Andrew Kaplan - US grants

The project outlined in this proposal is designed to complement earlier research I have performed on the biology of the replication of the human immunodeficiency virus, type 1 (HIV-1) with training in the techniques of molecular biology. My goal is to learn to apply these techniques to answer questions regarding the encapsidation of the viral genomic RNA. Specifically, I plan to subject both the putative RNA packaging signal as well as the nucleocapsid protein to random mutagenesis. Mutants will be pooled and selected for viability simply by their ability to grow in culture. Their genomes will be recovered by PCR amplification of infected cells. Sequence information will point to either nucleotides, in the case of the RNA packaging signal, or amino acids that are intolerant of substitution. In this way I hope to identify those residues which play important structural roles in RNA packaging. Given the impact of HIV infection and the crucial role RNA packaging plays in the formation of infectious particles, an understanding of the mechanisms by which HIV recognizes its genomic RNA is an important health-related aspect of this work.

The project outlined in this proposal is designed to complement earlier research I have performed on the biology of the replication of the human immunodeficiency virus, type 1 (HIV-1) with training in the techniques of molecular biology. My goal is to learn to apply these techniques to answer questions regarding the encapsidation of the viral genomic RNA. Specifically, I plan to subject both the putative RNA packaging signal as well as the nucleocapsid protein to random mutagenesis. Mutants will be pooled and selected for viability simply by their ability to grow in culture. Their genomes will be recovered by PCR amplification of infected cells. Sequence information will point to either nucleotides, in the case of the RNA packaging signal, or amino acids that are intolerant of substitution. In this way I hope to identify those residues which play important structural roles in RNA packaging. Given the impact of HIV infection and the crucial role RNA packaging plays in the formation of infectious particles, an understanding of the mechanisms by which HIV recognizes its genomic RNA is an important health-related aspect of this work.

The objectives of this proposal are twofold: First, to identify and characterize resistant variants of the Human immunodeficiency virus, type 1 (HIV-1) as they appear in patients treated with an inhibitor of the viral protease. Second, to assess the impact these isolates might have on disease progression. The HIV-1 protease plays a crucial role in viral replication. The core particle of the virus is composed of structural and enzymatic proteins which are translated as polyprotein precursors; these precursors are cleaved by the protease during core assembly. Accurate and complete processing is essential in the formation of infectious particles and, for this reason, the protease has been intensively studied as a potential target for therapeutic intervention. Several protease inhibitors have been developed which prevent precursor processing in vitro. HIV-1 infected cells treated with these inhibitors in culture produce non-infectious particles composed of unprocessed precursors, and, recently, several inhibitors of the protease have entered clinical trials. Among the likely obstacles facing this new class of anti-retroviral agents is the development of resistance. Virus variants resistant to inhibitors of the HIV-1 reverse transcriptase are readily recovered from patients on therapy. In order to evaluate the potential for resistance to the protease inhibitors, we initiated a cell culture selection scheme to produce variants of HIV-1 which are less susceptible to the effects of one of these inhibitors. We identified several mutations in the protease coding domain which, when introduced into the purified enzyme, decreased its sensitivity by as much as 60-fold. We have also carried out an analysis of the role played by the protease in assembly of the viral core particle and demonstrated that subtle defects in protease activity result in profound deficits in virus assembly and infectively. Finally, we attempted to define the mechanism by which protease-deficient HIV-1 particles lose infectively. We found that although virus particles composed of unprocessed precursors do not synthesize viral DNA upon infection of susceptible cells, the are capable of efficiently reverse transcribing their RNA genome. This suggests that the infection of these cells is inhibited at a step before reverse transcription. Although, our preliminary results indicate that viruses encoding proteases with decreased susceptibility appear after a limited period of selection in cell culture, the role for these variants in disease progression is uncertain. We will define the range of mutations which result in protease inhibitor resistance in a group of HIV-1 infected patients enrolled in a Phase II trial of a new inhibitor. In addition, we will clarify the impact these substitutes have on HIV-1 pathogenicity. We believe that the information gained by these studies will be useful in understanding the mechanism of protease inhibitor resistance, in the rational design of new protease inhibitors, and in optimizing therapy for individual patients.

DESCRIPTION: The applicant has designed a Listeria monocytogenes-based vector system that expresses heterologous proteins. Unlike other bacterial vaccine systems, Listeria replicates in the cytoplasm of infected cells. This aspect of its life cycle, combined with the organism's ability to induce IL-12 secretion by macrophages leads to the induction of a strong CTL response. The applicant has used this system to elicit protective immunity in three animal models of viral disease, lymphocytic choriomeningitis virus infection of mice as well as HTLV-1 and cottontail rabbit papilloma virus infection of rabbits. Through a collaborative effort composed of researchers at the UCLA School of Medicine, the UC Davis Regional Primate Center, and Duke University, the applicant now proposes to apply insights gained from these preliminary studies to an evaluation of the rhesus macaque immune response to recombinant attenuated strains of Listeria that express a cocktail of SIV proteins. Despite the almost uniformly fatal course of HIV infection, animal models and studies of infected humans suggest that immune responses are capable of altering the tempo of viral replication. However, demonstration of an effective response has been largely limited to early, acute infection and in small trials of immune modulating therapies. Therefore, an understanding of the immune response to retroviral proteins is critical in attempts to design vaccines and anti-retroviral immune based interventions. The generation of CTL plays an important role in controlling retroviral infection: the ability to elicit a cell mediated immune response is an important component of any vaccine strategy. The applicant will focus on optimizing immunization protocols, both in terms of attenuation strategy and route of delivery, with the goal of inducing a mucosal immune response that limits viral replication following vaginal challenge. Specifically, the applicant will : (1) optimize attenuated L. Monocytogenes vectors expressing SIV proteins; (2) evaluate the immune response of rhesus macaques infected with the recombinant Listeria vectors; and (3) assess the ability of immunization strategies to alter virus replication.

DESCRIPTION: The applicant has designed a Listeria monocytogenes-based vector system that expresses heterologous proteins. Unlike other bacterial vaccine systems, Listeria replicates in the cytoplasm of infected cells. This aspect of its life cycle, combined with the organism's ability to induce IL-12 secretion by macrophages leads to the induction of a strong CTL response. The applicant has used this system to elicit protective immunity in three animal models of viral disease, lymphocytic choriomeningitis virus infection of mice as well as HTLV-1 and cottontail rabbit papilloma virus infection of rabbits. Through a collaborative effort composed of researchers at the UCLA School of Medicine, the UC Davis Regional Primate Center, and Duke University, the applicant now proposes to apply insights gained from these preliminary studies to an evaluation of the rhesus macaque immune response to recombinant attenuated strains of Listeria that express a cocktail of SIV proteins. Despite the almost uniformly fatal course of HIV infection, animal models and studies of infected humans suggest that immune responses are capable of altering the tempo of viral replication. However, demonstration of an effective response has been largely limited to early, acute infection and in small trials of immune modulating therapies. Therefore, an understanding of the immune response to retroviral proteins is critical in attempts to design vaccines and anti-retroviral immune based interventions. The generation of CTL plays an important role in controlling retroviral infection: the ability to elicit a cell mediated immune response is an important component of any vaccine strategy. The applicant will focus on optimizing immunization protocols, both in terms of attenuation strategy and route of delivery, with the goal of inducing a mucosal immune response that limits viral replication following vaginal challenge. Specifically, the applicant will : (1) optimize attenuated L. Monocytogenes vectors expressing SIV proteins; (2) evaluate the immune response of rhesus macaques infected with the recombinant Listeria vectors; and (3) assess the ability of immunization strategies to alter virus replication.

Studies presented at the recent International AIDS Conference demonstrate that combination therapy with inhibitors of the viral protease and reverse transcriptase produce profound and durable declines in the viral RNA present in plasma and increases in CD4 counts. Several investigators have even proposed that long term suppression with agents now available may result in eradication of virus and cure of disease. However, although these regimens produce very powerful antiviral effects in the short term, several features of drug dosing and medication adherence, viral replication, as well as pharmacokinetic variability may prevent the realization of this goal. First, the regimens require the administration of between 20 to 30 pills/day and must be taken over the course of at least several years. Second, variants resistant to all of these drugs have been described. Third, there is considerable variability both between and within individuals with regard to the drug levels maintained with the same does. Therefore, a major concern is the ability of HIV-infected people to take this large number of medications regularly over the long term; less than perfect adherence will no doubt be manifested at some time by the majority of patients. Fluctuating drug levels due to variable pharmacokinetics are also expected. We believe that over the long course of therapy anticipated for these compounds, each of these variables will play a role in determining their overall effectiveness. It is the impact of these factors on treatment efficacy and the development of antiviral resistance that is the focus of the studies proposed in this application. Specifically, we will: I. Test the hypothesis that altered patterns of medication adherence with protease inhibitors lead to the development of resistance and define the level of medication adherence necessary to maintain a clinically relevant antiviral effect. II. Identify the barriers to medication adherence. III. Define patient factors that influence the concentrations of protease inhibitors using minimally-invasive strategies.

The UCLA CFAR Virology Core Laboratory has provided HIV-related tissue culture and molecular biology support for UCLA researchers since 1991. The Core has served as a resource for studies of HIV pathogenesis and the associated immune dysfunction. Specifically, the Core acts as a focal point for development and application of novel technologies in HIV molecular virologic assessment and as a bridge between basic and clinical studies conducted at UCLA. The Core also provides training to new investigators in HIV biosafety procedures. The overall purpose of the Core is to provide state of the art techniques of molecular virologic assessment as well as traditional cell culture methods to UCLA researchers. By making the assays and facilities of the Virology Core Facility available to all investigators at UCLA who are studying any aspect of HIV infection, we have successfully fostered interactions between scientists in several departments at the UCLA School of Medicine as well as the Schools of Public Health and Dentistry. Specifically, we will: 1. Maintain storage facilities and serum repositories for UCLA investigators. 2. Provide a biosafety level 3 facility for UCLA investigators. 3. Provide training in the procedures required for biosafety level facilities for UCLA investigators. 4. Introduce novel assays for use by UCLA investigators. 5. Promote the use of our assays and facilities by clinical, basic, public health and behavioral investigators.

DESCRIPTION: (Applicant's Abstract) The treatment of HIV-infected people within prison poses a significant challenge. A minimum of 5 percent of the HIV infected people in the United States are incarcerated (1,2) and a significant proportion of these have a history of drug, and/or alcohol dependence (13). Preliminary observations suggest that HIV-infected prison inmates have much higher AIDS-related mortality than matched populations outside of prison (3). Although highly active antiretroviral therapy (HAART) holds great promise in improving important clinical outcomes, difficulties with adherence appear to play a significant role in treatment failure. Directly observed therapy (DOT) is a potential mechanism to promote adherence. In the North Carolina Department of Corrections, all inmates on HAART receive HIV protease inhibitors by DOT. Although DOT is an attractive strategy, neither the level of adherence to HAART in prison nor the efficacy of DOT has been extensively evaluated. Our preliminary studies demonstrate that DOT has significant untoward effects for inmates and may be of limited utility in promoting adherence in a prison setting. The potential for a dramatic virologic response as well as the consequences of inadequate adherence for both the individual inmate and for transmission of resistant viruses makes an understanding of the adherence in this setting especially urgent. In a collaborative effort between investigators at the Centers for AIDS Research of UNC and UCLA, we will perform a comprehensive assessment of DOT among inmates with a history of substance abuse. We will determine the impact of a history of substance abuse on adherence and access to appropriate antiretroviral therapy. We will also evaluate the effect of a comprehensive adherence-enhancing intervention and document the associations among adherence, treatment failure, and the development of resistance. Specifically, we will: Aim 1. Characterize adherence to HAART among antiretroviral-naive inmates with a history of drug and/or alcohol use incarcerated in the North Carolina Department of Corrections. Aim II. Characterize the effect of a history of drug and/or alcohol use on adherence and the access of inmates to optimal antiretroviral therapy. Aim III. Define the relationship between adherence and viral load in antiretroviral-naive inmates.

DESCRIPTION (provided by applicant): A unique feature of all retroviruses studied to date is that they encapsidate a dimer of identical positive-strand genomic RNAs. The dimerization of retroviral genomic RNA plays a critical role in viral replication; mutations that interfere with the ability of the viral RNA to form dimers have a dramatic effect on infectivity. Despite the invariant nature of this highly specific phenomenon across all retroviral systems, important biological and structural questions remain unanswered. First, the precise role(s) played by dimerization in the viral life cycle is obscure. The region of the viral genome that encompasses the dimer linkage structure contains cis-acting signals that are indispensable for splicing, translation and packaging of the full length viral RNA. Studies performed in our laboratories as well as those reported by other groups suggest that the ability of the RNA to form dimers may impact on any or all of these steps in viral replication. Second, neither the mechanism by which dimers form nor the structure of the final dimer has been elucidated. Although several groups have suggested that dimerization is a dynamic process involving several determinants, there is significant disagreement regarding the nature of the RNA determinants themselves, the order in which these determinants interact, as well as the structure of the mature dimer. Finally, given these gaps in our knowledge, there is a very limited understanding of the interaction between the process by which dimers arise, the final structure they achieve and the ultimate impact of dimer formation on biological function. We believe our studies will provide important insights into RNA structure and retroviral biology. Specifically, we will: I. Characterize the role played by dimerization of the viral genomic RNA in viral replication. II. Establish a detailed molecular understanding of the RNA structures required for dimerization of the Moloney murine sarcoma virus (MuSV) genomic RNA and the mechanism by which these structures assemble into an active dimer. III. Define the interaction between the structure of the dimer and its function in the viral life cycle.

DESCRIPTION (provided by applicant): Activation of the HIV-1 protease is an essential step in viral replication. As is the case for all retroviral proteases, enzyme activity requires the formation of protease homodimers. Mutations that block dimerization interfere with the protease function; viral variants encoding non-functional enzymes are aberrantly assembled and non-infectious. In vitro studies of compounds that inhibit the enzyme from dimerizing produce similar results. Despite the invariant nature of protease dimerization across all retroviral systems, important biological and structural questions remain unanswered. First, although structural studies have identified the residues involved in the dimer interface, the role of specific amino acids in promoting and maintaining dimer formation has not been examined. Second, a wealth of structural and biological information suggests that there is a close association between ordered, protease-mediated precursor processing, particle assembly and infectivity. However, the precise effect of protease dimerization and subsequent enzyme activation on particle assembly and infectivity is unclear. Finally, although clinically available substrate-based inhibitors of the HIV protease have made a dramatic impact on disease progression, resistant variants frequently arise in patients treated with these active site-directed compounds. Novel approaches to inhibitor design are urgently needed to develop additional effective therapeutic agents. In the studies described below, we will define the interactions critical for maintenance of the HIV protease dimer and the role that the final structure plays in viral replication. Further, we will explore novel strategies for inhibition of the viral protease through disrupting dimer formation. Overall, our studies will provide important insights into protease structure, enzyme function and retroviral biology. Specifically, we will: I. Define the role of individual amino acids in dimer formation. II. Characterize the effect of substitutions in the F-IIV protease dimer interface on viral replication. IlI. Characterize HIV protease activation within GagPol as a potential target for inhibition.

DESCRIPTION (provided by applicant): The introduction of combination anti-HIV therapy has produced dramatic declines in mortality and morbidity among people with HIV. Unfortunately, a significant number of patients will experience a rebound in their disease within two years of starting therapy. In many cases this virologic failure is associated with the appearance of HIV variants encoding mutations that render them resistant to the available therapies. Of additional concern is the observation that these resistant variants are found with some frequency in newly infected individuals who have never received therapy, indicating that they can spread from one person to another. This development and subsequent dissemination of resistant viruses reflects a complicated interplay among aspects of the molecular biology of HIV, the behavior of HIV-infected people and the economic and social conditions in which people with H/V/AIDS find themselves. The research goals of this proposal are to address the question "What types of behaviors are most likely to lead to the failure of combination antiretroviral therapy?" Through these studies, I will begin to integrate my laboratory, health services and social science research interests. My mentoring goals are to: 1) Provide training and support to trainees and junior faculty as they advance to research independence 2) Develop a research curriculum related to behavioral medicine. I will devote particular attention to advancing my expertise in psychometric studies. The K24 award will allow me protected time and resources to explore this series of projects and to mentor the next generation of clinical researchers.

Description: The primary focus of this proposal is the identification of amino acid interactions that are critical for the formation of stable HIV-1 reverse transcriptase (RT) heterodimers. A detailed understanding of these interactions will potentially provide a basis for the rational design of new classes of therapeutically useful inhibitors for the processes involved in forming an active HIV-1 RT. The proposed experiments will distinguish between interactions critical for 1) protein folding, 2) stability of the folded protein structure, and 3) formation of the p66/p51 heterodimer. Approaches to the identification of peptide inhibitors for various stages in the synthesis and maturation of the RT heterodimer will also be investigated. The experimental approach will rely on efficient methods for production and screening of specific amino acid replacement mutations. The pro-pol-int coding sequence of HIV-1 is expressed in E. coli where it is processed by the encoded protease to produce p66/p51 heterodimer that is indistinguishable from that produced in the infected human cell. Mutant design will be guided by the 3-dimensional X-ray structure of the RT, and by previous mutational analysis which has identified amino acid residues critical for folding and/or stability. To identify interactions critical for dimerization, we have developed a simple assay for stable heterodimers which can be performed on crude extracts of E. coil expressing the RI. The C-terminus of the p66 subunit is tagged with six His residues so that it can be retained on nickel spin columns. The bound RT is eluted, analyzed by Western blotting, and visualized with an anti-RI monoclonal antibody. The wild-type RT shows both p66 and also p51 which is retained on the column in a stable dimer with the His tagged p66. Mutants that destabilize the dimer show little or no bound p51. Mutants at critical residues that produce reduced amounts of correctly folded active RT will be studied to determine whether the residue is critical only during the folding process, or for stability of the correctly folded product. It is expected that residues involved in important steps in the folding process can be identified in this way. Computer modeling of the structures of related RTs (HIV-2, SIV and others) will be performed to study the degree of evolutionary conservation of interactions critical for folding, stability, and dimerization of RT. These RTs will be cloned in our expression system to allow direct study of conserved critical interactions.

DESCRIPTION (provided by applicant): Activation of the HIV-1 protease (PR) is an essential step in viral replication. Although clinically available substrate-based PR inhibitors have made a dramatic impact on disease progression, resistant variants frequently arise in patients treated with these active site-directed compounds. The utility of these agents is further limited by the phenomenon of cross-resistance between members of the same class. This problem appears to be especially acute for the agents that target the PR active site. Given the increasing prevalence of antiviral resistance, the somewhat limited efficacy of salvage regimens and the problems related to cross-resistance, novel approaches to inhibitor design are urgently needed. Although the available inhibitors of the HIV PR were developed using the 99 amino acid mature protease as a target, the PR is initially translated as part of the 160 kDa GagPol precursor. Processing of this precursor by the viral PR is a critical step in viral replication. As is the case for all retroviral PRs, HIV PR is only active as a homodimer and this embedded, immature PR is sufficient for precursor processing. The functional consequence of this arrangement is that the PR domains of two GagPol precursors must dimerize, become activated and begin to process the precursor in order for the viral life cycle to proceed. Mutations that interfere with the activation of the PR within GagPol have a profound effect on infectivity. We propose to develop an ELISA-based high throughput screen (HTS) for identifying compounds that inhibit the activation of the HIV PR embedded within the GagPol precursor. This system is based on our ability to express full length GagPol and monitor PR activation by measuring the release of an epitope tag. Specifically, we will: 1. Increase the sensitivity of our detection system for compounds that inhibit activity of the HIV PR embedded within GagPol. 2. Decrease the background activation of the GagPol PR.

DESCRIPTION (provided by applicant): Activation of the HIV-1 protease (PR) is an essential step in viral replication. Although clinically available substrate-based PR inhibitors have made a dramatic impact on disease progression, resistant variants frequently arise in patients treated with these active site-directed compounds. The utility of these agents is further limited by the phenomenon of cross-resistance between members of the same class. This problem appears to be especially acute for the agents that target the PR active site. Given the increasing prevalence of antiviral resistance, the somewhat limited efficacy of salvage regimens and the problems related to cross-resistance, novel approaches to inhibitor design are urgently needed. Although the available inhibitors of the HIV PR were developed using the 99 amino acid mature protease as a target, the PR is initially translated as part of the 160 kDa GagPol precursor. Processing of this precursor by the viral PR is a critical step in viral replication. As is the case for all retroviral PRs, HIV PR is only active as a homodimer and this embedded, immature PR is sufficient for precursor processing. The functional consequence of this arrangement is that the PR domains of two GagPol precursors must dimerize, become activated and begin to process the precursor in order for the viral life cycle to proceed. Mutations that interfere with the activation of the PR within GagPol have a profound effect on infectivity. We propose to develop an ELISA-based high throughput screen (HTS) for identifying compounds that inhibit the activation of the HIV PR embedded within the GagPol precursor. This system is based on our ability to express full length GagPol and monitor PR activation by measuring the release of an epitope tag. Specifically, we will: 1. Increase the sensitivity of our detection system for compounds that inhibit activity of the HIV PR embedded within GagPol. 2. Decrease the background activation of the GagPol PR.