An In Vitro Model of Blood-Brain Barrier for Studies on HIV Neuroinflammation and CNS Antibody Penetration.

The blood-brain barrier (BBB) is one of several barriers between the brain and the peripheral blood system to maintain homeostasis. Understanding the interactions between infectious agents such as human immunodeficiency virus type 1 (HIV-1), which are capable of traversing the BBB and causing neuroinflammation requires modeling an authentic BBB in vitro. Such an in vitro BBB model also helps develop means of targeting viruses that reside in the brain via natural immune effectors such as antibodies. The BBB consists of human brain microvascular endothelial cells (HBMECs), astrocytes, and pericytes. Here we report in vitro methods to establish a dual-cell BBB model consisting of primary HBMECs and primary astrocytes to measure the integrity of the BBB and antibody penetration of the BBB, as well as a method to establish a single cell BBB model to study the impact of HIV-1 infected medium on the integrity of such a BBB.

CCL2 mobilizes ALIX to facilitate Gag-p6 mediated HIV-1 virion release.

Cellular ESCRT machinery plays pivotal role in HIV-1 budding and release. Extracellular stimuli that modulate HIV-1 egress are currently unknown. We found that CCL2 induced by HIV-1 clade B (HIV-1B) infection of macrophages enhanced virus production, while CCL2 immuno-depletion reversed this effect. Additionally, HIV-1 clade C (HIV-1C) was refractory to CCL2 levels. We show that CCL2-mediated increase in virus production requires Gag late motif LYPX present in HIV-1B, but absent in HIV-1C, and ALIX protein that recruits ESCRT III complex. CCL2 immuno-depletion sequestered ALIX to F-actin structures, while CCL2 addition mobilized it to cytoplasm facilitating Gag-ALIX binding. The LYPX motif improves virus replication and its absence renders the virus less fit. Interestingly, novel variants of HIV-1C with PYRE/PYKE tetrapeptide insertions in Gag-p6 conferred ALIX binding, CCL2-responsiveness and enhanced virus replication. These results, for the first time, indicate that CCL2 mediates ALIX mobilization from F-actin and enhances HIV-1 release and fitness.

A Naturally Occurring Polymorphism in the HIV-1 Tat Basic Domain Inhibits Uptake by Bystander Cells and Leads to Reduced Neuroinflammation.

HIV-1 Tat protein contributes to HIV-neuropathogenesis in several ways including its ability to be taken up by uninfected bystander CNS cells and to activate inflammatory host genes causing synaptic injury. Here, we report that in the globally dominant HIV-1 clade C, Tat displays a naturally occurring polymorphism, R57S, in its basic domain, which mediates cellular uptake. We examined the effect of this polymorphism on Tat uptake and its consequences for cellular gene transactivation. In decapeptides corresponding to the basic domain, a R57S substitution caused up to a 70% reduction in uptake. We also used a transcellular Tat transactivation assay, where we expressed Tat proteins of HIV-1 clade B (Tat-B) or C (Tat-C) or their position 57 variants in HeLa cells. We quantified the secreted Tat proteins and measured their uptake by TZM-bl cells, which provide readout via an HIV-1 Tat-responsive luciferase gene. Transactivation by Tat-B was significantly reduced by R57S substitution, while that of Tat-C was enhanced by the reciprocal S57R substitution. Finally, we exposed microglia to Tat variants and found that R57 is required for maximal neuroinflammation. The R57S substitution dampened this response. Thus, genetic variations can modulate the ability of HIV-1 Tat to systemically disseminate neuroinflammation.

Association of a 3' untranslated region polymorphism in proprotein convertase subtilisin/kexin type 9 with HIV viral load and CD4+ levels in HIV/hepatitis C virus coinfected women.

OBJECTIVE: To assess variation in genes that regulate cholesterol metabolism in relation to the natural history of HIV infection. DESIGN: Cross-sectional and longitudinal analysis of the Women's Interagency HIV Study. METHODS: We examined 2050 single nucleotide polymorphisms (SNPs) in 19 genes known to regulate cholesterol metabolism in relation to HIV viral load and CD4 T-cell levels in a multiracial cohort of 1066 antiretroviral therapy-naive women. RESULTS: Six SNPs were associated with both HIV viral load and CD4 T-cell levels at a false discovery rate of 0.01. Bioinformatics tools did not predict functional activity for five SNPs, located in introns of nuclear receptor corepressor 2, retinoid X receptor alpha (RXRA), and tetratricopeptide repeat domain 39B. Rs17111557 located in the 3' untranslated region of proprotein convertase subtilisin/kexin type 9 (PCSK9) putatively affects binding of hsa-miR-548t-5p and hsa-miR-4796-3p, which could regulate PCSK9 expression levels. Interrogation of rs17111557 revealed stronger associations in the subset of women with HIV/hepatitis C virus (HCV) coinfection (n = 408, 38% of women). Rs17111557 was also associated with low-density lipoprotein cholesterol levels in HIV/HCV coinfected (ß: -10.4; 95% confidence interval: -17.9, -2.9; P = 0.007), but not in HIV monoinfected (ß:1.2; 95% confidence interval: -6.3, 8.6; P = 0.76) women in adjusted analysis. CONCLUSION: PCSK9 polymorphism may affect HIV pathogenesis, particularly in HIV/HCV coinfected women. A likely mechanism for this effect is PCSK9-mediated regulation of cholesterol metabolism. Replication in independent cohorts is needed to clarify the generalizability of the observed associations.

FISHing Out the Hidden Enemy: Advances in Detecting and Measuring Latent HIV-Infected Cells.

The indomitable aspect of HIV-1 infection is not that HIV-1 proviral DNA is integrated into host DNA but that it can also turn itself off, remaining invisible to drug or immune surveillance. Thus, the goals of eradication include ways to precisely excise HIV-1 DNA or wake up the silent HIV-1 provirus and eliminate the infected cells thus identified. Methods to identify and fish out the latently infected cells or to delineate their characteristics are being rapidly developed. In 2016, Baxter et al. (A. E. Baxter, J. Niessl, R. Fromentin, J. Richard, F. Porichis, R. Charlebois, M. Massanella, N. Brassard, N. Alsahafi, G. G. Delgado, J. P. Routy, B. D. Walker, A. Finzi, N. Chomont, and D. E. Kaufmann, Cell Host Microbe 20:368-380, 2016, https://doi.org/10.1016/j.chom.2016.07.015) and Martrus et al. (G. Martrus, A. Niehrs, R. Cornelis, A. Rechtien, W. García-Beltran, M. Lütgehetmann, C. Hoffmann, and M. Altfeld, J Virol 90:9018-9028, 2016, https://doi.org/10.1128/JVI.01448-16) reported using the fluorescence in situ hybridization-flow cytometry technique to identify and quantify cells expressing HIV-1 RNA and Gag protein, as well as bearing unique cell surface markers. In a recent article in mBio, Grau-Expósito et al. (J. Grau-Expósito, C. Serra-Peinado, L. Miguel, J. Navarro, A. Curran, J. Burgos, I. Ocaña, E. Ribera, A. Torrella, B. Planas, R. Badía, J. Castellví, V. Falcó, M. Crespo, and M. J. Buzon, mBio 8:e00876-17, 2017, https://doi.org/10.1128/mBio.00876-17) reported a similar method that they claim to be more sensitive. With these methods, researchers are one step closer to measuring latent reservoirs and eliminating critical barriers to HIV eradication.

The Evolving Profile of the Signature Amino Acid Residues in HIV-1 Subtype C Tat.

Using several HIV-1 tat exon 1 amino acid sequences available from public databases and additional sequences derived from a southern Indian clinical cohort, we compared the profile of the signature amino acid residues (SAR) between two different time periods, 1986-2004 and 2005-2014. The analysis identified eight positions as signature residues in subtype C Tat and demonstrated a changing pattern at four of these positions between the two periods. At three locations (histidine 29, serine 57, and proline 60), there appears to be a nonuniform negative selection against the SAR. The negative selection appears to be severe, especially against histidine 29 (p < .0001) and moderate against proline 60 (p < .0001). The negative selection against serine 57 is statistically insignificant and appears to have begun recently. At position 63, the frequency of signature residue glutamic acid increased over the past decade, although the difference was not significant. Importantly, at the three locations where the negative selection is in progress, the substitute amino acids are the generic residues present in most of the other HIV-1 subtypes. Our data demonstrate that viral evolution can subject specific amino acid residues to subtle and progressive selection pressures without affecting the prevalence of other amino acid residues.

Measuring the Uptake and Transactivation Function of HIV-1 Tat Protein in a Trans-cellular Cocultivation Setup.

HIV-1 Tat protein is secreted from infected cells and is endocytosed by uninfected bystander cells. Subsequently, Tat is translocated to the nucleus and binds to promoters of host cell genes, increasing the production of inflammatory host cytokines and chemokines. This inflammatory activation of uninfected cells by HIV-1 Tat protein contributes to the overall inflammatory burden in the central nervous system (CNS) that leads to the development of HIV-associated neurocognitive disorders (HAND). Here we describe methods to evaluate the uptake and transcriptional impact of HIV-1 Tat on uninfected cells by using a trans-cellular transactivation system. Cell lines transiently transfected with Tat expression constructs secrete Tat into the culture medium. Trans-cellular uptake and transactivation caused by secreted Tat can be measured by co-culturing LTR-responsive reporter cells with Tat-transfected cells. Such Tat-producer cells can also be co-cultured with immune cell lines, such as monocytic THP-1 cells or lymphocytic Jurkat T-cells, to evaluate transcriptional changes elicited by Tat taken up by the uninfected cells.

Evaluating the Role of Viral Proteins in HIV-Mediated Neurotoxicity Using Primary Human Neuronal Cultures.

Despite the inability of HIV-1 to infect neurons, over half of the HIV-1-infected population in the USA suffers from neurocognitive dysfunction. HIV-infected immune cells in the periphery enter the central nervous system by causing a breach in the blood-brain barrier. The damage to the neurons is mediated by viral and host toxic products released by activated and infected immune and glial cells. To evaluate the toxicity of any viral isolate, viral protein, or host inflammatory protein, we describe a protocol to assess the neuronal apoptosis and synaptic compromise in primary cultures of human neurons and astrocytes.

Multivariable analysis to determine if HIV-1 Tat dicysteine motif is associated with neurodevelopmental delay in HIV-infected children in Malawi.

BACKGROUND: HIV-1 Tat protein is implicated in HIV-neuropathogenesis. Tat C31S polymorphism (Tat(CS)) has been associated with milder neuropathology in vitro and in animal models but this has not been addressed in a cohort of HIV-infected adults or children. METHODS: HIV viral load (VL) in plasma and cerebrospinal fluid (CSF) were determined and plasma HIV tat gene was sequenced. Neurodevelopmental assessment was performed using Bayley Scales of Infant Development III (BSID-III), with scores standardized to Malawian norms. The association between Tat(CS) and BSID-III scores was evaluated using multivariate linear regression. RESULTS: Neurodevelopmental assessment and HIV tat genotyping were available for 33 children. Mean age was 19.4 (SD 7.1) months, mean log VL was 5.9 copies/mL (SD 0.1) in plasma and 3.9 copies/mL (SD 0.9) in CSF. The prevalence of Tat(CC) was 27 %. Z-scores for BSID-III subtests ranged from -1.3 to -3.9. Tat(CC) was not associated with higher BSID-III z-scores. CONCLUSIONS: The hypothesis of milder neuropathology in individuals infected with HIV Tat(CS) was not confirmed in this small cohort of Malawian children. Future studies of tat genotype and neurocognitive disorder should be performed using larger sample sizes and investigate if this finding is due to differences in HIV neuropathogenesis between children and adults.

High-affinity RNA Aptamers Against the HIV-1 Protease Inhibit Both In Vitro Protease Activity and Late Events of Viral Replication.

HIV-1 aspartyl protease (PR) plays a key role in virion morphogenesis, underscoring the effectiveness of protease inhibitors (PI). Despite their utility, side effects and drug-resistance remains a problem. We report the development of RNA aptamers as inhibitors of HIV-1 PR for potential use in anti-HIV gene therapy. Employing Systematic Evolution of Ligands by Exponential Enrichment (SELEX), we isolated four unique families of anti-HIV-1 PR RNA aptamers displaying moderate binding affinities (Kd = 92-140 nmol/l) and anti-PR inhibitory activity (Kis = 138-647 nmol/l). Second-generation RNA aptamers selected from partially randomized pools based on two of the aptamer sequences displayed striking enhancements in binding (Kds = 2-22 nmol/l) and inhibition (Kis = 31-49 nmol/l). The aptamers were specific in that they did not bind either the related HIV-2 protease, or the cellular aspartyl protease, Cathepsin D. Site-directed mutagenesis of a second-generation aptamer to probe the predicted secondary structure indicated that the stem-loops SL2 and SL3 and the stem P1 were essential for binding and that only the 3'-most 17 nucleotides were dispensable. Anti-PR aptamers inhibited HIV replication in vitro and the degree of inhibition was higher for second-generation aptamers with greater affinity and the inhibition was abrogated for a nonbinding aptamer variant.

The lysine 65 residue in HIV-1 reverse transcriptase function and in nucleoside analog drug resistance.

Mutations in HIV-1 reverse transcriptase (RT) that confer nucleoside analog RT inhibitor resistance have highlighted the functional importance of several active site residues (M184, Q151 and K65) in RT catalytic function. Of these, K65 residue is notable due to its pivotal position in the dNTP-binding pocket, its involvement in nucleoside analog resistance and polymerase fidelity. This review focuses on K65 residue and summarizes a substantial body of biochemical and structural studies of its role in RT function and the functional consequences of the K65R mutation.

The gp120 protein is a second determinant of decreased neurovirulence of Indian HIV-1C isolates compared to southern African HIV-1C isolates.

Regional differences in neurovirulence have been documented among subtype/clade-C HIV-1 isolates in India and Southern Africa. We previously demonstrated that a C31S substitution in Clade-C Tat dicysteine motif reduces monocyte recruitment, cytokine induction and direct neurotoxicity. Therefore, this polymorphism is considered to be a causative factor for these differences in neurovirulence. We previously reported on the genotypic differences in Tat protein between clade-C and rest of the clades showing that approximately 90% of clade-C HIV-1 Tat sequences worldwide contained this C31S polymorphism, while 99% of non-clade C isolates lacked this Tat polymorphism at C31 residue (Ranga et al. (2004) J Virol 78:2586-2590). Subsequently, we documented intra-clade-C differences in the frequency of Tat dicysteine variants between India and Southern Africa, as the basis for differential disease severity and showed the importance of the Tat dicysteine motif for neuropathogenesis using small animal models. We have now examined if determinants of neurovirulence besides Tat are different between the clade-C HIV-1 isolates from Southern Africa and India. Envelope glycoprotein gp120 is a well-documented contributor to neurotoxicity. We found that gp120 sequences of HIV-1 isolates from these two regions are genetically distinct. In order to delineate the contribution of gp120 to neurovirulence, we compared direct in vitro neurotoxicity of HIV-infected supernatants of a representative neurovirulent US clade-B isolate with two isolates each from Southern Africa and India using primary human neurons and SH-SY5Y neuroblastoma cells. Immunodepletion of gp120 of both US clade B and the Southern African clade C isolates revealed robust decreases in neurotoxicity, while that of the Indian isolates showed minimal effect on neurotoxicity. The gp120 as a cause of differential neurotoxicity was further confirmed using purified recombinant gp120 from HIV isolates from these regions. We conclude that gp120 is one of the key factors responsible for the decreased neurovirulence of Indian clade C HIV-1 isolates when compared to South African clade C HIV-1.

Novel tetra-peptide insertion in Gag-p6 ALIX-binding motif in HIV-1 subtype C associated with protease inhibitor failure in Indian patients.

A novel tetra-peptide insertion was identified in Gag-p6 ALIX-binding region, which appeared in protease inhibitor failure Indian HIV-1C sequences (odds ratio=17.1, P < 0.001) but was naturally present in half of untreated Ethiopian HIV-1C sequences. The insertion is predicted to restore ALIX-mediated virus release pathway, which is lacking in HIV-1C. The clinical importance of the insertion needs to be evaluated in HIV-1C dominating regions wherein the use of protease inhibitor drugs are being scaled up.

New clues to understanding HIV nonprogressors: low cholesterol blocks HIV trans infection.

A small percentage of HIV-infected subjects (2 to 15%) are able to control disease progression for many years without antiretroviral therapy. Years of intense studies of virologic and immunologic mechanisms of disease control in such individuals yielded a number of possible host genes that could be responsible for the preservation of immune functions, from immune surveillance genes, chemokines, or their receptors to anti-HIV restriction factors. A recent mBio paper by Rappocciolo et al. (G. Rappocciolo, M. Jais, P. Piazza, T. A. Reinhart, S. J. Berendam, L. Garcia-Exposito, P. Gupta, and C. R. Rinaldo, mBio 5:e01031-13, 2014) describes another potential factor controlling disease progression: cholesterol levels in antigen-presenting cells. In this commentary, we provide a brief background of the role of cholesterol in HIV infection, discuss the results of the study by Rappocciolo et al., and present the implications of their findings.

Viral and cellular factors underlying neuropathogenesis in HIV associated neurocognitive disorders (HAND).

As the HIV-1 epidemic enters its fourth decade, HIV-1 associated neurological disorders (HAND) continue to be a major concern in the infected population, despite the widespread use of anti-retroviral therapy. Advancing age and increased life expectancy of the HIV-1 infected population have been shown to increase the risk of cognitive dysfunction. Over the past 10 years, there has been a significant progress in our understanding of the mechanisms and the risk factors involved in the development of HAND. Key events that lead up to neuronal damage in HIV-1 infected individuals can be categorized based on the interaction of HIV-1 with the various cell types, including but not limited to macrophages, brain endothelial cells, microglia, astrocytes and the neurons. This review attempts to decipher these interactions, beginning with HIV-1 infection of macrophages and ultimately resulting in the release of neurotoxic viral and host products. These include: interaction with endothelial cells, resulting in the impairment of the blood brain barrier; interaction with the astrocytes, leading to metabolic and neurotransmitter imbalance; interactions with resident immune cells in the brain, leading to release of toxic cytokines and chemokines. We also review the mechanisms underlying neuronal damage caused by the factors mentioned above. We have attempted to bring together recent findings in these areas to help appreciate the viral and host factors that bring about neurological dysfunction. In addition, we review host factors and viral genotypic differences that affect phenotypic pathological outcomes, as well as recent advances in treatment options to specifically address the neurotoxic mechanisms in play.

Phylogenetic characterization of six full-length HIV-1 subtype C molecular clones from three patients: identification of rare subtype C strains containing two NF-κB motifs in the long terminal repeat.

Molecular surveillance is the backbone of HIV-1 vaccinology. Full-length HIV-1 sequences are useful tools that can provide a better understanding of the epidemiology in a given region. A limited number of full-length HIV-1 sequences are available from India, where >95% of the HIV infections are due to HIV-1 subtype C (HIV-1C), which is distinct from the prototype African HIV-1C. In this study, we sequenced six full-length clones isolated from three patients. Extensive phylogenetic analyses of the full-length viral sequences using bioinformatic tools identified a separate cluster of Indian strains, thus confirming the distinct phylogenetic identity of the Indian HIV-1C. Notably, the long terminal repeat (LTR) of two of the six molecular clones contained only two NF-κB binding sites. The sequences also displayed features characteristic of HIV-1C including a Tat dicysteine motif, a shortened Rev open reading frame, and a predicted CCR5 coreceptor tropism for gp120 of three of the proviral sequences.

Clade C HIV-1 isolates circulating in Southern Africa exhibit a greater frequency of dicysteine motif-containing Tat variants than those in Southeast Asia and cause increased neurovirulence.

BACKGROUND: HIV-1 Clade C (Subtype C; HIV-1C) is responsible for greater than 50% of infections worldwide. Unlike clade B HIV-1 (Subtype B; HIV-1B), which is known to cause HIV associated dementia (HAD) in approximately 15% to 30% of the infected individuals, HIV-1C has been linked with lower prevalence of HAD (0 to 6%) in India and Ethiopia. However, recent studies report a higher prevalence of HAD in South Africa, Zambia and Botswana, where HIV-1C infections predominate. Therefore, we examined whether Southern African HIV-1C is genetically distinct and investigated its neurovirulence. HIV-1 Tat protein is a viral determinant of neurocognitive dysfunction. Therefore, we focused our study on the variations seen in tat gene and its contribution to HIV associated neuropathogenesis. RESULTS: A phylogenetic analysis of tat sequences of Southern African (South Africa and Zambia) HIV isolates with those from the geographically distant Southeast Asian (India and Bangladesh) isolates revealed that Southern African tat sequences are distinct from Southeast Asian isolates. The proportion of HIV - 1C variants with an intact dicysteine motif in Tat protein (C30C31) was significantly higher in the Southern African countries compared to Southeast Asia and broadly paralleled the high incidence of HAD in these countries. Neuropathogenic potential of a Southern African HIV-1C isolate (from Zambia; HIV-1C 1084i), a HIV-1C isolate (HIV-1 IndieC1) from Southeast Asia and a HIV-1B isolate (HIV-1 ADA) from the US were tested using in vitro assays to measure neurovirulence and a SCID mouse HIV encephalitis model to measure cognitive deficits. In vitro assays revealed that the Southern African isolate, HIV-1C 1084i exhibited increased monocyte chemotaxis and greater neurotoxicity compared to Southeast Asian HIV-1C. In neurocognitive tests, SCID mice injected with MDM infected with Southern African HIV-1C 1084i showed greater cognitive dysfunction similar to HIV-1B but much higher than those exposed to Southeast Asian HIV - 1C. CONCLUSIONS: We report here, for the first time, that HIV-1C from Southern African countries is genetically distinct from Southeast Asian HIV-1C and that it exhibits a high frequency of variants with dicysteine motif in a key neurotoxic HIV protein, Tat. Our results indicate that Tat dicysteine motif determines neurovirulence. If confirmed in population studies, it may be possible to predict neurocognitive outcomes of individuals infected with HIV-1C by genotyping Tat.

INI1/hSNF5-interaction defective HIV-1 IN mutants exhibit impaired particle morphology, reverse transcription and integration in vivo.

BACKGROUND: Retroviral integrase catalyzes integration of viral DNA into the host genome. Integrase interactor (INI)1/hSNF5 is a host factor that binds to HIV-1 IN within the context of Gag-Pol and is specifically incorporated into HIV-1 virions during assembly. Previous studies have indicated that INI1/hSNF5 is required for late events in vivo and for integration in vitro. To determine the effects of disrupting the IN-INI1 interaction on the assembly and infectivity of HIV-1 particles, we isolated mutants of IN that are defective for binding to INI1/hSNF5 and tested their effects on HIV-1 replication. RESULTS: A reverse yeast two-hybrid system was used to identify INI1-interaction defective IN mutants (IID-IN). Since protein-protein interactions depend on the surface residues, the IID-IN mutants that showed high surface accessibility on IN crystal structures (K71R, K111E, Q137R, D202G, and S147G) were selected for further study. In vitro interaction studies demonstrated that IID-IN mutants exhibit variable degrees of interaction with INI1. The mutations were engineered into HIV-1(NL4-3) and HIV-Luc viruses and tested for their effects on virus replication. HIV-1 harboring IID-IN mutations were defective for replication in both multi- and single-round infection assays. The infectivity defects were correlated to the degree of INI1 interaction of the IID-IN mutants. Highly defective IID-IN mutants were blocked at early and late reverse transcription, whereas partially defective IID-IN mutants proceeded through reverse transcription and nuclear localization, but were partially impaired for integration. Electron microscopic analysis of mutant particles indicated that highly interaction-defective IID-IN mutants produced morphologically aberrant virions, whereas the partially defective mutants produced normal virions. All of the IID-IN mutant particles exhibited normal capsid stability and reverse transcriptase activity in vitro. CONCLUSIONS: Our results demonstrate that a severe defect in IN-INI1 interaction is associated with production of defective particles and a subsequent defect in post-entry events. A partial defect in IN-INI1 interaction leads to production of normal virions that are partially impaired for early events including integration. Our studies suggest that proper interaction of INI1 with IN within Gag-Pol is necessary for proper HIV-1 morphogenesis and integration.

Lys66 residue as a determinant of high mismatch extension and misinsertion rates of HIV-1 reverse transcriptase.

A major factor contributing to the high mutation rate of HIV-1 reverse transcriptase (RT) is its high propensity for misincorporation. Misincorporation requires both deoxyribonucleotide triphosphate (dNTP) misinsertion and the subsequent extension of the mismatched terminus thus formed. We hypothesized that Lys66 is a determinant of mismatch extension based on its position near the primer terminus. This hypothesis was tested by steady-state kinetic studies using wild-type HIV-1 RT and four Lys66 substitution mutants: Lys66Arg, Lys66Ala, Lys66Asn and Lys66Thr. The mismatch extension efficiency was reduced for all mutants, with Lys66Ala, Lys66Asn and Lys66Thr showing a four- to six-fold reduction compared with wild-type HIV-1 RT. Surprisingly, the nonconservative substitutions also led to large decreases in misinsertion efficiency, ranging from as low as three-fold to values much higher than 23-fold. Thus, the Lys66Arg mutant was akin to wild-type HIV-1 RT, whereas all nonconservative mutants displayed significantly decreased efficiency for both events. Our results suggest that Lys66, much like Lys65, is a determinant of both dNTP misinsertion and mismatch extension efficiency. While Lys65 is known to contact the γ-phosphate of incoming dNTP, the Lys66 side chain is in the vicinity of the primer terminus. However, our results suggest that both residues have a similar influence on dNTP misinsertion and mispair extension efficiencies of HIV-1 RT. When we tested the mutants for susceptibility to selected nucleoside analog and non-nucleoside analog drugs, similarly to Lys65Arg, the Lys66Ala and Lys66Asn mutants displayed mild resistance to the nucleoside analog drug 3'-azido-3'-deoxythymidine-5'-triphosphate (AZTTP).

Pre-steady state kinetic analysis of cyclobutyl derivatives of 2'-deoxyadenosine 5'-triphosphate as inhibitors of HIV-1 reverse transcriptase.

Pre-steady state kinetic analysis was utilized for biochemical evaluation of a series of cyclobutyl adenosine nucleotide analogs with HIV-1 RT(WT). The phosphonyl-diphosphate form of the cyclobutyl nucleotide, 5, was the most efficiently incorporated of the series. Nucleotide 5 was fourfold more efficiently incorporated than the FDA approved TFV-DP by RT(WT). The kinetics of incorporation for 5 using the drug resistant mutant enzyme K65R was also determined. Compound 5 was threefold more efficiently incorporated compared to TFV-DP with RT(K65R). These results demonstrate cyclobutyl adenosine analogs can act as substrates for incorporation by HIV-1 RT and be a potential scaffold for HIV inhibitors.