Download or read book Investigating Nucleic Acid protein and Protein protein Interactions that Govern HIV 1 Replication Using Fluorescence Techniques written by Minh Kim Hong and published by . This book was released on 2004 with total page 356 pages. Available in PDF, EPUB and Kindle. Book excerpt:

Download or read book Footprinting of Nucleic Acid Protein Complexes written by and published by Academic Press. This book was released on 2014-05-19 with total page 216 pages. Available in PDF, EPUB and Kindle. Book excerpt: Footprinting of Nucleic Acid-Protein Complexes provides protocols for studying the stoichiometry, binding site size and location, and structural changes in nucleic acids caused by their interaction with proteins. The methods are central to studying key biological processes, such as transcription and translation. The techniques are important to experiments in vivoand in vitro, in eukaryotes and in prokaryotes, at qualitative and quantitative levels,and across many disciplines. This book is a laboratory manual of footprinting techniques for studying nucleic acid-protein interactions. It contains clear and concise descriptions of the most important methodologies, and includes in vivo as well as in vitro applications. It is aimed at bench scientists from graduate students on, and should be of value in industrial labs as well as in academic settings. Use of different footprinting approaches can provide unique insights into DNA-protein systems. The protocols containedin this handbook are written to be"user-friendly,"and thus should be conducive to extending the use of footprinting to new systems. The section on quantitative analysis of DNAse I footprints should prove especially useful for in depth evaluation of cooperative interactions. (For the End User) Provides clear exposition of footprinting techniques for characterizing DNA-protein interactions Covers both protection methods for identifying sites of protein binding and interference methods for determining points of contact between DNA and protein Includes approaches for both in vitro and in vivo measurements High quality, timely, and of lasting practical value in the laboratory

Download or read book Single molecule Studies on the Role of HIV 1 Nucleocapsid Protein nucleic Acid Interaction in the Viral Replication Cycle written by Hsiao-Wei Liu and published by . This book was released on 2007 with total page 232 pages. Available in PDF, EPUB and Kindle. Book excerpt: The discovery of the crucial intermediates and pathway in the process of the reverse transcription was reported using single-molecule spectroscopy and related techniques including single-molecule fluorescence resonance energy transfer, fluorescence correlation spectroscopy and confocal imaging. Reverse transcription of the HIV-1 RNA genome involves several complex nucleic acid rearrangement steps that are catalyzed by the HIV-1 nucleocapsid protein (NC), including for example, the annealing of the transactivation response (TAR) region of the viral RNA to the complementary region (TAR DNA) in minus-strand strong-stop DNA. In this dissertation, the research focused on elucidating the mechanism of NC-facilitated TAR DNA/RNA annealing. The single molecule spectroscopic measurements reported that the crucial intermediates as well as the mechanistic insight into the annealing of TAR RNA with TAR DNA mediated by viral NC proteins. The data reveal that NC partially melted the secondary structure of TAR DNA (termed the "YTAR") as well as TAR RNA. In the subsequent studies, various short DNA oligonucleotdies were applied to anneal with the TAR to mimic the initial annealing steps. The data support that the YTAR serves as a nucleation center for the annealing to occur through the multiple sites along the TAR structure. Two major nucleation pathways were observed, which are the annealing through the 3'/5' termini, namely "zipper" pathway and the annealing through the hairpin loop region, namely "kissing" pathway. The annealing mechanism was further explored by performing the annealing of wild-type TAR DNA with wild-type TAR RNA in the presence of NC in vitro. The annealing kinetic data suggest that the nucleation of TAR DNA/RNA annealing occurs in an encounter complex form in which one or two DNA/RNA strands in the "Y" form associated with multiple NC molecules. This encounter complex leads to the multiple nucleation complexes, i.e. zipper or kissing intermediates. The data further indicate that although the two complementary strands nucleate at multiple sites, i.e. any single-strand region of TAR, the annealing of two TAR complements occurs through a common mechanism.

Download or read book DNA Protein Interactions written by G. Geoff Kneale and published by Springer Science & Business Media. This book was released on 2008-02-02 with total page 428 pages. Available in PDF, EPUB and Kindle. Book excerpt: The study of protein-nucleic acid interactions is currently one of the most rapidly growing areas of molecular biology. DNA binding proteins are at the very heart of the regulation and control of gene expression, replication, and recombination: Enzymes that recognize and either modify or cleave specific DNA sequences are equally important to the cell. Some of the techniques reported in this volume can be used to identify previously unknown DNA binding proteins from crude cell extracts. Virtually all are capable of giving direct information on the molecular basis of the interaction—the location of the DNA binding site; the strength and specificity of binding; the identities of individual groups on specific bases involved in binding; the specific amino acid residues of the protein that interact with the DNA; or the effects of protein binding on gross conformation and local structure of DNA. The recognition of DNA sequences by proteins is a complex phenomenon, involving specific hydrogen bonding contacts to the DNA bases ("direct readout") and/or interactions with the sugar-phos phate backbone ("indirect readout"). The latter interactions can also be highly specific because of sequence-dependent conformational changes in the DNA. In addition, intercalation of planar aromatic amino acid side-chains between the DNA bases can occur, most notably with single-stranded DNA binding proteins. Furthermore, when bound, many DNA binding proteins induce drastic structural changes in the DNA as an integral part of their function.

Download or read book Probing HIV 1 NC induced Nucleic Acid Structural Rearrangement by Single molecule Fluorescence Spectroscopy written by Yining Zeng and published by . This book was released on 2007 with total page 300 pages. Available in PDF, EPUB and Kindle. Book excerpt: Reverse transcription of HIV-1 genome involves multiple nucleic acids structural rearrangements chaperoned by nucleocapsid protein (NC). One such critical step is that the DNA transactivation response element (TAR) anneals to it complementary RNA region on the genome. It has been challenging to investigate mechanistic details on the annealing process because of the involvement of heterogeneous sets of protein/nucleic acid complexes. Here, we use single-molecule spectroscopy to study the NC induced melting of nucleic acid structure and the annealing activity of different regions along TAR structure. We find that NC induced secondary fluctuations are limited to the terminal stems, and the mechanism for the fluctuations is complex. By employing complementary targeted oligomers, we kinetically trap and investigate stable states of the putative nucleation complexes for the annealing process. This single molecule spectroscopy method directly probes kinetic reversibility and the chaperone role of NC at various stages along the reaction sequence. The new results lead to detailed understanding of NC chaperoned reverse-annealing and the partially annealed conformational sub-states. Argininamide, because of its specific binding to the loop regions of TAR, was studied on its specific inhibition to strand transfer. The loopmediated annealing is found to be more potentially inhibited than stem-mediated one.

Download or read book Investigating the Impact of Sequence and Structural Elements of the Hiv 1 5 Utr on Genomic Rna Conformation and Function written by Jonathan P. Kitzrow and published by . This book was released on 2021 with total page 0 pages. Available in PDF, EPUB and Kindle. Book excerpt: Human immunodeficiency virus type-1 (HIV-1) preferentially infects CD4 positive T-cells and, if left untreated, leads to immunodeficiency and death from AIDS-related illness. HIV-1 provirus produces over 40 differentially spliced viral transcripts during transcription. Of these mRNA splice variants, only the full-length transcript can act as either mRNA, encoding the Gag and Gag-Pol polyproteins, or as genetic material that is selected by Gag from the milieu of host and viral RNAs for packaging into the budding HIV-1 virion. The 5′ untranslated region (5′ UTR) of HIV-1 RNA transcripts is known to regulate many important processes during the HIV-1 lifecycle, including RNA splicing, translation, genome dimerization, and viral packaging. These processes are tightly regulated by host/viral protein interactions with conserved structural elements of the 5′ UTR. Biochemical and nuclear magnetic resonance spectroscopy experiments support a model in which the 5′ UTR can adopt at least two mutually exclusive conformational states. In one state, the genome remains a monomer, as the palindromic dimerization initiation site (DIS) is sequestered via base pairing interactions with upstream RNA regions. In the second state, the DIS is exposed and the genome is competent for kissing loop dimerization and packaging into assembling virions, where an extended dimer is formed during the virion maturation process. According to this model the conformation of the 5′ UTR determines the fate of the genome. I hypothesize that these different conformations and conformational equilibria are modulated by specific host/viral protein:RNA interactions. In this work, the dynamics of this proposed conformational switch and the factors that regulate it were probed using multiple single-molecule and in-gel ensemble Forster resonance energy transfer (FRET) assays. Our results show that a 238-nucleotide (nt) HIV-1 5′ UTR construct intrinsically samples conformations that are stabilized by both viral and host factor binding. Annealing of tRNALys3, the primer for initiation of reverse transcription, can promote the kissing dimer but not the extended dimer. In contrast, HIV-1 nucleocapsid protein (NC) promotes formation of the extended dimer in both the absence and presence of tRNALys3. Our data are consistent with an ordered series of events that involves primer annealing, genome dimerization, and virion assembly. The number of 5′ guanosines has also been implicated in the localization of HIV-1 genomic RNA (gRNA); transcripts with three 5′ guanosines (3G) plus a 5′-5′ triphosphate-linked 7-methylguanosine (7MeG) cap are abundant in the cytoplasm, whereas transcripts with a single 5′ guanosine (1G) and a 7MeG cap are preferentially packaged into budding virions. The impact of the number of 5′ guanosines on the ensemble of RNA conformations and their dynamics is unknown. RNA chemical probing using selective 2′-hydroxyl acylation analyzed by primer extension (SHAPE), native gel analysis, and viral packaging assays revealed that 2G and 4G 5′ UTRs (designed to mimic 1G/3G plus a 7MeG cap) adopt a different ensemble of RNA conformations, with the 1G RNAs adopting an ensemble of structures that favor selective packaging. Structural and viral packaging studies performed on mutants designed to disrupt known HIV-1 5′ UTR structures strongly suggest the stability of the PolyA hairpin and subsequent upstream 5′ UTR structural rearrangements in the Psi packaging signal are responsible for differential 1G packaging. Preliminary results of in-gel FRET experiments using the FH.238-nt 5′ UTR system to characterize the effects of Gag on the 5′ UTR and tRNALys3 annealing suggest Gag promotes tRNALys3 annealing and kissing dimer formation, but unlike NC, does not chaperone extended dimer formation. We also began investigating the 355- and 357-nt 1G and 3G 5′ UTR RNAs using ensemble in-gel FRET. This was accomplished using a bifacial peptide nucleic acid strategy to position internal FRET dyes. Results support the feasibility of this labeling technique to monitor structural states/equilibria in the full HIV-1 5′ UTR.

Download or read book Labeling HIV 1 Nucleic Acids During Infection and Effect of MOV10 in HBV Replication written by Maritza N. Puray Chavez and published by . This book was released on 2018 with total page 0 pages. Available in PDF, EPUB and Kindle. Book excerpt: "Technical limitations in simultaneous microscopic visualization of RNA, DNA, and proteins of HIV have curtailed progress in this field. To address this need we have developed a microscopy approach, multiplex immunofluorescent cell-based detection of DNA, RNA and Protein (MICDDRP), which is based on branched DNA in situ hybridization technology. MICDDRP enables simultaneous single-cell visualization of HIV spliced and unspliced RNA, cytoplasmic and nuclear DNA, and Gag. We use MICDDRP to visualize incoming capsid cores containing RNA and/or nascent DNA and follow reverse transcription kinetics. We have also reported transcriptional "bursts" of nascent RNA from integrated proviral DNA. The major barrier towards a cure is the ability of the virus to remain dormant in long-lived cells known as the latent HIV reservoirs; however, the mechanisms that regulate latency are poorly understood. HIV-1 integration is directed to regions of the genome by the chromatin binding protein lens epithelium derived growth factor (LEDGF/p75, referred to here as LEDGF). In the absence of LEDGF, HIV-1 infection is markedly less efficient. We report for the first time that knocking out LEDGF results in upregulation of antisense HIV-1 RNA transcription. Antisense HIV-1 transcripts are abundant in a higher proportion of cells when the Integrase-LEDGF interaction is lost, whether by knocking out LEDGF or by pharmacological inhibition. We also demonstrate that intriguingly, antisense RNA is rapidly lost from infected cells. Using a dual reporter virus to investigate the association between HIV-1 antisense RNA and latency, we have determined that antisense RNA has low expression in "latently" infected cells. Hepatitis B virus infection is a major health problem, there are more than 350 million people living with chronic HBV. Immune system can clear infection during the acute phase in immune-competent patients. A better understanding of the immune response against HBV infection may lead to new insights a find a cure. RNA helicases contribute to the immune response and some also have antiviral effects. MOV10 is a helicase RNA protein that has detrimental effect in HIV infection and HCV replication under MOV10 depletion or overexpression. Considering that HBV replication rely on reverse transcription to form DNA from pre-genomic RNA, we study the effect of MOV10 in HBV replication. Our data suggest that while MOV10 overexpression leads to HBV reduction, suppression of MOV10 enhances HBV replication."--MoSpace.

Download or read book A Laboratory Guide to in Vitro Studies of Protein DNA Interactions written by Jean Pierre Jost and published by Biomethods. This book was released on 1991 with total page 336 pages. Available in PDF, EPUB and Kindle. Book excerpt: A Safety Considerations Many techniques described here involve a number of hazards, such as high electrical current and voltage, radioactivity and highly toxic chemicals. It is absolutely essential that the instructions of equipment manufacturers be followed, and that particular attention be paid to the local and federal safety regulations. B Introduction The expression of prokaryotic and eukaryotic genes has been shown most often to be regulated at the level of mRNA synthesis. Thanks to the rapid development of methods for dissecting DNA sequences, cis-acting regulatory elements such as promoters and enhancers have been recognised. More recently, the widely expressed intuition that discrete sequences within these elements constitute binding sites for sequence-specific binding proteins has been confirmed, especially through the use of "footprinting" assays (for examples, Galas and Schmitz, 1978). This and similar assays have already resulted in the recognition, isolation and analysis of DNA-bind ing proteins for several genes. Excellent reviews exist of the structural studies on these transcription regulatory proteins and related DNA elements (for example, Glover, 1989 and Johnson and McKnight, 1989), to which the reader is referred for detailed information. To set the scene for applications of the techniques described in this volume, only the barest outline of previous studies is presented here. Protein-DNA interactions are dependent on very specific tertiary configurations of the binding protein which allow the closest contact with the DNA helix.

Download or read book Experimental and Computational Investigations Into Interactions Between HIV 1 and the RNA Interference Pathway written by Owen Dunkley and published by . This book was released on 2022 with total page pages. Available in PDF, EPUB and Kindle. Book excerpt: "As an obligate intracellular pathogen, the human immunodeficiency virus type 1 (HIV-1) targets and co-opts a diverse set of host processes to overcome cellular barriers to infection, including pathways involved in the regulation of gene expression. It remains controversial whether HIV-1 affects the RNA interference (RNAi) pathway, a key post-transcriptional regulatory mechanism that is being used to develop a new class of gene therapies. To better understand the replication cycle of the virus and inform the development of future antiviral therapies, we investigated the hypothesis that HIV-1 changes the substrates and functionality of the RNAi system in specific contexts. To investigate our hypothesis, we first aimed to identify regulatory networks associated with blocks in virus replication during latency. We characterized a novel cellular model for HIV-1 infection that was designed to study events in HIV-1 latency reversion that follow transcription initiation. We then reactivated this model using differently acting latency reversing agents, to then sequence long and short RNA transcriptomes associated with latency maintenance and reversion. These data contributed new genes and regulatory RNA networks to our understanding of latency, and possibly new targets for RNA therapies. We next analyzed an interaction between the HIV-1 protein Gag and the RNAi enzyme Dicer, which leads to the specific enrichment of three microRNAs (miRNAs) on Dicer. A combination of bioinformatic analyses were used to identify the targets of these miRNAs and to define a target-specific hypothesis for the function of this interaction. Using gene reporter assays, Western blots and reverse transcription quantitative polymerase chain reactions, we explored one miRNA-target interaction in depth, showing that this miRNA inhibits HIV-1 expression and that Gag promotes viral expression by increasing expression of the target, possibly by inhibiting the antiviral miRNA. Finally, we sought to develop a testing platform to distinguish between RNAi substrates that could be used in combination therapies against HIV-1. We designed a novel protocol that can score RNAi substrates with four primary measures: cellular toxicity, inhibition of HIV expression, inhibition of HIV replication, and ability to lock proviruses in a latent state. Due to delays related to cloning, this protocol could not be fully employed in this manuscript. However, we individually tested the first three endpoints and used a surrogate fourth endpoint to make preliminary assessments of the therapeutic potential of several molecules. Here, we describe two workflows used to gain better insight into interactions between HIV-1 and the RNAi pathway, and further designed a protocol for testing small RNAs for their potential to lock the HIV-1 in a latent state"--

Download or read book Single Molecule Force Spectroscopy Studies of DNA Binding and Chaperone Proteins written by and published by . This book was released on 2008 with total page 155 pages. Available in PDF, EPUB and Kindle. Book excerpt: The nucleocapsid protein (NC) plays an important role in retroviral replication, in part, by facilitating numerous nucleic acid rearrangements throughout the reverse transcription process. The nucleic acid chaperone activity of the human immunodeficiency virus type-1 (HIV-1) NC has been extensively studied, and duplex destabilization, nucleic acid aggregation, and rapid protein binding kinetics have been identified as major components of the activity of this highly basic protein (pI ~10). The chaperone activity of other NC proteins is not well understood. We used single molecule DNA stretching to characterize the activity of HIV-1, RSV, and MMLV NC. We found distinct differences in the chaperone activities of each protein, which reflect the requirements for nucleic acid chaperone activity in each retroviral replication system. HTLV-1 NC exhibited overall poor nucleic acid chaperone acitivity. This result is explained by its poor aggregating activity and slow dissociation from single-stranded DNA. This NC protein is overall neutral at pH=7.5 and possesses a unique, acidic C-terminal domain. By studying different HTLV-1 NC mutants, the role of C-terminal domains to the chaperone activity was elucidated. The results suggest that the electrostatic interaction between HTLV-1 NC and nucleic acids is the major factor determining the kinetics. We also examine the nucleic acid interaction properties of the Apolipoprotein B mRNA editing enzyme, a catalytic polypeptide-like 3G (APOBEC3G/A3G) that is known to inhibit HIV-1 reverse transcription in absence of viral infectivity factor (Vif). Our stretching experiments suggested a novel mechanism for deaminase-independent inhibition of reverse transcription due to vital differences of nucleic acid binding kinetics between NC, A3G and reverse transcriptase (RT). Finally, Long interspersed nucleic elements (LINE) are highly repeated nucleic acids sequences in mammal genomes. Our single DNA molecule stretching experiments characterized the nucleic acid chaperone function of ORF1p in the mouse LINE-1 retrotransposon. We found that a single amino acid substitution altered retrotransposition efficiency by a factor of 15 due to a reduction in nucleic acid chaperone activity exhibited by ORF1p. For all of the studies presented here, we used single molecule methods to characterize the nucleic acid interactions of proteins involved in reverse transcription in retroviruses or retrotransposons. In each case, complementary bulk experiments were done by collaborators. The results are presented together in each chapter of the thesis.

Download or read book A frame shift in Perspective written by Bayleigh Esther Benner and published by . This book was released on 2020 with total page 246 pages. Available in PDF, EPUB and Kindle. Book excerpt: Viruses have universally evolved RNA structures as key regulators of viral replication, utilizing cis-acting features (RNA motifs which regulate nucleic acid regions on the same RNA molecule) to modulate production of viral proteins. Retroviruses such as human immunodeficiency virus (HIV) face the challenge of producing two distinct polyproteins, Gag and Gag-Pol (GP), from the same 9 kilobase unspliced RNA (US RNA) transcript. A conserved RNA stem-loop and heptanucleotide slippery sequence (U_UUU_UUA) within the gag reading frame dictate the proportions of Gag and Gag-Pol (GP) synthesized. The pol gene is translated due to a -1 programmed ribosomal frameshift (PRF) near the end of the gag coding region, producing the GP polyprotein ~5% of the time. Frameshifting events are tightly regulated because GP encodes enzymes required for virion maturation and genome replication. Utilizing previously published frameshift site mutants, we have shown that increasing stem-loop thermodynamic stability increases frameshifting efficiency, causing large reductions in infectivity. Herein, we first investigated the mechanisms underpinning this infectivity loss. Increased frameshifting resulted in decreased GP cleavage products in virions, suggesting that excess GP causes premature Gag cleavage and release of viral proteins into the cytoplasm during virion budding. Interestingly, although over-frameshifting mutants incorporate excess GP into virions, we could not reach these levels by providing GP in trans, thus demonstrating that GP incorporation into virions exhibits a strong cis preference and that GP incorporation is non-stochastic contrary to what was previously thought. These results demonstrate how frameshifting fidelity leads to compartmentalized protein interactions which facilitate a preference for cis-packaging of GP, indicating that HIV translation and virion assembly are coupled processes. Second, we show that creation of mutants that are predicted to disrupt RNA pseudoknot formation at the frameshift site do not impair frameshifting efficiency, but that the GGG frameshift site spacer sequence is essential for viral infectivity. Third, we used fluorescently tagged RNA and live-cell imaging techniques to determine, for the first time, whether translated US RNAs are capable of being packaged into virions. Fourth, we utilized a fluorescence-based HIV-1 reporter virus to create a cell-based assay to identify frameshift site inhibitors for HIV-1 and SARS-CoV-2. Taken together, these studies underline the importance of understanding viral programmed ribosomal frameshifting as a potential target for therapeutics aimed at treating current and future pathogens.

Download or read book Inhibition of Protein protein Interactions written by Susan Northfield and published by . This book was released on 2012 with total page 434 pages. Available in PDF, EPUB and Kindle. Book excerpt: Over 34 million people worldwide are currently infected with human immunodeficiency virus 1 (HIV-1), which is the etiologic agent of acquired immunodeficiency syndrome (AIDS). This virus is becoming resistant to current drug therapy. Consequently, there is a need for new drugs, which work alone or in combination with existing therapies, to treat HIV-positive patients and limit the spread of the disease. More than 30 drugs are currently approved by the United States Food and Drug Administration (FDA) to treat HIV. These include inhibitors of HIV-1 enzymes reverse transcriptase and protease, as well as entry inhibitors and fusion inhibitors. However, there is currently only one drug on the market that targets HIV integrase. In recent years, new approaches to inhibit HIV replication have been sought. These have included attempts to prevent enzymes in the replication cycle from binding to endogenous cofactors important for enzyme activity. Lens epithelium-derived growth factor (LEDGF) is a cellular protein, essential for efficient integration of viral DNA into the host chromosome, and consequently for successful viral replication. LEDGF functions include aiding nuclear import of the pre-integration complex (PIC) and directing the PIC to active transcription sites on the chromatin. The tetrapeptide sequence Lys364-Ile365-Asp366-Asn367, present in LEDGF, has been shown to be crucial for LEDGF binding to integrase.This thesis describes the design, synthesis and binding analysis of cyclic peptides designed to mimic LEDGF binding to HIV integrase. The peptides incorporate the Lys-Ile-Asp-Asn sequence in conjunction with a turn-inducing scaffold and were designed to block the protein-protein interaction between LEDGF and integrase. Analysis of peptide binding to integrase was achieved using a combination of techniques including HSQC NMR, X-ray crystallography and SPR. The first body of work describes the initial design and synthesis of our cyclic peptides. Disulfide cyclised and head-to-tail cyclised peptides were initially explored. Head-to-tail cyclic peptides, incorporating the LEDGF tetrapeptide sequence and a D-Xaa-L-Pro dipeptide scaffold, showed the ability to readily mimic the conformation of LEDGF. These initial analogues spurred the development of a head-to-tail cyclic peptide library.The head-to-tail cyclic peptides were optimised through investigation of alternate turn-inducing motifs. These included a range of D-Xaa-L-Pro dipeptide scaffolds in addition to a selection of constrained [beta]-amino acids. Additionally, the Ile365 side chain was known to protrude into a hydrophobic pocket on the surface of integrase. This provided an opportunity to increase hydrophobic interactions by substituting alternate residues at this position. Following development of the peptide library, a means to introduce conjugates to the peptides was sought. This involved the development of modified proline amino acids that included an alkyne linker, which allowed conjugation of the peptides using 'click chemistry' to couple any azide conjugate of our choice. A conjugate library was efficiently and rapidly developed, incorporating a mixture of carbohydrates, fluorophores and biotin labels to our peptides.

Download or read book Visualization of the Intracellular HIV 1 Replication Cycle written by Ryan David Stultz and published by . This book was released on 2017 with total page 167 pages. Available in PDF, EPUB and Kindle. Book excerpt: The sequence of events from HIV-1 entry through transcription of proviral DNA is not well understood. While many studies have attempted to investigate these post-fusion events, the unstable nature of intracellular HIV-1 particles has made biochemical analyses difficult. Developments in imaging techniques have given a glimpse of intracellular HIV-1 particles during active infection, but few of these attempts have connected both the early and late events of HIV-1 infection in primary human target cells. Even fewer of these attempts effectively visualized actively transcribing viruses that contribute to productive infection. Here, we developed a method to visualize HIV DNA reverse transcription (RT) products by incorporation and fluorescent labeling of the thymidine analog 5-ethynyl-2’-deoxyuridine (EdU) in infected monocyte-derived macrophages (MDM). We observed distinct EdU puncta in the cytoplasm and nuclei of infected cells which increased in number following depletion of the dNTPase SAMHD1. Interestingly, we found HIV-1 capsid associated with HIV-1 DNA in the cytoplasm and nuclei of infected MDM. Our assay also represented improvements upon current qPCR techniques in measuring the efficiency of nuclear import of two CA mutants, N74D and Q63A/67A, and showed marked sensitivity in detecting intranuclear HIV-1 genomes in productively and non-productively infected MDM. Because we were able to track nuclear HIV-1 DNA through productive infection of MDM, we were interested in whether we could identify actively transcribing proviruses. Using RNA fluorescence in situ hybridization (FISH) we were for the first time able to image actively transcribing proviruses through colocalization of both HIV-1 DNA and HIV-1 RNA. These transcribing proviruses were further confirmed through colocalization of nuclear HIV-1 DNA and RNA with specific HIV-1 transcription factors. Modification of our newly developed RNA FISH technique to monitor HIV-1 transcription in productively infected and reactivated, HIV-latently infected CD4 T cells identified specific intranuclear foci of HIV-1 RNAs. These foci correlated with the number of proviral integration sites in latently infected cell lines, suggesting RNA FISH detects sites of proviral transcription in these cells. Overall, our novel HIV-1 imaging techniques allow for stable labeling and monitoring of functional HIV-1 complexes within infected cells during cytoplasmic transit, nuclear import, and transcription.

Download or read book 2 Aminopurine and Pyrenylethyne Modified Uridine as Fluorescent Probes for the Investigation of Nucleic Acids and Their Interactions written by and published by . This book was released on 2006 with total page pages. Available in PDF, EPUB and Kindle. Book excerpt: The present thesis investigates RNA-RNA and RNA-protein interactions as well as energy and charge transfer through DNA using fluorescence detected methods. The base analogs 2-aminopurine and 5-(1-pyrenylethynyl)-2'-deoxyuridine were used as fluorescent probes, which were incorporated into RNA and DNA by phosphoramidite solid phase synthesis. Using these fluorescent probes it was possible for one to determine kinetics and mechanisms of two antisense RNA systems from the plasmid replication system ColE1 of E. coli and the dimerization initiation site of HIV-1. In addition energy and charge transfer in DNA could be shown by excimer emission, and fluorescence quenching, respectively.

Download or read book HIV 1 Integrase written by Nouri Neamati and published by John Wiley & Sons. This book was released on 2011-08-10 with total page 710 pages. Available in PDF, EPUB and Kindle. Book excerpt: This book comprehensively covers the mechanisms of action and inhibitor design for HIV-1 integrase. It serves as a resource for scientists facing challenging drug design issues and researchers in antiviral drug discovery. Despite numerous review articles and isolated book chapters dealing with HIV-1 integrase, there has not been a single source for those working to devise anti-AIDS drugs against this promising target. But this book fills that gap and offers a valuable introduction to the field for the interdisciplinary scientists who will need to work together to design drugs that target HIV-1 integrase.

Download or read book Functional Analysis on the Interactions of the Human Immunodeficiency Virus Type 1 Integrase with Its Cofactors that Regulate Viral Replication written by Yingfeng Zheng and published by . This book was released on 2008 with total page 0 pages. Available in PDF, EPUB and Kindle. Book excerpt: Like all viruses, the replication of HIV-1 relies heavily on host proteins due to its limited genome products. HIV-1 integrase (IN) catalyzes the integration of viral DNA into host genome and also impacts other steps of viral replication cycle, all of which are assisted by various cellular proteins. Among them, LEDGF/p75 acts as the IN-to-chromatin tethering factor. However, whether other cellular cofactors also participate in this process still remains elusive. To gain insight into the mechanism of action of HIV-1 IN during viral integration, we used a previously described IN/yeast lethality system and our results revealed that the HIV-1 IN-induced yeast lethality absolutely required its chromatin binding ability. Since there is no yeast homolog of LEDGF/p75, it raises the possibility that IN may recruit other cellular cofactors for its chromatin targeting. Consistently, further analysis in mammalian cells indicated that HIV-1 IN was able to mediate chromatin binding independent of IN-LEDGF/p75 interaction and that HIV-1 fitness relied more on chromatin binding than LEDGF/p75 binding of IN. These data greatly enrich our current knowledge on the dynamic interplay within the ternary complex IN/LEDGF/chromatin. HIV-1 exploits multiple cellular cofactors not only to facilitate viral replication, but also to evade the host defense system in favor of the virus. IN is known to be an unstable protein, degraded by the host ubiquitin-proteasome pathway. To investigate how IN avoids the host degradation machinery in the context of viral infection, we showed that IN interacted with host protein Ku70 and protected itself from the Lys48-linked polyubiquitination proteasomal pathway. More importantly, Ku70 was shown to be incorporated into the progeny virus in an IN-dependent manner, and both cell- and virus- associated Ku70 were essential for HIV-1 replication. Finally, the data demonstrated that the interactions between HIV-1 IN and host cofactors can be regulated through its SUMO-interacting motifs (SIMs). Three putative SIMs (72VILV75; 200IVDI203 and 257IKII260) in IN were examined and shown to be essential for IN-LEDGF/p75 but not IN-Ku70 interaction. In summary, this study advances our knowledge of the interaction network between IN and its cofactors, which would have important implications for the design of anti-HIV drugs.

Download or read book Enhancing the Solubility of Intrinsically Disordered HIV 1 Tat Protein at Physiological PH and Structural Investigation by NMR Spectroscopy written by Kiran Krishnamurthy and published by . This book was released on 2021 with total page 0 pages. Available in PDF, EPUB and Kindle. Book excerpt: Human Immunodeficiency Virus-1 (HIV-1) Transactivator of transcription (Tat) protein is a 101-residue intrinsically disordered protein, responsible for enhancing the transcription process and ultimately viral replication. To understand its mechanistic role in enhancing transcription and viral replication, a study of its structure and dynamics in the presence of its binding partners is required. However, the protein is soluble only under acidic conditions (pH 4) and it precipitates at pH 7, which precludes the determination of its structure, dynamics and interactions under physiological conditions. Hence, the primary objective of this research was to solubilize Tat-protein near pH 7 so that further studies may be carried out to discern the mechanistic role of the Tat protein in viral replication. Multiple approaches were employed to improve the solubility of the Tat protein at pH 7. A sequence-specific nickel-assisted cleavage (SNAC) approach that involves cleaving the polyhistidine-tagged Tat was employed to produce full-length Tat protein minus the purification tag, but this was unsuccessful owing to an unpredictable internal cleavage site. Poly-anionic RNA from Torula yeast was used as a solubilizing agent and it was found to increase the solubility of Tat but Nuclear Magnetic Resonance (NMR) spectra were only marginally improved. Tat was observed to be insoluble at pH 4−7 in the presence of TAR (TransActivation Response) RNA, one of the binding partners of Tat which is vital for the functioning of the protein. Moreover, the solubility of Tat was tested in a solution containing HIV-1 TAR RNA and Torula yeast RNA. Here too, the protein was soluble but no major improvement in the NMR spectra of Tat was observed. Tat protein tagged with a supercharged sequence at the N-terminal was genetically engineered and expressed to test the effect of a high net-charge on Tat's solubility. The increased net charge did not improve Tat's solubility. Genetic engineering was used to replace seven cysteine residues of iii the Tat protein with aspartic acid to study the role of cysteine residues in the aggregation of the protein at neutral pH. This approach produced highly soluble Tat at pH 7 with well-resolved NMR spectra leading to the realization of the important role that Cys oxidation plays in the solubility of the protein. The carboxy terminal domain of RNA polymerase II (RNAP II) undergoes liquid-liquid phase separation in the presence of a crowding agent. With the expectation that Tat might be soluble in a liquid-liquid phase separated medium at physiological pH, a RNAP II domain fusion protein was expressed, but poor yields of proteolyzed protein precluded Tat solubility studies. Several analytical techniques were employed to characterize the structure of Tat protein. Fluorescence studies were carried out to understand the structural changes taking place as the pH is elevated. Somewhat surprisingly, the fluorescence spectra indicated that the single Trp residue resides in a solvent-restricted region of intrinsically-disordered Tat. Infrared spectroscopy was used to study the secondary structure of the protein and to quantify the fractions of different secondary structures comprising the Tat protein in the range of pH 4−7. Multi-dimensional solution- and solid-state NMR spectroscopy were employed as the primary analytical tool in the structural analysis of Tat protein. In addition to conventional indirect-detection NMR methods, 15N-direct-detection NMR experiments were attempted to help monitor solubility, structure and dynamics.