ABSTRACT
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.
Subject(s)
Developmental Disabilities/virology , HIV Infections/psychology , HIV Infections/virology , HIV-1/pathogenicity , tat Gene Products, Human Immunodeficiency Virus/chemistry , tat Gene Products, Human Immunodeficiency Virus/metabolism , Amino Acid Motifs , Cross-Sectional Studies , Female , Genotype , HIV-1/metabolism , Humans , Infant , Malawi , Male , Viral LoadABSTRACT
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.
ABSTRACT
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.
Subject(s)
Astrocytes , Blood-Brain Barrier , Endothelial Cells , HIV Infections , HIV-1 , Blood-Brain Barrier/virology , Blood-Brain Barrier/metabolism , Humans , Astrocytes/virology , Astrocytes/metabolism , Astrocytes/immunology , Endothelial Cells/virology , Endothelial Cells/metabolism , Endothelial Cells/immunology , HIV-1/immunology , HIV-1/physiology , HIV Infections/virology , HIV Infections/immunology , Pericytes/virology , Pericytes/metabolism , Pericytes/immunology , Neuroinflammatory Diseases/virology , Neuroinflammatory Diseases/immunology , Coculture Techniques/methods , Cells, Cultured , Brain/virology , Brain/immunology , Brain/metabolismABSTRACT
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.
Subject(s)
Chromosomal Proteins, Non-Histone/metabolism , DNA-Binding Proteins/metabolism , HIV Integrase/metabolism , HIV-1/physiology , Host-Pathogen Interactions , Reverse Transcription/physiology , Transcription Factors/metabolism , Virus Assembly/physiology , Virus Integration/physiology , Cell Line , HIV Integrase/genetics , HIV-1/genetics , HIV-1/ultrastructure , Humans , Microscopy, Electron, Transmission , SMARCB1 Protein , Virion/ultrastructureABSTRACT
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.
Subject(s)
AIDS Dementia Complex/epidemiology , AIDS Dementia Complex/virology , HIV Infections/complications , HIV Infections/virology , HIV-1/genetics , tat Gene Products, Human Immunodeficiency Virus/genetics , Adult , Africa, Southern/epidemiology , Animals , Asia, Southeastern/epidemiology , Female , Genotype , HIV-1/classification , HIV-1/isolation & purification , HIV-1/pathogenicity , Humans , Male , Mice , Middle Aged , Molecular Sequence Data , Prevalence , Sequence Analysis, DNAABSTRACT
Gag orchestrates the assembly and release of human immunodeficiency virus type 1 (HIV-1) particles. We explored here the potential of anti-Gag RNA aptamers to inhibit HIV-1 replication. In vitro, RNA aptamers raised against an HIV-1 Gag protein, lacking the N-terminal myristate and the C-terminal p6 (DP6-Gag), could bind to matrix protein (MA), nucleocapsid protein (NC), or entire DP6-Gag protein. Upon cotransfection with pNL4-3.Luc molecular clone into 293T cells, six of the aptamers caused mild inhibition (2- to 3-fold) in the extracellular capsid levels, and one aptamer displayed 20-fold inhibition. The reduction was not due to a release defect but reflected Gag mRNA levels. We hypothesized that the aptamers influence genomic RNA levels via perturbation of specific Gag-genomic RNA interactions. Binding studies revealed that the "NC-binders" specifically compete with the packaging signal (ψ) of HIV-1 for binding to DP6-Gag. Therefore, we tested the ability of two NC-binders to inhibit viruses containing ψ-region deletions (ΔSL1 or ΔSL3) and found that the NC-binders were no longer able to inhibit Gag synthesis. The inability of these aptamers to inhibit ψ-deleted viruses correlated with the absence of competition with the corresponding ψ transcripts lacking SL1 or SL3 for binding DP6-Gag in vitro. These results indicate that the NC-binding aptamers disrupt Gag-genomic RNA interaction and negatively affect genomic RNA transcription, processing, or stability. Our results reveal an essential interaction between HIV-1 Gag and the ψ-region that may be distinct from that which occurs during the encapsidation of genomic RNA. Thus, anti-Gag aptamers can be an effective tool to perturb Gag-genomic RNA interactions.
Subject(s)
Aptamers, Nucleotide/metabolism , Gene Products, gag/metabolism , HIV Antigens/metabolism , HIV-1/drug effects , Nucleocapsid Proteins/metabolism , gag Gene Products, Human Immunodeficiency Virus/metabolism , Aptamers, Nucleotide/pharmacology , Cell Line , Gene Products, gag/chemistry , HIV Antigens/chemistry , HIV-1/chemistry , HIV-1/genetics , HIV-1/metabolism , Humans , Nucleocapsid Proteins/chemistry , Protein Binding , Virus Assembly/drug effects , Virus Replication/drug effects , gag Gene Products, Human Immunodeficiency Virus/chemistryABSTRACT
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.
Subject(s)
Anti-HIV Agents/chemical synthesis , Cyclobutanes/chemical synthesis , Deoxyadenine Nucleotides/chemical synthesis , HIV Reverse Transcriptase/antagonists & inhibitors , HIV-1/drug effects , Reverse Transcriptase Inhibitors/chemical synthesis , Adenine/analogs & derivatives , Adenine/pharmacology , Anti-HIV Agents/pharmacology , Cyclobutanes/pharmacology , DNA Primers , Deoxyadenine Nucleotides/pharmacology , Drug Resistance, Viral/drug effects , Drug Resistance, Viral/genetics , HIV Reverse Transcriptase/chemistry , HIV Reverse Transcriptase/genetics , HIV-1/genetics , Humans , Kinetics , Mutation , Nucleic Acid Amplification Techniques , Organophosphonates/pharmacology , Reverse Transcriptase Inhibitors/pharmacology , TenofovirABSTRACT
Human immunodeficiency virus (HIV)-associated dementia (HAD) is common among clade B HIV-infected individuals, but less common and less severe among individuals infected with clade C HIV-1, suggesting clade-specific differences in neuropathogenicity. Although differences in neuropathogenicity have been investigated in vitro using viral proteins responsible for HAD, to date there are no virological studies using animal models to address this issue. Therefore, we investigated neuropathogenesis induced by HIV-1 clades using the severe combined immune deficiency (SCID) mouse HIV encephalitis model, which involves intracranial injection of macrophages infected with representative clade B (HIV-1(ADA)) or clade C (HIV-1(Indie-C1)) HIV-1 isolates into SCID mice. In cognitive tests, mice exposed to similar inputs of HIV-1 clade C made fewer memory errors than those exposed to HIV-1 clade B. Histopathological analysis of mice exposed to clade B exhibited greater astrogliosis and increased loss of neuronal network integrity. In vitro experiments revealed differences in a key characteristic of HIV-1 that influences HAD, increased monocyte infiltration. HIV-1(Indie-C1)-infected macrophages recruited monocytes poorly in vitro compared with HIV-1(ADA)-infected macrophages. Monocyte recruitment was HIV-1 Tat and CCL2 dependent. This is the first demonstration, ever since HIV neuropathogenesis was first recognized, that viral genetic differences between clades can affect disease severity and that such studies help identify key players in neuropathogenesis by HIV-1.
Subject(s)
HIV Infections/pathology , HIV Infections/virology , HIV-1/physiology , AIDS Dementia Complex/etiology , AIDS Dementia Complex/pathology , AIDS Dementia Complex/virology , Animals , Cells, Cultured , Gene Products, tat/physiology , HIV Infections/etiology , HIV-1/isolation & purification , Humans , Mice , Mice, Inbred C57BL , Mice, SCIDABSTRACT
The unintegrated viral DNA synthesized during human immunodeficiency virus type 1 infection includes linear and circular forms. Circular forms of viral DNA are surrogate markers for nuclear import of viral DNA during virus replication as well as events surrounding the completion of reverse transcription. Analysis of 2-LTR circles is convenient and the quantity of 2-LTR circle formed is directly proportional to the amount of viral DNA imported into the cell nucleus. In addition, correct synthesis of 2-LTR circles is an outcome of HIV-1 Gag-Pol function. Thus, quantitation and sequence analysis of 2-LTR circles have been very important in studying the structure and function relationship of key viral proteins. In this chapter, we describe the methods of quantitation and analysis of 2-LTR circle junctions isolated from HIV-1 infected cells.
Subject(s)
DNA, Circular/analysis , DNA, Circular/genetics , DNA, Viral/analysis , DNA, Viral/genetics , HIV Long Terminal Repeat/genetics , HIV-1/genetics , Cell Line , HIV-1/physiology , Humans , T-Lymphocytes/chemistry , T-Lymphocytes/virology , Virus Replication/physiologyABSTRACT
HIV-associated dementia (HAD) is a multi-factorial disease set in motion by the presence of HIV-infected cells in the brain. A characteristic feature of HAD is the infiltration of mononuclear phagocytes into the brain, which is aided by HIV-1 Tat protein and other chemokines secreted by both HIV-infected cells and uninfected cells in their vicinity. Both direct and indirect chemokine activity of HIV-1 Tat protein has been demonstrated employing purified recombinant Tat protein. However, a corroboration of a key role for Tat or other chemokines in monocyte migration, in the context of HIV-infection, has not yet been demonstrated. Here we describe methods, to measure the role of soluble factors, such as chemokines and Tat, released by HIV-infected cells or uninfected cells in their vicinity, in monocyte migration in vitro.
Subject(s)
Cell Migration Assays, Leukocyte/methods , Cell Movement , HIV-1/immunology , Monocytes/immunology , Cell Line , Cells, Cultured , Chemokines/immunology , Humans , tat Gene Products, Human Immunodeficiency Virus/immunologyABSTRACT
Human telomerase reverse transcriptase (hTERT), the catalytic subunit of human telomerase, contains conserved motifs common to retroviral reverse transcriptases and telomerases. Within the C motif of hTERT is the Leu866-Val867-Asp868-Asp869 tetrapeptide that includes a catalytically essential aspartate dyad. Site-directed mutagenesis of Tyr183 and Met184 residues in HIV-1 RT, residues analogous to Leu866 and Val867, revealed that they are key determinants of nucleotide binding, processivity and fidelity. In this study, we show that substitutions at Val867 lead to significant changes in overall enzyme activity and telomere repeat extension rate, but have little effect on polymerase processivity. All Val867 substitutions examined (Ala, Met, Thr) led to reduced repeat extension rates, ranging from approximately 20 to 50% of the wild-type rate. Reconstitution of V867M hTERT and telomerase RNAs (TRs) with mutated template sequences revealed the effect on extension rate was associated with a template copying defect specific to template A residues. Furthermore, the Val867 hTERT mutants also displayed increased nucleotide incorporation fidelity, implicating Val867 as a determinant of telomerase fidelity. These findings suggest that by evolving to have a valine at position 867, the wild-type hTERT protein may have partially compromised polymerase fidelity for optimal and rapid repeat synthesis.
Subject(s)
Telomerase/chemistry , Telomerase/metabolism , Valine/chemistry , Amino Acid Sequence , Amino Acid Substitution , Binding Sites , Conserved Sequence , DNA/biosynthesis , Humans , Models, Molecular , Mutation , Nucleotides/metabolism , Sequence Alignment , Telomerase/genetics , Templates, Genetic , Thymine Nucleotides/metabolism , Valine/geneticsABSTRACT
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.
Subject(s)
Bystander Effect , HIV-1 , Microglia , Neurons , Polymorphism, Genetic , tat Gene Products, Human Immunodeficiency Virus , HIV-1/genetics , HIV-1/metabolism , HeLa Cells , Humans , Inflammation/genetics , Inflammation/metabolism , Inflammation/pathology , Inflammation/virology , Microglia/metabolism , Microglia/pathology , Neurons/metabolism , Neurons/pathology , Neurons/virology , Protein Domains , tat Gene Products, Human Immunodeficiency Virus/genetics , tat Gene Products, Human Immunodeficiency Virus/metabolismABSTRACT
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.
Subject(s)
Calcium-Binding Proteins/metabolism , Cell Cycle Proteins/metabolism , Chemokine CCL2/metabolism , Endosomal Sorting Complexes Required for Transport/metabolism , HIV-1/growth & development , Host-Pathogen Interactions , Virus Release , gag Gene Products, Human Immunodeficiency Virus/metabolism , Cells, Cultured , Humans , Macrophages/virologyABSTRACT
BACKGROUND: The pathogenic significance of coreceptor switch in the viral infection of HIV-1 is not completely understood. This situation is more complex in subtype C infection where coreceptor switch is either absent or extremely rare. To gain insights into the mechanisms that underlie coreceptor requirement of subtype C, we screened several primary viral isolates and identified a clinical sample that demonstrated a potential to grow on standard T-cell lines with no detectable CCR5 expression. The subject was diagnosed with HIV-1 associated dementia in the absence of opportunistic infections of the brain. To isolate molecular clones from this virus, we devised a novel strategy based on anchor primers that target a sequence in the reverse transcriptase, highly conserved among diverse subtypes of HIV-1. RESULTS: Using this strategy, we isolated 8 full-length molecular clones from the donor. Two of the eight molecular clones, 03In94_D17 and 03In94_D24, (D17 and D24) generated replication-competent viruses. Phylogenetic analysis of the full-length viral sequences revealed that both clones were non-recombinant subtype C viruses. They contain intact open reading frames in all the viral proteins. Both the viral clones are endowed with several unique molecular and biological properties. The viral promoter of the clones is characterized by the presence of four NF-kB binding elements, a feature rarely seen in the subtype C HIV-1 LTR. Interestingly, we identified the coexistence of two different forms of Rev, a truncated form common to subtype C and a full-length form less common for this subtype, in both proviral and plasma virus compartments. An exceptional property of the viruses, atypical of subtype C, is their ability to use a wide range of coreceptors including CCR5, CXCR4, and several others tested. Sequence analysis of Env of D17 and D24 clones identified differences within the variable loops providing important clues for the expanded coreceptor use. The V1, V2 and V4 loops in both of the molecular clones are longer due to the insertion of several amino acid residues that generated potential N-linked glycosylation sites. CONCLUSION: The exceptional biological and molecular properties of these clones make them invaluable tools to understand the unique pathogenic characteristics of subtype C.
Subject(s)
AIDS Dementia Complex/virology , HIV Seropositivity/virology , HIV-1/physiology , Receptors, HIV/metabolism , Adult , Cell Line , Cloning, Molecular , Genetic Variation , HIV Enhancer/genetics , HIV Long Terminal Repeat/genetics , HIV-1/chemistry , HIV-1/classification , HIV-1/isolation & purification , Humans , Leukocytes, Mononuclear/virology , Male , Phylogeny , Proviruses/genetics , Receptors, CCR5 , Sequence Analysis , T-Lymphocytes/immunology , T-Lymphocytes/virology , env Gene Products, Human Immunodeficiency Virus/genetics , rev Gene Products, Human Immunodeficiency Virus/geneticsABSTRACT
HIV-1 reverse transcriptase shares the key features of high fidelity polymerases, such as a closed architecture of the active site, but displays a level of fidelity that is intermediate to that of high fidelity, replicative polymerases and low fidelity translesion synthesis (TLS) polymerases. The beta3-beta4 loop of the HIV-1 RT fingers subdomain makes transient contacts with the dNTP and template base. To investigate the role of active site architecture in HIV-1 RT fidelity, we truncated the beta3-beta4 loop, eliminating contact between Lys65 and the gamma-phosphate of dNTP. The mutant, in a manner reminiscent of TLS polymerases, was only able to incorporate a nucleotide that was capable of base-pairing with the template nucleotide, but not a nucleotide shape-analog incapable of Watson-Crick hydrogen bonding. Unexpectedly, however, the deletion mutant differed from the TLS polymerases in that it displayed an increased fidelity. The increased fidelity was associated with reduced dNTP binding affinity as measured using the dead end complex formation. In an effort to delineate the specific amino acid residue in the deleted segment responsible for this phenotype, we examined the K65 residue. Two substitution mutants, K65R and K65A were studied. The K65A mutant behaved similarly to the deletion mutant displaying dependence on Watson-Crick hydrogen bonding, increased fidelity and reduced dNTP-binding, while the K65R was more akin to wild-type enzyme. These results underscore the key role of the K65 residue in the phenotype observed in the deletion mutant. Based on the well-known electrostatic interaction between K65 and the gamma-phosphate moiety of incoming dNTP substrate in the ternary complex structure of HIV-1 RT, we conclude that non-discriminatory interactions between beta3-beta4 loop and the dNTP in wild-type HIV-1 RT help lower dNTP selectivity. Our results show that the fidelity of dNTP insertion is influenced by protein interactions with the triphosphate moiety.
Subject(s)
Deoxyribonucleotides/metabolism , HIV Reverse Transcriptase/chemistry , HIV Reverse Transcriptase/genetics , HIV-1/enzymology , Alanine , Amino Acid Sequence , Amino Acid Substitution , Binding Sites , HIV Reverse Transcriptase/metabolism , HIV-1/genetics , Molecular Sequence Data , Mutagenesis, Site-Directed , Protein Structure, TertiaryABSTRACT
We reported previously that substitutions F61L, F61W, F61Y and F61A in human immunodeficiency virus type 1 (HIV-1) reverse transcriptase affect strand displacement synthesis [T. S. Fisher, T. Darden and V. R. Prasad (2003) J. Mol. Biol., 325, 443-459]. We have now determined the effect of these mutations on HIV replication. All mutant viruses were replication defective. Measuring replication intermediates in infected cells did not reveal a specific block as all mutants displayed reduced DNA synthesis (wild-type>F61L>F61W>F61Y>F61A). Analysis of 2-LTR circle junctions revealed that F61W and F61Y mutants generated increased aberrant circle junctions. Circle junctions corresponding to F61Y included 3'-PPT insertions suggesting ribonuclease H defect. In vitro assays mimicking PPT primer generation indicated that F61L, F61W and F61Y mutant RTs were unaffected, while F61A mutant cleaved both at PPT/U3 junction and at +6 with similar efficiencies. In assays measuring cleavage at the RNA/DNA junction to remove the PPT primer, all mutants were significantly affected with F61Y and F61A being most severely impaired. Our results show that (i) replication block of most mutants is due to more than one biochemical defect; (ii) mutations in polymerase domain can affect the function of a distal domain; and (iii) virological analyses of RT mutations can yield insight into structure-function relationship that is otherwise not obvious.
Subject(s)
HIV Reverse Transcriptase/genetics , HIV-1/enzymology , HIV-1/genetics , Ribonuclease H/metabolism , Virus Replication , Amino Acid Substitution , Cell Line, Tumor , DNA Primers , DNA Replication , DNA, Viral/biosynthesis , DNA, Viral/chemistry , HIV Long Terminal Repeat , HIV Reverse Transcriptase/chemistry , HIV Reverse Transcriptase/metabolism , HIV-1/physiology , Humans , Phenylalanine/genetics , Reverse Transcription , Sequence Analysis, DNAABSTRACT
BACKGROUND: Internal polymerase III promoters in retroviral vectors have been used extensively to express short RNA sequences, such as ribozymes, RNA aptamers or short interfering RNA inhibitors, in various positions and orientations. However, the stability of these promoters in the reverse orientation has not been rigorously evaluated. RESULTS: A series of retroviral vectors was generated carrying the U6+1 promoter with 3 different HIV-1 RT-specific RNA aptamers and one control aptamer, all in the reverse orientation. After shuttle packaging, the CD4+ cell line CEMx174 was transduced with each vector, selected for expression of GFP, and challenged with HIV-1. We did not observe inhibition of HIV-1 replication in these transduced populations. PCR amplification of the U6+1 promoter-RNA aptamer inhibitor cassette from transduced CEMx174 cells and RT-PCR amplification from transfected Phoenix (amphotropic) packaging cells showed two distinct products: a full-length product of the expected size as well as a truncated product. The sequence of the full-length PCR product was identical to the predicted amplicon sequence. However, sequencing of the truncated product revealed a 139 bp deletion in the U6 promoter. This deletion decreased transcriptional activity of the U6 promoter. Analysis of the deleted sequences from the U6 promoter in the antisense direction indicated consensus splice donor, splice acceptor and branch point sequences. CONCLUSION: The existence of a cryptic splice site in the U6 promoter when expressed in a retroviral vector in the reverse orientation generates deletions during packaging and may limit the utility of this promoter for expression of small RNA inhibitors.
ABSTRACT
Human and mouse telomerases show a high degree of similarity in both the protein and RNA components. Human telomerase is more active and more processive than the mouse telomerase. There are two key differences between hTR [human TR (telomerase RNA)] and mTR (mouse TR) structures. First, the mouse telomerase contains only 2 nt upstream of its template region, whereas the human telomerase contains 45 nt. Secondly, the template region of human telomerase contains a 5-nt alignment domain, whereas that of mouse has only 2 nt. We hypothesize that these differences are responsible for the differential telomerase activities. Mutations were made in both the hTR and mTR, changing the template length and the length of the RNA upstream of the template, and telomerase was reconstituted in vitro using mouse telomerase reverse transcriptase generated by in vitro translation. We show that the sequences upstream of the template region, with a potential to form a double-stranded helix (the P1 helix) as in hTR, increase telomerase activity. The longer alignment domain increases telomerase activity only in the context of the P1 helix. Thus the TR contributes to regulating the level of activity of mammalian telomerases.
Subject(s)
RNA/genetics , Telomerase/metabolism , Animals , Cell Extracts , Cell Line, Tumor , Humans , Mice , Mutation , Protein Structure, Secondary , Telomerase/chemistry , Telomerase/genetics , Templates, GeneticABSTRACT
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.
Subject(s)
CD4-Positive T-Lymphocytes/virology , HIV Infections/virology , HIV-1/physiology , In Situ Hybridization, Fluorescence/methods , Virus Latency , Animals , CD4-Positive T-Lymphocytes/ultrastructure , Cell Count , HIV Infections/diagnosis , HIV-1/genetics , HIV-1/ultrastructure , Humans , Proviruses/physiology , Proviruses/ultrastructure , RNA, Viral/analysisABSTRACT
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 (nâ=â408, 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; Pâ=â0.007), but not in HIV monoinfected (ß:1.2; 95% confidence interval: -6.3, 8.6; Pâ=â0.76) 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.