Lys3) into HIV-1 particles is dependent upon the viral incorporation of the Pr160gag-pol precursor protein. In order to map a tRNA(Lys3) binding site within this precursor, we have studied the effects of mutations in Pr160gag-pol upon the selective incorporation of tRNA(Lys3). Many of these mutations were placed in a protease-negative HIV-1 proviral DNA to prevent viral protease degradation of the mutant Gag-Pol protein. C-terminal deletions of protease-negative Gag-Pol that removed the entire integrase sequence and the RNase H and connection subdomains of reverse transcriptase did not inhibit the incorporation of either the truncated Gag-Pol or the tRNA(Lys3), indicating that these regions are not required for tRNA(Lys3) binding. On the other hand, larger C-terminal deletions, which also remove the thumb subdomain sequence, did prevent tRNA(Lys3) packaging, without inhibiting viral incorporation of the truncated Gag-Pol, indicating a possible interaction between thumb subdomain sequences and tRNA(Lys3). While point mutations K249E, K249Q, and R307E in the primer grip region of the thumb subdomain have been reported to inhibit the in vitro interaction of mature reverse transcriptase with the anticodon loop of tRNA(Lys3), we find that these mutations do not inhibit tRNA(Lys3) packaging into the virus, which supports other work indicating that the anticodon loop of tRNA(Lys3) is not involved in interactions with Pr160gag-pol during tRNA(Lys3) packaging.

An early step in the life cycle of human immunodeficiency virus type 1 is the reverse transcription of the viral RNA genome into double-stranded DNA, which is subsequently translocated to the cell nucleus. It is then integrated into host DNA and serves as a template for viral gene expression. Reverse transcription is catalyzed by the viral enzyme reverse transcriptase and is a complex process comprising a series of RNA-dependent DNA polymerization, DNA-dependent DNA polymerization, and RNase H reactions. Strand transfer reactions are required to complete the process. Reverse transcription is initiated when a molecule of host cell tRNA(lys3), which serves as a primer, is bound to the primer binding site of viral genomic RNA. The viral nucleocapsid protein is involved in each of the initiation of reverse transcription and in subsequent strand transfer or template-switching events. We review the interactions among reverse transcriptase, viral genomic RNA, the tRNA primer of reverse transcription, and viral nucleocapsid protein in the various steps of reverse transcription, including primer placement, initiation, and processive synthesis.

We have tested both wild-type and drug-resistant mutated, recombinant human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) molecules for sensitivity to each of two non-nucleoside RT inhibitors (NNRTI), (+)-calanolide A and nevirapine, in primer extension assays. We found that RT containing either the V106A or Y181C substitutions, associated with NNRTI resistance, displayed approximately 90-fold resistance to nevirapine but remained fully sensitive to (+)-calanolide A and that the Y181C mutation marginally enhanced susceptibility to the latter drug. In contrast, the Y188H substitution in RT resulted in about 30-fold resistance to (+)-calanolide A in these assays but did not result in diminished sensitivity to nevirapine. Tissue culture results indicated that the combination of (+)-calanolide A and nevirapine possessed an additive to weakly synergistic effect in blocking replication of HIV-1 in tissue culture. These results suggest that (+)-calanolide A and nevirapine might have rationale as a combination therapy for HIV disease.

Prolonged treatment with antiretroviral drugs results in the selection of HIV-1 variants with mutations conferring resistance to nucleoside and non-nucleoside reverse transcriptase inhibitors (NRTI and NNRTI) or to protease inhibitors (PI). There is serious concern about transmission of resistant viruses to newly infected persons. This study monitored the prevalence of resistant viruses in individuals undergoing primary HIV infection.Resistance testing was performed on 81 individuals infected between 1997 and 1999 by injecting drug use (n =21), sexual (n = 56), or unknown (n = 4) transmission.Automated sequencing was used to genotype the reverse transcriptase (RT) and protease regions of virus isolated from patients' plasma. The phenotypic susceptibility of stimulated peripheral blood mononuclear cells to antiretroviral drugs was assayed. Line probe assays detected quasispecies variations in wild-type and mutated RT codons.A high prevalence of PI and RT genotypic variants, associated with high-level resistance to antiretroviral drugs, was observed in individuals newly infected by injecting drug use (PI = 24%, RT = 24%) or sexual transmission (PI = 12%, RT = 22%). The PI mutations, L101, V82A, and L90M, were found in 10.5, 3 and 4% of cases, respectively; whereas for RT, primary mutations at positions T215Y (zidovudine), M184V (lamivudine), T69D/A (zalcitabine), and K103N (multi-NNRTI) were present in 8, 5, 4, and 4% of subjects, respectively. Resistance to NRTI was demonstrated by phenotypic, genotypic, and line probe analyses. Transmission of multidrug (NRTI/NNRTI/PI) resistance in eight subjects (9.9%) was confirmed by showing that source partners possessed viruses of similar genotype.The transmission of drug-resistant HIV is a serious problem that merits further attention by public health officials as well as virologists and clinicians.

To study the extent to which phenotypic resistance to stavudine occurs under therapy, we studied 18 pairs of human immunodeficiency virus type 1 (HIV-1) isolates from patients both prior to and following 24-48 weeks of treatment with stavudine monotherapy or stavudine in combination with either didanosine or lamivudine. We also used a nested polymerase chain reaction (PCR) assay to probe for the presence of specific mutations associated in culture with stavudine resistance. The results showed that resistance to stavudine (approximately 3-10 fold) was observed in nine of ten cases of monotherapy, in three of four cases of therapy involving both stavudine and didanosine, and in two of four cases involving stavudine and lamivudine. Viruses from the four patients receiving stavudine plus didanosine became resistant to didanosine in only one instance while the use of lamivudine plus stavudine yielded resistance to lamivudine each time. Whereas changes in the reverse transcriptase (RT) genes of resistant isolates were frequently observed, two mutations, previously identified with stavudine resistance in tissue culture (i.e., V75T and I50T), could not be identified in the clinical samples by either direct sequencing of the RT gene or by PCR amplification. Thus, resistance to stavudine can occur, albeit at low levels, in the context of prolonged therapy with this drug but is not associated with specific mutations in HIV RT at either codons 75 or 50 in clinical samples.

We have selectively mutagenized specific residues at the junction between the protease (PR) and reverse transcriptase (RT) genes of human immunodeficiency virus type 1 (HIV-1) to study the effects of PR-RT fusion proteins in the context of a full-length, infectious proviral construct. Mutant viruses derived from COS-7 cells transfected with this construct were analyzed in regard to each of viral replication, maturation, and infectivity. Immunoblot analysis revealed that the mutation prevented cleavage between the PR and RT proteins and that both existed as a PR-RT fusion protein in each of cellular and viral lysates. Interestingly, intracellular PR that existed within the PR-RT fusion protein remained functionally active, whereby HIV-1 precursor proteins were processed efficiently. Furthermore, the RT component of the fusion protein also retained its enzymatic activity as shown in RT assays. Electron microscopy revealed that the mutant viruses containing the PR-RT fusion protein possessed wild-type morphology. These viruses also displayed wild-type sensitivities to inhibitors of each of the HIV-1 PR and RT activities. However, viruses containing the PR-RT fusion protein were 20 times less infectious than wild-type viruses. This defect was further pronounced when mutated Gag-Pol proteins were overexpressed as a consequence of an additional mutation that interfered with frameshifting. Thus, unlike cleavage site mutations at the N terminus of PR, a cleavage site mutation between PR and RT did not affect the enzymatic activities of either PR or RT and viruses containing PR-RT fusion proteins were viable.

HIV-infected individuals, who received 3TC monotherapy over one year, generally had lower plasma viral burden than at base-line. This was in spite of high-level resistance to this compound and the appearance of the M184V substitution in the HIV reverse transcriptase (RT) gene, responsible for diminished sensitivity to 3TC. This apparent contradiction is explained by an increase in the fidelity of the HIV RT, conferred by the M184V mutation, on the basis of the following observations. First, titers of viral neutralizing antibodies, as measured against sequential autologous HIV isolates, remained stable in this population in contrast to rapid declines in patients treated with other drugs. This suggests that increased fidelity of M184V RT may limit variability in the HIV env gene and result in protracted effectiveness of anti-viral immune responsiveness. Second, recombinant HIV, that contained the M184V substitution in RT, could not replicate in the presence of d4T, AZT, Nevirapine, Delavirdine or Saquinavir, using previously described protocols for the generation of drug resistance in vitro.

We conducted detailed virological evaluations of 16 HIV-1-infected paediatric patients treated with 3TC (lamivudine) monotherapy. High-level phenotypic resistance against this compound (up to 2,500-fold) was seen in virtually all cases, usually within 8-12 weeks of initiation of therapy. This was concomitant with the appearance of the M184V mutation in viral reverse transcriptase, previously shown to be responsiblefor such resistance. Viral burden fell in virtually all cases after commencement of therapy, and remained below baseline in each instance studied, despite a rebound effect and the appearance of drug resistance. Viral isolates from some patients underwent a switch from a non-syncytium-inducing (NSI) to a syncytium-inducing (SI) phenotype during the course of the study, although no relationship was apparent between dose of drug employed, time to development of drug resistance or time of appearance of SI phenotype.

To determine the rate of development of in vitro HIV resistance to (-)2'-deoxy-3'-thiacytidine (3TC) and relate the effect of dose to emergence of resistance.HIV-infected men and non-pregnant women, agedor = 18 years, with a CD4 countor = 300 x 10(6)/l cells were followed in a Phase I/II study, in which they were evaluated for tolerance to 3TC and effect of this agent with regard to viral susceptibility. Peripheral blood and plasma samples were collected at regular intervals for analysis. HIV was isolated using umbilical cord blood mononuclear cells as targets. These cells were also used in determinations of median inhibitory drug concentration. Specific amplification of the 184 mutation site, associated with HIV resistance to 3TC, was performed by polymerase chain reaction, using specific primer pairs, on DNA harvested from infected peripheral blood mononuclear cells (PBMC) of donors or, alternatively, on DNA that had been reverse transcribed from plasma-associated HIV RNA.Phenotypic resistance was detected in approximately one-third of individuals studied, who were followed between 8 and 56 weeks. Development of 3TC resistance occurred independently of dose, although time of first appearance of resistant HIV-1 variants appeared reduced at high 3TC doses. Amino-acid changes at codon 184 in HIV-1 reverse transcriptase were associated with, and preceded, the development of phenotypic 3TC resistance. Most commonly, a Met to Ile substitution appeared transiently before being superceded by a Val substitution at codon 184.In vitro resistance to 3TC developed in a high proportion of subjects who received prolonged monotherapy with this drug. The development of resistance to 3TC was associated with appearance of mutated viral forms and the disappearance of wild-type virus, with regard to codon 184, in both patient plasma and PBMC.

The prolonged use of anti-viral nucleosides (ZDV, ddI, ddC) in HIV-infected patients has given rise to the isolation of viral variants that display resistance against these compounds. Tissue culture selection experiments, involving increasing concentrations of anti-viral compounds, have likewise been shown to select for drug-resistant strains of HIV. Cloning, sequencing and site-directed mutagenesis have shown that a series of point mutations in the viral reverse transcriptase (RT) are responsible for this phenomenon. A different series of mutations in RT are responsible for resistance against non-nucleoside inhibitors of this enzyme. These mutations are due to the error-prone nature of RT during viral replication. Mutated forms of recombinant RT, that derive from drug-resistant viruses, have reduced affinity for relevant triphosphorylated nucleosides.

The K65R mutation in human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) encodes cross-resistance to 2',3'-dideoxycytidine (ddC), 2',3'-dideoxy-3'-thiacytidine (3TC), and 2',3'-dideoxyinosine (ddI). We characterized the in vitro sensitivities of recombinant wild type (wt) and K65R mutant RT to dideoxynucleoside triphosphate (ddNTP) inhibitors, using a variety of primer-templates. With poly(rA)-oligo(dT), the K65R mutant showed slight increases in Ki for ddTTP and 3'-azido, 3'-deoxythymidine triphosphate (AZTTP) compared to wt RT, but neither wt nor K65R RT was inhibited by ddCTP or ddATP. With poly(rI)-oligo(dC), the K65R mutant showed a 2-fold increase in Km for dCTP and a 20-fold increase in Ki for ddCTP compared to wt, whereas ddATP, ddTTP, and AZTTP failed to inhibit either enzyme. With a heteropolymeric primer-template, the K65R mutant showed 10-fold reduced sensitivities to ddCTP, 3TCTP, and ddATP, and 4-fold reduced sensitivity to AZTTP, compared to wt. In contrast, both enzymes were equally inhibited by ddTTP and ddGTP. HIV-1 cross-resistance to ddC/3TC/ddI resulting from the K65R mutation may therefore involve selective alterations in substrate/inhibitor recognition. Additionally, competitive inhibition by ddNTPs noncomplementary to the template base appears to be unimportant in the mechanism of inhibition of HIV-1 RT by dideoxynucleoside analogs.

Nucleoside antagonists of human immunodeficiency virus (HIV) reverse transcriptase (RT) activity have been commonly used in the therapy of HIV-associated disease. The prolonged use of such drugs has led to the development of HIV variants that display resistance against these compounds. HIV drug resistance has been documented clinically for each of the following nucleosides: 3'-azido-3'-deoxythymidine (AZT; zidovudine, ZDV), 2',3'-dideoxyinosine (ddI; didanosine), and 2',3'-dideoxycytidine (ddC; zalcitabine). In addition, resistance has been demonstrated against a series of non-nucleoside inhibitors of the viral RT. Several groups have documented that a series of point mutations within the HIV pol gene, that encodes the RT enzyme, is responsible for HIV drug resistance. Diminished sensitivity to anti-viral drugs results from both the selective pressure exerted by these compounds in individuals receiving prolonged therapy and the error-prone nature of the viral RT itself, thus permitting the outgrowth of mutated forms. Patients suffering from both disease progression and/or low CD4 counts are most likely to develop HIV drug resistance.

We developed an endogenous in vitro reverse transcription assay to study the properties of priming and template switching during human immunodeficiency virus (HIV) replication. Reactions were primed with HIV reverse transcriptase (RT) and either a deoxyoligonucleotide primer (dPR) or tRNA(Lys-3), the natural primer for reverse transcription. The RNA templates utilized were the actual HIV sequences involved in the first template switch, namely a primer binding sequence (PBS)/U5/R RNA donor template and a R/U3 RNA acceptor template. Reverse transcription reactions using the latter templates and dPR or tRNA(Lys-3) as primers yielded four major products: (-)-strong-stop DNA, a partial template-switched DNA, full template-switched DNA, and a pseudo-PBS-primed product. Use of dPR resulted in three times less template switching than was obtained with tRNA(Lys-3). When reactions were primed with either dPR or tRNA(Lys-3), increases in acceptor:donor template ratios resulted in augmented template switching. Increasing the concentration of RT resulted in increased priming from the PBS but had no effect on the efficiency of template switching. Decreasing the extent of R region overlap resulted in a drop in efficiency of template switching. Decreases in the R region on the donor template also caused a drop in initiation of transcription that was primed by tRNA(Lys-3) from the PBS. In contrast, a corresponding reduction of the R region on the acceptor template had no effect on priming. We conclude that a transcriptional complex of tRNA(Lys-3) and RT may be associated not only with the PBS but also with other cis RNA sequences and secondary structures in a manner essential for efficient priming and template switching.

We generated variants of the human immunodeficiency virus type 1 (HIV-1) that are resistant to 2',3'-dideoxycytidine (ddC) and 2',3'-didehydro-3'-deoxythymidine (d4T) by in vitro selection in MT-4 cells. Portions of flanking protease and integrase sequences as well as the complete reverse transcriptase (RT) open-reading frame of these viruses were cloned and sequenced, using polymerase chain reaction (PCR)-based methods. Mutations were observed at amino acid position 65 (Lys--Arg; AAA--AGA) when ddC was employed in the selection procedure and at site 50 (Ile--Thr; ATT--ACT) when d4T was used. We confirmed the ability of these mutations to confer diminished sensitivity for these compounds by site-directed mutagenesis, in which these mutations were inserted into the pol gene of infectious recombinant HXB2-D DNA. Viruses that contained the site 65 mutation possessed approximately 5-10 fold resistance against ddC when compared with wild-type HXB2-D. The site 50 mutation conferred approximately 30-fold resistance to d4T in these same assays. Similar results were obtained using primary cord blood lymphocytes in drug resistance assays, indicating that these mutations could confer drug resistance in more than one cell type and that the respective mutations could be expressed in cells of primary origin. No cross-resistance against 3'-azido-3'-deoxythymidine (AZT) was noted for either the site 65 or 50 mutations.

A series of monoclonal antibodies against p51/p66 human immunodeficiency virus-1 (HIV-1) reverse transcriptase (RT) were prepared by immunizing mice with the native enzyme immobilized on nitrocellulose. One of these antibodies, designated 1E8, was found to inhibit both RNA-dependent and DNA-dependent polymerase activities of RT but had no effect on the RNase H activity of the enzyme. This inhibition was noncompetitive with respect to primer/template and competitive with respect to deoxynucleoside triphosphate (dNTP). The extent of 1E8 inhibition of RT polymerase activity decreased with increasing concentrations of dNTP in the incubation but was not affected by changes in primer/template concentration. 1E8 bound equally well in solution to both free RT and to the RT-primer/template complex. However, binding to the latter was significantly reduced by the addition of increasing concentrations of dNTP. The ability of dNTP to inhibit the interaction of 1E8 with the RT-primer/template complex was dependent on the identity of the homopolymeric primer/template used; only that dNTP complementary to the template was effective in this respect. 1E8 bound to the p51/p66 reverse transcriptase heterodimer in solution and reacted with both p51 and p66 subunits of reverse transcriptase on Western blots. The antibody is therefore presumed to recognize a linear surface epitope on the enzyme. 1E8 was found to specifically recognize a peptide with the sequence KKDSTKWRK. This sequence corresponds to residues 65-73 of HIV-1 reverse transcriptase, a region identified as highly antigenic by several computer algorithms. Two mutations within this sequence have been identified with resistance to 3'-azido,3'-deoxythymidine. We conclude that residues 65-73 of HIV-1 reverse transcriptase may be at or near the polymerase active site of the enzyme, and may form part of the deoxynucleoside triphosphate binding domain of the enzyme.

The use of zidovudine (ZDV) and other forms of nucleoside therapy, including dideoxyinosine (ddI), to treat HIV-infected individuals has led to both longer survival and improved quality of life. However, ZDV-resistant variants of HIV-1 can be isolated from patients undergoing prolonged therapy with this drug. HIV drug resistance against ZDV, ddI and other nucleosides is attributable to a series of point mutations within the pol gene of HIV-1 that encodes the viral enzyme, reverse transcriptase (RT). This is not surprising, since the virus is known to replicate at high rates in infected individuals; moreover the RT which mediates transcription of proviral DNA from viral genomic RNA is known to be highly error-prone. Thus, mutants of HIV-1, which possess a drug resistance phenotype and genotype, may be expected to emerge under the selective pressure of long-term anti-viral chemotherapy. HIV drug resistance occurs most commonly in individuals with low CD4 counts, who have progressed to more serious forms of disease. Moreover, viruses obtained from patients with AIDS generally display higher levels of resistance, relative to pre-treatment isolates, than do viruses from patients with more limited illness. Although observations of drug resistance can be correlated with disease progression and a weakened immune system, it is still unclear whether a cause and effect relationship exists. Current clinical research is designed to answer this question while testing the notion that combinations of nucleosides and immuno-stimulatory drugs may provide important clinical benefits.

Infection by HIV-1 of monocyte cell lines, in contrast to T lymphocytes, did not lead to decreased steady-state levels of CD4 mRNA. Similar results were also obtained using clonal derivatives of infected U-937 cells that produced either competent, highly replicative progeny viruses or defective non-infectious particles. In each case, the infected U-937 cells or clonal derivatives were found to be significantly deficient with regard to surface representation of CD4 protein, in spite of the presence of high levels of CD4 mRNA. However, both HIV-1-infected U-937 cells, as well as clonal derivatives which produced high levels of viral env mRNA and non-infectious viral structures that lacked envelope glycoproteins, contained diminished levels of OKT4-immunoprecipitable CD4 protein, in comparison with uninfected U-937 cells. Thus, expression of viral env mRNA but neither the efficient synthesis or packaging of viral glycoproteins or viral assembly is required for disappearance of cell surface CD4 to occur. Furthermore, viral gp160 co-precipitated with CD4 in both the parental and cloned cell lines. We have also shown that the generation of intracellular complexes of gp160 and CD4 is directly responsible for the disappearance of cell surface CD4 in HIV-1-infected U-937 cells. In this system, expression of gp160 was both necessary and sufficient to result in CD4 receptor down-modulation. Finally, in vitro co-translation studies revealed that the presence or synthesis of viral gp160 led to a failure to efficiently generate CD4 protein.

It has been widely reported that zidovudine (ZDV)-resistant variants of human immunodeficiency virus type 1 (HIV-1) can be isolated from patients undergoing prolonged therapy with this drug. At the same time, treatment of HIV-infected individuals with ZDV and other forms of nucleotide therapy, including didanosine (ddI), have enabled patients to live longer than would otherwise be the case and to enjoy improved quality of life. HIV resistance to ZDV, ddI, and other nucleosides is attributable to a series of point mutations within the pol gene of HIV-1 that encodes the viral enzyme, reverse transcriptase (RT). This is not surprising as the virus is known to replicate at high rates in infected individuals; moreover the RT that mediates transcription of proviral DNA from viral genomic RNA is known to be highly error prone. Thus, mutants of HIV-1, which possess a drug-resistance phenotype and genotype, may be expected to emerge under the selective pressure of long-term antiviral chemotherapy. This article describes a novel mutation at site 184 within the pol gene that accounts for resistance against both ddI and zalcitibine (ddC). HIV drug resistance occurs most commonly in individuals with low CD4 cell counts who have progressed to more serious forms of disease. Moreover, viruses obtained from patients with AIDS generally display higher levels of resistance, relative to pretreatment isolates, than do viruses from patients with more-limited illness. Although observations of drug resistance can be correlated with disease progression and a weakened immune system, it is still unclear whether a cause-and-effect relationship exists. Because of the error-prone nature of viral RT and the fact that the HIV-1 genome can mutate efficiently, it can be anticipated that viral drug resistance may emerge for all forms of nucleotide therapy to be offered in the future. In addition, resistance may also become apparent with regard to drugs that block HIV replication by acting at sites within the viral replication cycle other than RT.

The replication cycle of any virus involves a number of steps, beginning with specific attachment to a cell surface receptor leading eventually to production of progeny viruses by infected cells. In the case of the immunodeficiency virus type-1 (HIV-1), the first step involves a specific interaction between the gp120 viral envelope surface protein and specific CD4 receptor sites at the cell surface. This is followed by penetration of the virus into cells and the formation of proviral double-stranded DNA from single-stranded viral RNA, a process mediated through the action of the viral enzyme called reverse transcriptase. This, in turn, leads to the migration of proviral DNA into the nucleus of the cell and the integration of such DNA within the host cell genome. Finally both viral RNA and viral proteins are produced by the cell's genetic apparatus and new viruses are assembled at the cell surface. The fact that integration of viral DNA into host cell chromosomes occurs means that any cellular replication event will be accompanied by replication of viral DNA. Each of these steps represents a potential target for anti-viral chemotherapy. To date, most efforts to treat HIV-associated disease have focused on the reverse transcription step. In this respect, zidovudine (AZT) has been the most widely used anti-viral drug studied. However, the relative toxicity and lack of efficiency of this drug means that our efforts to develop new therapeutic strategies to combat HIV infection must continue.(ABSTRACT TRUNCATED AT 250 WORDS)