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Thesis (Ph.D.)--University of Rochester. School of Medicine & Dentistry. Dept. of Microbiology and Immunology, 2014., Human immunodeficiency virus type-1 (HIV-1) and acquired immunodeficiency syndrome (AIDS) remain subjects of global importance. There is currently no vaccine approved to protect against HIV-1/AIDS and the emergence of drug resistance mutants to existing antiretroviral therapies poses a serious challenge demanding the need for newer strategies. Like other retroviruses, HIV-1 converts its RNA genome into double-stranded DNA by reverse transcription and then integrates this DNA into the host genome. These processes are mediated by the viral enzymes reverse transcriptase (RT) and integrase (IN). Reverse transcription involves obligatory strand transfer events. Strand transfers are unique processes that allow both completion of reverse transcription and genetic recombination. Transfers are potential drug targets, and so it is absolutely vital to understand their mechanisms. To accomplish transfer, a growing DNA primer shifts from a donor to an acceptor template. We designed a unique primer-template system allowing measurements of strand transfer without ribonuclease H (RNase H) activity. Using it, we showed that RT dissociates during transfers, allowing the critical primer shift step to occur before RT re-binds to complete the process. Additional experiments defined the mechanisms by which the non-nucleoside RT inhibitor Efavirenz (EFV) stimulates RT ribonuclease H activity. EFV had been shown to stimulate growth of a number of drug resistant virus mutants in cell culture. We examined the mechanism of EFV stimulation in vitro. EFV stimulated viral RNase H activity of both wild type and EFV resistant mutants by increasing primary and secondary cuts. EFV increased RT binding to its RNA/DNA substrate and appeared to change the binding conformation of RT so it slid more efficiently to positions of secondary cuts. The coordinated stimulation of primary and secondary cuts most likely accelerates removal of genomic RNA, facilitating minus strand transfer and making way for plus

Thesis (Ph.D.)--University of Rochester. School of Medicine & Dentistry. Dept. of Pathology, 2014., HIV-1 is known to be genetically diverse due to the frequent mutations caused by the low fidelity HIV-1 reverse transcriptase (RT). The diversity comes in part from the recombination that occurs between the two single-stranded viral RNA genomes during reverse transcription. These recombination events can contribute to the accumulation of multiple mutations, which may result in the virus having multiple antiretroviral drug resistance. Therefore, it is crucial to understand the factors that influence HIV-1 recombination events during replication. A multi-drug resistant HIV-1 containing a dipeptide insertion mutation within the template and primer (T/P) binding domain of RT was investigated. The questions asked were: 1) what biochemical changes does this insertion mutation induce on RT for nucleoside reverse transcriptase inhibitor drugs (NRTI) resistance 2) the effect this insertion has on HIV-1 recombination rate. We hypothesize that this mutation will affect T/P binding affinity of RT, therefore influencing drug removal and strand transfer efficiency. We found that: 1) the dipeptide insertion mutation was responsible for the removal of a thymidine analog chain terminator Zidovudine (AZT) from the terminated primer and 2) the insertion mutation had higher strand transfer efficiency due to a lower KD. The tighter binding to T/P gives more time for RNase H domain of RT to cleave the template RNA for strand invasion to occur. HIV recombination events can also be influenced by dynamic copy choice strand transfer events. Dynamic choice strand transfer occurs when slower kinetics of RT allows RNase H to exert its activity and degrade the RNA template thereby enabling strand invasion to occur. It has been shown that lower polymerase processivity and lowering dNTP concentration in in vitro strand transfer assays resulted in higher strand transfer events. From these results, we hypothesize that a deoxynucleotide triphosphohydrolase enzyme called the sterile alpha mot

HIV undergoes high rates of mutation and recombination during reverse transcription, but it is not known whether these events occur independently or are linked mechanistically. Here we used a system of silent marker mutations in HIV and a single round of infection in primary T lymphocytes combined with a high-throughput sequencing and mathematical modeling approach to directly estimate the viral recombination and mutation rates. From >7 million nucleotides (nt) of sequences from HIV infection, we observed 4,801 recombination events and 859 substitution mutations (≈1.51 and 0.12 events per 1,000 nt, respectively). We used experimental controls to account for PCR-induced and transfection-induced recombination and sequencing error. We found that the single-cycle virus-induced mutation rate is 4.6 × 10(-5) mutations per nt after correction. By sorting of our data into recombined and nonrecombined sequences, we found a significantly higher mutation rate in recombined regions (P = 0.003 by Fisher's exact test). We used a permutation approach to eliminate a number of potential confounding factors and confirm that mutation occurs around the site of recombination and is not simply colocated in the genome. By comparing mutation rates in recombined and nonrecombined regions, we found that recombination-associated mutations account for 15 to 20% of all mutations occurring during reverse transcription.

Thesis (Ph.D.)--University of Rochester. School of Medicine and Dentistry. Dept. of Biochemistry and Biophysics, 2009., Reverse transcription of the HIV-1 genome from ssRNA to dsDNA is a necessary step of the viral life cycle. Transfer of the cDNA strand between non-identical, copackaged RNA genomes during reverse transcription can lead to recombination of genetically advantageous traits. RT hydrolyzes RNA in the DNA-RNA hybrid generating a gap where another RNA template invades and hybridizes with the DNA. The newly formed hybrid propagates out from the site of invasion toward the DNA 3' terminus. Transfer is completed when the DNA terminus switches to the next RNA template allowing RT to resume synthesis. Factors that influence individual steps of the strand transfer reaction during reverse transcription were examined. A DNA-RNA hybrid reconstituting a gapped intermediate was used to evaluate strand transfer efficiency. The hybrid substrate had a 20 nt DNA overhang functioning as a pre-created invasion site (PCIS). A second, homologous RNA template containing sequence complementary with the PCIS was used as an invading strand to initiate the transfer reaction. Results indicated that the PCIS affected transfer approximately 17 - 32 bp downstream suggesting strand exchange is limited by the distance separating the invasion site and DNA primer terminus. The chaperone properties of HIV-1 nucleocapsid (NC) protein extended the effective distance from 17 bp to 24 bp, and when combined with RNase H activity of RT the distance of the PCIS influence increased to 32 bp. As directed by enzyme-substrate binding, RT cleaves the RNA in primary, secondary, polymerase-dependent or polymerase-independent modes. Results showed that in the presence or absence of NC all modes of cleavage facilitated invasion site formation. In contrast, in the presence of NC, only polymerase-dependent, primary cleavages were necessary to support expansion of the DNA-RNA hybrid emanating from the PCIS. These results are consistent with a model in which NC and nicks in the RNA increase the distance over which a specific

Thesis (Ph.D.)--University of Rochester. School of Medicine and Dentistry. Dept. of Biochemistry and Biophysics, 2008., HIV-1 can recombine at least three times each replication cycle. Recombination can disperse mutations to generate virus resistant to multiple drugs. Each HIV-1 virus contains two copies of the positive sense, single strand RNA genome, which makes recombination possible. During HIV-1 virus replication, DNA synthesis by viral reverse transcriptase (RT) can switch from the original RNA template (donor) to the other (acceptor), a process called strand transfer. When the templates are different, genetic recombination will occur. In addition to the minus and plus strong stop transfers needed for viral replication, strand transfer recombination can occur throughout the whole viral genome. Previous research in vitro showed that synthesis pausing promotes strand transfer by concentrating RNase H cleavages at the pause site on the donor. Some donor segments dissociate and expose the newly synthesized cDNA that can interact with the acceptor, which is called invasion. Then the cDNA/acceptor interaction propagates to catch up with the DNA terminus downstream of the pause site, a step called terminus transfer. Using a similar system with a stable hairpin on the templates, extended homology was found to shift the peak of recombination to the pause site, the base of the hairpin. This and other results suggest that RT pausing also allows cDNA/acceptor interactions initiated upstream to catch up with the DNA terminus to complete strand transfer. A preferred site for recombination was identified previously using a cell-culture system. The recombination profile can be reproduced in vitro. The sequence of the hot spot is unusually G-rich (38%) compared to the rest of HIV-1 genome (24%). Several evenly spaced pauses that correspond to G runs in the sequence occur in the hot spot region. Altering the G runs often decreased RT pausing and sometimes decreased transfers. Results suggest that a hot spot could be created by appropriately spaced pause sites that allow efficient invasion and th

The retroviral reverse transcription reaction normally occurs in capsid-like structures in the cytoplasm of infected cells. Reverse transcription can also be carried out in vitro in totally reconstituted reactions with purified enzymes and model RNA templates. However, in this case fully synthesized DNAs are rarely generated from genomic RNA. This could be because the capsid creates an extremely concentrated and specific environment that cannot be completely reproduced in vitro. An in vitro system that closely mimics replication and that can be easily manipulated would enhance our understanding of the replication process. In this thesis report, in vitro reaction conditions that allowed efficient synthesis of DNA products up to 4 kb from genomic RNA segments of Human Immunodeficiency Virus (HIV) were generated. The reactions required high amounts of HIV reverse transcriptase enzyme (RT) and nucleocapsid protein (NC) sufficient to completely coat the RNA template in the reaction. Synthesis of long DNA products required the formation of high molecular weight aggregates with nucleic acids, RT and NC. Removal of the dimerization region did not affect synthesis of long DNA products in vitro. Processivity of RT does not play a role in the synthesis of long DNA products. NC finger mutants lacking either finger or with the finger positions switched were all effective in synthesizing long DNA products suggesting that the aggregation/condensation activity but not the unwinding activity of NC is required for the synthesis of long DNAs in vitro. These results taken together, we propose that high molecular weight aggregates promote synthesis of long reverse transcription products in vitro by concentrating nucleic acids, RT enzyme and NC into a smaller area, thereby mimicking the role of the capsid environment within the host cell. In addition, strand transfer assays indicate that strand transfer is the molecular mechanism involved in the synthesis of long DNAs and the rate of tra

HIV-1 recombination between different subtypes has a major impact on the global epidemic. The generation of these intersubtype recombinants follows a defined set of events starting with dual infection of a host cell, heterodiploid virus production, strand transfers during reverse transcription, and then selection. In this study, recombination frequencies were measured in the C1-C4 regions of the envelope gene in the presence (using a multiple cycle infection system) and absence (in vitro reverse transcription and single cycle infection systems) of selection for replication-competent virus. Ugandan subtypes A and D HIV-1 env sequences (115-A, 120-A, 89-D, 122-D, 126-D) were employed in all three assay systems. These subtypes co-circulate in East Africa and frequently recombine in this human population. Increased sequence identity between viruses or RNA templates resulted in increased recombination frequencies, with the exception of the 115-A virus or RNA template. Analyses of the recombination breakpoints and mechanistic studies revealed that the presence of a recombination hotspot in the C3/V4 env region, unique to 115-A as donor RNA, could account for the higher recombination frequencies with the 115-A virus/template. Single-cycle infections supported proportionally less recombination than the in vitro reverse transcription assay but both systems still had significantly higher recombination frequencies than observed in the multiple-cycle virus replication system. In the multiple cycle assay, increased replicative fitness of one HIV-1 over the other in a dual infection dramatically decreased recombination frequencies. Sequence variation at specific sites between HIV-1 isolates can introduce unique recombination hotspots, which increase recombination frequencies and skew the general observation that decreased HIV-1 sequence identity reduces recombination rates. These findings also suggest that the majority of intra- or intersubtype A/D HIV-1 recombinants, generated w

A series of point mutations was constructed in domain IIIα of the Mu A protein. The mutant transposases were purified and assayed for their ability to promote various aspects of the in vitro Mu DNA strand transfer reaction. All mutants with discernable phenotypes were inhibited in stable synapsis (Type 0 or Type 1 complex formation). In contrast, these mutant proteins were capable of LER formation (a transient early reaction intermediate in which the Mu left and right ends have been synapsed with the enhancer), at levels comparable to wild-type transposase. These proteins therefore comprise a novel class of transposase mutants, which are specifically inhibited in stable transpososome assembly. The defect in these proteins was also uniformly suppressed by either Mn2+, or the Mu B protein in the presence of ATP and target DNA. Striking phenotypic similarities were recognized between the domain IIIα transposase mutant characteristics noted above, and those for substrate mutants carrying a terminal base-pair substitution at the point of cleavage on the donor molecule. This phenotypic congruence suggests that the alterations in either protein or DNA are exerting an effect on the same step of the reaction i.e., engagement of the terminal nucleotide by the active site. We suggest that domain IIIα of the transposase comprises the substrate binding pocket of the active site which interacts with the Mu-host junction.

A tetramer of the Mu transposase is the structural and functional core in all three stable higher-order nucleoprotein complexes (Type 0, Type 1 and Type 2 transpososomes) generated in a defined in vitro strand transfer reaction. Although functional in donor cleavage, we report here that contrary to previous belief, the Mu A tetramer is incapable of unassisted strand transfer. The Mu B protein is required to stimulate the tetramer for intermolecular strand transfer. In the absence of Mu B protein we show that additional Mu A molecules must be added to the core tetramer to stimulate intramolecular strand transfer. Mapping experiments indicate that domain II of the assisting Mu A mediates functional interactions with the core tetramer. The recipient site for Mu A stimulated strand transfer on the A tetramer is likely in domain II and is clearly different from the domain IIIb site used by the Mu B protein. The Mu accessory end binding sites and the Mu enhancer are not required in the Mu A assisted strand transfer, suggesting that helper A molecules in solution can interact with the core tetramer to stimulate the reaction. Finally, we argue that the strand transfer activity and protein sites for target interaction reside within the core tetramer; hence the role of the stimulatory A molecules appears to be limited to that of an auto-allosteric effector.

A tetramer of the Mu transposase is the structural and functional core in all three stable higher-order nucleoprotein complexes (Type 0, Type 1 and Type 2 transpososomes) generated in a defined in vitro strand transfer reaction. Although functional in donor cleavage, we report here that contrary to previous belief, the Mu A tetramer is incapable of unassisted strand transfer. The Mu B protein is required to stimulate the tetramer for intermolecular strand transfer. In the absence of Mu B protein we show that additional Mu A molecules must be added to the core tetramer to stimulate intramolecular strand transfer. Mapping experiments indicate that domain II of the assisting Mu A mediates functional interactions with the core tetramer. The recipient site for Mu A stimulated strand transfer on the A tetramer is likely in domain II and is clearly different from the domain IIIb site used by the Mu B protein. The Mu accessory end binding sites and the Mu enhancer are not required in the Mu A assisted strand transfer, suggesting that helper A molecules in solution can interact with the core tetramer to stimulate the reaction. Finally, we argue that the strand transfer activity and protein sites for target interaction reside within the core tetramer; hence the role of the stimulatory A molecules appears to be limited to that of an auto-allosteric effector.

A tetramer of the Mu transposase is the structural and functional core in all three stable higher-order nucleoprotein complexes (Type 0, Type 1 and Type 2 transpososomes) generated in a defined in vitro strand transfer reaction. Although functional in donor cleavage, we report here that contrary to previous belief, the Mu A tetramer is incapable of unassisted strand transfer. The Mu B protein is required to stimulate the tetramer for intermolecular strand transfer. In the absence of Mu B protein we show that additional Mu A molecules must be added to the core tetramer to stimulate intramolecular strand transfer. Mapping experiments indicate that domain II of the assisting Mu A mediates functional interactions with the core tetramer. The recipient site for Mu A stimulated strand transfer on the A tetramer is likely in domain II and is clearly different from the domain IIIb site used by the Mu B protein. The Mu accessory end binding sites and the Mu enhancer are not required in the Mu A assisted strand transfer, suggesting that helper A molecules in solution can interact with the core tetramer to stimulate the reaction. Finally, we argue that the strand transfer activity and protein sites for target interaction reside within the core tetramer; hence the role of the stimulatory A molecules appears to be limited to that of an auto-allosteric effector.