This laboratory has been studying the cellular and molecular processes and host factors that control chemokine structure and function with an emphasis on chemokines and cytokines that effect HIV-1 replication. These studies have identified key cytokines that regulate chemokine and chemokine receptor expression and function that ultimately influence both susceptibility to and outcome of HIV infection in T cells and macrophages. Natural forms of chemokines secreted from normal lymphocytes and macrophages under different cell activation conditions are being purified from cells and culture supernatants and then biochemically characterized. The fine structure of chemokines were determined using several techniques including gel electrophoresis, peptide mapping and mass spectrometry. From these studies we have characterized several common modifications to chemokines and have identified key enzyme dependent pathways that are responsible for the N-terminal processing. Processed forms of chemokines show changes in receptor interactions compared with full-length forms. Chemokines can either lose activity or in some cases gain activity after processing. One key enzyme controlling chemokine structure is the dipeptidase CD26, which removes two amino acids from the protein. We have studied the cellular expression pattern of this enzyme and have correlated enzyme expression with changes in secreted chemokine structure. Other peptidases are being studied which function together with CD26 in regulating chemokine activity. These pathways regulate differential cell migration during inflammatory and immune responses as well as control the structure and function of chemokines that inhibit HIV-1 infection. Understanding the natural processing mechanisms that metabolize endogenous and therapeutic chemokines which may change the biological functions of these proteins and is important to evaluating chemokines proposed for therapeutic and vaccine strategies. Chemokines have a wide spectrum of biological activities that are relevant to cancer and AIDS therapies and vaccines. These studies help to identify key structural parameters that need to be monitored in therapeutic chemokine clinical trials. In addition, these biochemical studies may identify new forms of chemokines with either enhanced or antagonistic biological activities. Truncated chemokine antagonists may constitute a novel class of anti-inflammatory agents. This laboratory is interested in host factors that regulate early steps in HIV entry. In addition to host factors that control chemokine and chemokine receptor expression, we have studied a novel pathway mediated by amino acid transporters that inhibits HIV replication. Currents effects are focused on understanding the molecule events that mediate these effects. Molecular Mechanisms of HIV-1 Infection and Pathogenesis. After entry of HIV-1 nucleoprotein into the cytoplasm of a target cell, viral DNA is synthesized by the virally encoded viral reverse transcriptase, and forms a preintegration complex that is translocated to the host cell nucleus. The level of virus production is determined to a large extent by the efficiency of these early steps in viral replication. We have continued our studies to understand how antibodies that bind and signal through amino-acid (AA) transporters inhibit early events in HIV-1 infection of T cells. We have focused on two monoclonal antibodies to AA transporters that inhibit virus replication, but with slightly different potencies and effects. These antibodies do not inhibit cell surface CD4, CXCR4 or CCR5 expression and do not block virus binding. Both antibodies decrease the amount of DNA that enters the nucleus and reduce the amount of 2 LTR circles. We have continued to investigate the role of the cytoskeleton in this effect and to work out the mechanism of inhibition. We find that the HIV pre-integration complex is associated with a large cytoskeletal complex that is removed by centrifugation from detergent lysates of the cells. Initial experiments found that HIV DNA was associated with actin and intermediate filaments(IF), but not with tubulin. Following antibody treatment, viral DNA remained associated with actin, but did not co-precipitate with anti-IF antibodies. Experiments to dissect the cytoskeletal role in this phenomenon have been technically difficult experiments to perform, because of the physical properties of an intact cytoskeleton. Presently using conditions that allow the cytoskeleton to be more easily manipulated, we find that HIV DNA is mainly associated with IFs, but does show some binding to microtubular proteins. The results confirm a relationship between AA transporters and intermediate filaments, which controls completion of reverse transcription and nuclear translocation of HIV-1 viral DNA. We have examined the structural properties of IFs following antibody treatment and find that they are not degraded and do not change in total phosphorylation. In addition to experiments on the role of the cytoskeleton, we are beginning to use pseudotyped virus constructs to study this phenomenon. We find that HIV-GFP vectors pseudotyped with VSV G protein are also sensitive to inhibition by anti-AA transporter antibodies. The pseudotyped system should provide a valuable tool to dissect the mechanism of how these antibodies interfere with reverse transcription and nuclear translocation, as well as to gain new insight into cellular host factors involved in these processes. The regulation of reverse transcription and nuclear translocation by a cell surface amino acid transporter identifies a novel pathway impacting early steps of virus replication, which has potential for both therapeutic and prevention strategies. Molecular Regulation of Chemokine Structure and Function. We have continued to study the cellular and molecular processes that control chemokine/receptor structure and function with an emphasis on chemokines that effect HIV-1 replication. Natural forms of chemokines secreted from normal lymphocytes and macrophages under different cell activation conditions are being purified from cells and culture supernatants for further characterization. The fine structure of these proteins are then analyzed using several techniques including gel electrophoresis, peptide mapping and mass spectrometry. We have found that native MIP-1b secreted by activated peripheral blood lymphocytes (PBLs) is a truncated form missing two amino-terminal amino acids MIP-1b(3-69) suggesting that it is processed by dipeptidyl peptidase IV (DPPIV)/CD26. CD26 is a membrane-bound ectopeptidase with dipeptidyl peptidase IV (DPPIV) activity that has diverse functional properties in T cell physiology and in regulation of bioactive peptides. We have recently documented that full length recombinant MIP-1b is processed by CD26/DPPIV to the truncated form and that cleavage can be blocked by DPPIV inhibitory peptides derived from HIV Tat (1-9) or the thromboxane A2 receptor, TAX2-R (1-9). Addition of Tat (1-9) or TAX2-R (1-9) peptides to PBL cultures partially blocks endogenous MIP-1 processing. The kinetics of conversion of MIP-1 to the truncated form in activated PBLs correlates with cell surface expression of CD26. Our results demonstrate that NH2-terminal processing of MIP-1 and possibly other chemokines depends on the balance between CD26/DPPIV enzymatic activity and inhibitory cellular and viral proteins, which ultimately controls cell recruitment and anti-HIV activity during inflammatory and antiviral responses. Novel Vaccine Strategies for Prevention and Treatment of Infectious Disease and Bioterrorism. We have continued to study molecular adjuvants that will potentiate the quality and kinetics of immune responses to vaccines against bioterrorism agents and infectious diseases. We have characterized natural chemokines and have identified modified forms that in combination have potent chemoattractive effects on antigen presenting cells. One particular combination composed of MIP-1 alpha and a MIP-1 beta variant dimer efficiently attracts monocytes and macrophages, the cells that initially engulf vaccine antigens. Cytokines in combination with DNA adjuvants have also been tested for effects on potentiating differentiation and activation of monocytes into antigen presenting dendritic cells. Certain combinations of cytokines and oligonucleotides have been identified that rapidly induce expression of surface antigens that correlate with dendritic cell differentiation. These mixtures also stimulate high level expression of chemokines that should attract immune cells into vaccination sites. These potent chemokine, cytokine, oligonucleotide adjuvant mixtures will next be combined with vaccines and then tested in small animals. Antibody and cell mediated responses will be evaluated using ELISA assays for specific antibody and cytokine release assays for cell responses. Adjuvants that more effectively attract and activate antigen present cells should have utility for both vaccines strategies targeting both bioterrorism agents and other infectious diseases.
