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Papers by David Davis

Research paper thumbnail of Activity of Human Immunodeficiency Virus Type 1 Protease Inhibitors against the Initial Autocleavage in Gag-Pol Polyprotein Processing

Antimicrobial Agents and Chemotherapy, 2012

Inhibitors of HIV protease have proven to be important drugs in combination anti-HIV therapy. The... more Inhibitors of HIV protease have proven to be important drugs in combination anti-HIV therapy. These inhibitors were designed to target mature protease and prevent viral particle maturation by blocking Gag and Gag-Pol processing by mature protease. Currently there are few data assessing the ability of these protease inhibitors to block the initial step in autoproteolytic processing of Gag-Pol. This unique step involves the dimerization of two Gag-Pol polyproteins and autocleavage of the Gag-Pol polyprotein by the embedded dimeric protease. We developed a plasmid encoding a modified form of Gag-Pol that can undergo autoprocessing only at the initial cleavage site between p2 and nucleocapsid. Using an in vitro transcription/translation system, we assessed the ability of six different approved protease inhibitors (darunavir, indinavir, nelfinavir, ritonavir, saquinavir, and tipranavir) to block this initial autocleavage step. Of these inhibitors, darunavir and saquinavir were the most effective. Darunavir and saquinavir were also the most effective at blocking the initial autoprocessing of full-length Gag-Pol in HIV-1-infected T cells. Thus, we have identified at least two HIV-1 protease inhibitors that have activity against the primary autocatalytic step of the embedded HIV-1 protease in Gag-Pol at concentrations that may be attained in HIV-1-infected patients. Due to unique aspects of the initial processing step, it may be possible to develop inhibitors with greater potency against this step, thus halting viral maturation at the earliest stages. The transcription/translation assay could be used to develop more potent inhibitors of this essential first step in viral maturation.

Research paper thumbnail of Identification of Caspase Cleavage Sites in KSHV Latency-Associated Nuclear Antigen and Their Effects on Caspase-Related Host Defense Responses

PLoS pathogens, 2015

Kaposi's sarcoma-associated herpesvirus (KSHV), also known as human herpesvirus-8, is the cau... more Kaposi's sarcoma-associated herpesvirus (KSHV), also known as human herpesvirus-8, is the causative agent of three hyperproliferative disorders: Kaposi's sarcoma, primary effusion lymphoma (PEL) and multicentric Castleman's disease. During viral latency a small subset of viral genes are produced, including KSHV latency-associated nuclear antigen (LANA), which help the virus thwart cellular defense responses. We found that exposure of KSHV-infected cells to oxidative stress, or other inducers of apoptosis and caspase activation, led to processing of LANA and that this processing could be inhibited with the pan-caspase inhibitor Z-VAD-FMK. Using sequence, peptide, and mutational analysis, two caspase cleavage sites within LANA were identified: a site for caspase-3 type caspases at the N-terminus and a site for caspase-1 and-3 type caspases at the C-terminus. Using LANA expression plasmids, we demonstrated that mutation of these cleavage sites prevents caspase-1 and caspase...

Research paper thumbnail of Next-Generation Sequencing Analysis Reveals Differential Expression Profiles of MiRNA-mRNA Target Pairs in KSHV-Infected Cells

PLOS ONE, 2015

Kaposi&am... more Kaposi's sarcoma associated herpesvirus (KSHV) causes several tumors, including primary effusion lymphoma (PEL) and Kaposi's sarcoma (KS). Cellular and viral microRNAs (miRNAs) have been shown to play important roles in regulating gene expression. A better knowledge of the miRNA-mediated pathways affected by KSHV infection is therefore important for understanding viral infection and tumor pathogenesis. In this study, we used deep sequencing to analyze miRNA and cellular mRNA expression in a cell line with latent KSHV infection (SLKK) as compared to the uninfected SLK line. This approach revealed 153 differentially expressed human miRNAs, eight of which were independently confirmed by qRT-PCR. KSHV infection led to the dysregulation of ~15% of the human miRNA pool and most of these cellular miRNAs were down-regulated, including nearly all members of the 14q32 miRNA cluster, a genomic locus linked to cancer and that is deleted in a number of PEL cell lines. Furthermore, we identified 48 miRNAs that were associated with a total of 1,117 predicted or experimentally validated target mRNAs; of these mRNAs, a majority (73%) were inversely correlated to expression changes of their respective miRNAs, suggesting miRNA-mediated silencing mechanisms were involved in a number of these alterations. Several dysregulated miRNA-mRNA pairs may facilitate KSHV infection or tumor formation, such as up-regulated miR-708-5p, associated with a decrease in pro-apoptotic caspase-2 and leukemia inhibitory factor LIF, or down-regulated miR-409-5p, associated with an increase in the p53-inhibitor MDM2. Transfection of miRNA mimics provided further evidence that changes in miRNAs are driving some observed mRNA changes. Using filtered datasets, we also identified several canonical pathways that were significantly enriched in differentially expressed miRNA-mRNA pairs, such as the epithelial-to-mesenchymal transition and the interleukin-8 signaling pathways. Overall, our data provide a more detailed understanding of KSHV latency and guide further studies of the biological significance of these changes.

Research paper thumbnail of A conserved hydrogen-bonding network of P2 bis-tetrahydrofuran-containing HIV-1 protease inhibitors (PIs) with a protease active-site amino acid backbone aids in their activity against PI-resistant HIV

Antimicrobial agents and chemotherapy, 2014

In the present study, GRL008, a novel nonpeptidic human immunodeficiency virus type 1 (HIV-1) pro... more In the present study, GRL008, a novel nonpeptidic human immunodeficiency virus type 1 (HIV-1) protease inhibitor (PI), and darunavir (DRV), both of which contain a P2-bis-tetrahydrofuranyl urethane (bis-THF) moiety, were found to exert potent antiviral activity (50% effective concentrations [EC50s], 0.029 and 0.002 μM, respectively) against a multidrug-resistant clinical isolate of HIV-1 (HIVA02) compared to ritonavir (RTV; EC50, >1.0 μM) and tipranavir (TPV; EC50, 0.364 μM). Additionally, GRL008 showed potent antiviral activity against an HIV-1 variant selected in the presence of DRV over 20 passages (HIVDRV(R)P20), with a 2.6-fold increase in its EC50 (0.097 μM) compared to its corresponding EC50 (0.038 μM) against wild-type HIV-1NL4-3 (HIVWT). Based on X-ray crystallographic analysis, both GRL008 and DRV showed strong hydrogen bonds (H-bonds) with the backbone-amide nitrogen/carbonyl oxygen atoms of conserved active-site amino acids G27, D29, D30, and…

Research paper thumbnail of Systemic neutralizing antibodies induced by long interval mucosally primed systemically boosted immunization correlate with protection from mucosal SHIV challenge

This article appeared in a journal published by Elsevier. The attached copy is furnished to the a... more This article appeared in a journal published by Elsevier. The attached copy is furnished to the author for internal non-commercial research and education use, including for instruction at the authors institution and sharing with colleagues. Other uses, including reproduction and distribution, or selling or licensing copies, or posting to personal, institutional or third party websites are prohibited. In most cases authors are permitted to post their version of the article (e.g. in Word or Tex form) to their personal website or institutional repository. Authors requiring further information regarding Elsevier's archiving and manuscript policies are encouraged to visit: http://www.elsevier.com/copyright Immune correlates of vaccine protection from HIV-1 infection would provide important milestones to guide HIV-1 vaccine development. In a proof of concept study using mucosal priming and systemic boosting, the titer of neutralizing antibodies in sera was found to correlate with protection of mucosally exposed rhesus macaques from SHIV infection. Mucosal priming consisted of two sequential immunizations at 12-week intervals with replicating host range mutants of adenovirus type 5 (Ad5hr) expressing the HIV-1 89.6p env gene. Following boosting with either heterologous recombinant protein or alphavirus replicons at 12-week intervals animals were intrarectally exposed to infectious doses of the CCR5 tropic SHIV SF162p4. Heterologous mucosal prime systemic boost immunization elicited neutralizing antibodies (Nabs), antibody-dependent cytotoxicity (ADCC), and specific patterns of antibody binding to envelope peptides. Vaccine induced protection did not correlate with the type of boost nor T-cell responses, but rather with the Nab titer prior to exposure.

Research paper thumbnail of Analysis of Memory B Cell Responses and Isolation of Novel Monoclonal Antibodies with Neutralizing Breadth from HIV-1-Infected Individuals

PLoS ONE, 2010

The isolation of human monoclonal antibodies (mAbs) that neutralize a broad spectrum of primary H... more The isolation of human monoclonal antibodies (mAbs) that neutralize a broad spectrum of primary HIV-1 isolates and the characterization of the human neutralizing antibody B cell response to HIV-1 infection are important goals that are central to the design of an effective antibody-based vaccine. We immortalized IgG(+) memory B cells from individuals infected with diverse clades of HIV-1 and selected on the basis of plasma neutralization profiles that were cross-clade and relatively potent. Culture supernatants were screened using various recombinant forms of the envelope glycoproteins (Env) in multiple parallel assays. We isolated 58 mAbs that were mapped to different Env surfaces, most of which showed neutralizing activity. One mAb in particular (HJ16) specific for a novel epitope proximal to the CD4 binding site on gp120 selectively neutralized a multi-clade panel of Tier-2 HIV-1 pseudoviruses, and demonstrated reactivity that was comparable in breadth, but distinct in neutralization specificity, to that of the other CD4 binding site-specific neutralizing mAb b12. A second mAb (HGN194) bound a conserved epitope in the V3 crown and neutralized all Tier-1 and a proportion of Tier-2 pseudoviruses tested, irrespective of clade. A third mAb (HK20) with broad neutralizing activity, particularly as a Fab fragment, recognized a highly conserved epitope in the HR-1 region of gp41, but showed striking assay-dependent selectivity in its activity. This study reveals that by using appropriate screening methods, a large proportion of memory B cells can be isolated that produce mAbs with HIV-1 neutralizing activity. Three of these mAbs show unusual breadth of neutralization and therefore add to the current panel of HIV-1 neutralizing antibodies with potential for passive protection and template-based vaccine design.

Research paper thumbnail of The Initial Step in Human Immunodeficiency Virus Type 1 GagProPol Processing Can Be Regulated by Reversible Oxidation

PLoS ONE, 2010

Background: Maturation of human immunodeficiency virus type 1 (HIV-1) occurs upon activation of H... more Background: Maturation of human immunodeficiency virus type 1 (HIV-1) occurs upon activation of HIV-1 protease embedded within GagProPol precursors and cleavage of Gag and GagProPol polyproteins. Although reversible oxidation can regulate mature protease activity as well as retrovirus maturation, it is possible that the effects of oxidation on viral maturation are mediated in whole, or part, through effects on the initial intramolecular cleavage event of GagProPol. In order assess the effect of reversible oxidation on this event, we developed a system to isolate the first step in protease activation involving GagProPol.

Research paper thumbnail of Reversible Oxidative Modification as a Mechanism for Regulating Retroviral Protease Dimerization and Activation

Journal of Virology, 2003

Human immunodeficiency virus protease activity can be regulated by reversible oxidation of a sulf... more Human immunodeficiency virus protease activity can be regulated by reversible oxidation of a sulfurcontaining amino acid at the dimer interface. We show here that oxidation of this amino acid in human immunodeficiency virus type 1 protease prevents dimer formation. Moreover, we show that human T-cell leukemia virus type 1 protease can be similarly regulated through reversible glutathionylation of its two conserved cysteine residues. Based on the known three-dimensional structures and multiple sequence alignments of retroviral proteases, it is predicted that the majority of retroviral proteases have sulfur-containing amino acids at the dimer interface. The regulation of protease activity by the modification of a sulfurcontaining amino acid at the dimer interface may be a conserved mechanism among the majority of retroviruses.

Research paper thumbnail of Transmission of Simian Immunodeficiency Virus Carrying Multiple Cytotoxic T-Lymphocyte Escape Mutations with Diminished Replicative Ability Can Result in AIDS Progression in Rhesus Macaques

Journal of Virology, 2008

Cytotoxic T-lymphocyte (CTL) responses frequently select for immunodeficiency virus mutations tha... more Cytotoxic T-lymphocyte (CTL) responses frequently select for immunodeficiency virus mutations that result in escape from CTL recognition with viral fitness costs. The replication in vivo of such viruses carrying not single but multiple escape mutations in the absence of the CTL pressure has remained undetermined. Here, we have examined the replication of simian immunodeficiency virus (SIV) with five gag mutations selected in a macaque possessing the major histocompatibility complex haplotype 90-120-Ia after its transmission into 90-120-Ia-negative macaques. Our results showed that even such a "crippled" SIV infection can result in persistent viral replication, multiple reversions, and AIDS progression.

Research paper thumbnail of Design and evaluation of an in-house HIV-1 (group M and O), SIVmnd and SIVcpz antigen capture assay

Journal of Virological Methods, 1998

... To design an in-house antigen capture ... positive sample signals were obtained when using pu... more ... To design an in-house antigen capture ... positive sample signals were obtained when using purified IgG 1 for coating at a concentration of 4 μg/well and purified and conjugated IgG 2 for detection at a concentration of 5 μg ml −1 . The anti-p24 titer for IgG 1 was 1/10 000 and 1 ...

Research paper thumbnail of The HIV-2 genotype and the HIV-1 syncytium-inducing phenotype are associated with a lower virus replication in dendritic cells

Journal of Medical Virology, 2000

During sexual transmission, HIV infects the mucosal dendritic cells and is transferred to CD4 T c... more During sexual transmission, HIV infects the mucosal dendritic cells and is transferred to CD4 T cells. Whether HIV variants of a particular genetic (sub)type or phenotype selectively infect dendritic cells (DC) or are preferentially transferred to T cells remains highly controversial. To avoid the cumbersome use of primary dendritic cells, in vitro dendritic cell models were generated from precursors, either hematopoietic progenitor cells (HPC) or monocytes (MO). Productive infection in the dendritic cells and transfer of the virus to T cells was assessed for a range of HIV variants. HPC-derived dendritic cells (HPC-DC) were more susceptible to HIV-1 than to HIV-2 isolates. The HIV-1 group O strains were more productive in HPC-DC than group M, but amongst the latter, no subtype-related difference was observed. Both non-syncytium-inducing (NSI) and SI HIV isolates and lab strains could productively infect HPC-DC, albeit with a different efficiency. Adding blocking antibodies confirmed that both CCR-5 and CXCR-4 co-receptors were functional. Biological HIV-1 clones of the NSI/R5 phenotype infected more readily HPC-DC than SI/X4 clones. MO-derived dendritic cells were, however, more exclusive in their preference for NSI/R5 clones. Some HIV variants, that did not grow readily in HPC-DC alone, could be rescued by adding resting or pre-activated T cells. The present data show that HIV-2 isolates and SI clones replicate less in model-DC, but no preference for a particular HIV-1 subtype was evident. Co-culture with T cells could "correct" a limited growth in dendritic cells. Clearly, both intrinsic dendritic cell susceptibility and enhancement by T cells are explained only partly by HIV genotype and phenotype. The in vitro dendritic cell models seem useful tools to further unravel interactions between HIV, DC, and T cells.

Research paper thumbnail of Serum inactivation contributes to the failure of stromal-derived factor-1 to block HIV-I infection in vivo

Journal of Leukocyte Biology, 2003

The chemokine stromal-derived factor-1 (SDF-1) can block human immunodeficiency virus type 1 (HIV... more The chemokine stromal-derived factor-1 (SDF-1) can block human immunodeficiency virus type 1 (HIV-1) infection in vitro by binding to the CXC chemokine receptor, CXCR-4, which serves as a coreceptor for T cell tropic HIV-1. In spite of being constitutively expressed in vivo, SDF-1 does not appear to block HIV-1 infection and spread in vivo. We report that SDF-1 is consistently measured in normal serum (15.4؎3.0 ng/ ml; mean؎SD) and in serum from AIDS patients (16.6؎3.7 ng/ml). However, we find that circulating SDF-1 is modified to an inactive form. When exposed to serum, recombinant SDF-1 is specifically and rapidly altered to yield an apparently smaller chemokine that does not bind to SDF-1 receptor-expressing cells, does not have chemoattractive or pre-B cell stimulatory activity, and does not block HIV-1 infection. Thus, serum modification and inactivation contribute to the failure of SDF-1 to block HIV-1 infection and spread in man. The inactivation of circulating SDF-1 may be critical in permitting local gradients to develop and direct cell trafficking. J. Leukoc. Biol. 74: 000 -000; 2003.

Research paper thumbnail of Detection of Serum Antibodies to a Kaposi's Sarcoma‐Associated Herpesvirus‐Specific Peptide

The Journal of Infectious Diseases, 1997

Kaposi's sarcoma (KS) -associated herpesvirus/human herpesvirus type 8 (KSHV/HHV-8) may play an e... more Kaposi's sarcoma (KS) -associated herpesvirus/human herpesvirus type 8 (KSHV/HHV-8) may play an etiologic role in the pathogenesis of KS. In an attempt to assess KSHV/HHV-8 infection, an ELISA was developed using an 18 -amino acid peptide from a putative minor capsid protein of KSHV/HHV-8 conjugated to bovine serum albumin. Overall, sera from human immunodeficiency virus type 1 (HIV-1) -positive patients with KS had a higher reactivity in the assay than did sera from HIV-1 -positive patients without KS (P Å .018). Of 35 HIV-1 -positive patients with KS, 60% were antibody positive, compared with 27% of 33 HIV-1 -positive patients without KS. Of 30 healthy blood donors, 20% were antibody positive. The ELISA responses did not correlate with antibody titers to Epstein-Barr virus.

Research paper thumbnail of Kaposi's sarcoma-associated herpesvirus (human herpesvirus 8): epidemiology and pathogenesis

Journal of Antimicrobial Chemotherapy, 2000

The complete ORF54 of the Kaposi's sarcoma-associated herpesvirus (KSHV) (human herp... more The complete ORF54 of the Kaposi's sarcoma-associated herpesvirus (KSHV) (human herpes-virus-8; HHV-8) was cloned and expressed in E. coli. The results show that KSHV/HHV-8 ORF54 encodes a functional dUTPase which specifically hydrolyses dUTP ...

Research paper thumbnail of 130 Inhibition of HIV-1 Release by Cell Permeable Peptides

JAIDS Journal of Acquired Immune Deficiency Syndromes, 2009

Research paper thumbnail of Identification of carboxypeptidase N as an enzyme responsible for C-terminal cleavage of stromal cell-derived factor-1  in the circulation

Blood, 2005

The chemokine stromal-derived factor-1␣ (SDF-1␣) is an essential regulator of hematopoiesis, lymp... more The chemokine stromal-derived factor-1␣ (SDF-1␣) is an essential regulator of hematopoiesis, lymphocyte homing, pre-Bcell growth, and angiogenesis. As SDF-1␣ is constitutively expressed in many tissues, chemokine function is mostly regulated by proteolytic degradation. Human serum cleaves the 68-amino acid chemokine, SDF-1␣, at both termini. The enzyme or enzymes responsible for the removal of the carboxy-terminal lysine from SDF-1␣, leading to significant reduction in biologic activity, have not been identified.

Research paper thumbnail of Regulation of HIV-1 Protease Activity through Cysteine Modification

Biochemistry, 1996

The homodimeric protease of the human immunodeficiency virus 1 contains two cysteine residues per... more The homodimeric protease of the human immunodeficiency virus 1 contains two cysteine residues per monomer which are highly conserved among viral isolates. However, these cysteine residues are not essential for catalytic activity which raises the question of why they are conserved. We have found previously that these cysteine residues are unusually susceptible to oxidation by metal ions, and this results in inhibition of protease activity. Recombinant protease mutants (C67A, C95A, and the double mutant C67A,C95A) were prepared to assess the possible role of these cysteines in redox regulation of the enzyme. Mixed disulfides were formed between the cysteine residues of the enzymes and low molecular weight thiols. Enzyme activity was lost when a mixed disulfide was formed between 5,5′-dithiobis(2nitrobenzoic acid) and cysteine 95, while the same mixed disulfide at cysteine 67 reduced activity by 50%. This effect was reversible as normal activity could be restored when the enzyme was treated with dithiothreitol. The cysteines could also be modified with the common cellular thiol glutathione. Modification with glutathione was verified by mass spectrometry of the protein peaks obtained from HPLC separation. Glutathiolation of cysteine 95 abolished activity whereas modification at cysteine 67 increased the k cat by more than 2-fold with no effect on K m . In addition, glutathiolation at cysteine 67 markedly stabilized the enzyme activity presumably by reducing autoproteolysis. These results demonstrate one possible mechanism for regulation of the HIV-1 protease through cysteine modification and identify additional targets for affecting protease activity other than the active site.

Research paper thumbnail of HIV-2 protease is inactivated after oxidation at the dimer interface and activity can be partly restored with methionine sulphoxide reductase

Biochemical Journal, 2000

Human immunodeficiency viruses encode a homodimeric protease that is essential for the production... more Human immunodeficiency viruses encode a homodimeric protease that is essential for the production of infectious virus. Previous studies have shown that HIV-1 protease is susceptible to oxidative inactivation at the dimer interface at Cys-95, a process that can be reversed both chemically and enzymically.

Research paper thumbnail of Inhibition of HIV-1 replication by a peptide dimerization inhibitor of HIV-1 protease

Antiviral Research, 2006

Peptides based on the amino (N) and carboxy (C)-terminal regions of human immunodeficiency virus ... more Peptides based on the amino (N) and carboxy (C)-terminal regions of human immunodeficiency virus type-1 (HIV-1) protease and on the Cterminus of p6 * can inhibit HIV-1 protease activity by preventing dimerization. We developed a peptide dimerization inhibitor, P27, that included these domains and a cell permeable domain derived from HIV-1 Tat. P27 inhibited wild type (WT) and protease inhibitor (PI)-resistant HIV-1 protease (IC 50 : 0.23-0.32 M). Kinetic and biochemical assays confirmed that P27 inhibits protease dimerization. Fluorescein-labeled peptide accumulated in MT-2 cells and protected acutely infected MT-2 cells from HIV-1-induced cytotoxicity (IC 50 : 5.1 M). P27 also inhibited p24 accumulation from H9 and U937 cells chronically infected with WT or PI-resistant HIV-1. Immunoblot analysis on the supernatants and infected cells revealed a block in virus release by P27 rather than an inhibition of polyprotein processing. However, inhibition of p55 Gag processing by active-site inhibitors was enhanced when combined with P27, suggesting that P27 can affect protease function in maturing virions. Although P27 was rationally designed to block dimerization of the mature HIV-1 protease, the effects of P27 on HIV-1 replication may be related to partial inhibition of Gag-Pol processing leading to a disruption in virus release. Published by Elsevier B.V.

Research paper thumbnail of Intravirion Processing of the Human Immunodeficiency Virus Type 1 Vif Protein by the Viral Protease May Be Correlated with Vif Function

Journal of Virology, 2002

The human immunodeficiency virus type 1 (HIV-1) Vif protein is specifically packaged into virus p... more The human immunodeficiency virus type 1 (HIV-1) Vif protein is specifically packaged into virus particles through an interaction with viral genomic RNA in which it associates with the viral nucleoprotein complex. We now demonstrate for the first time that virus-associated Vif is subject to proteolytic processing by the viral protease (Pr). Pr-dependent processing of Vif was observed both in vivo and in vitro. In vivo processing of Vif was cell type independent and evident by the appearance of a 7-kDa processing product, which was restricted to cell-free virus preparations. Processing of Vif required an active viral Pr and was sensitive to Pr inhibitors such as ritonavir. The processing site in Vif was characterized both in vivo and in vitro and mapped to Ala 150 . Interestingly, the Vif processing site is located in a domain that is highly conserved among HIV-1, HIV-2, and simian immunodeficiency virus Vif isolates. Mutations at or near the processing site did not affect protein stability or packaging efficiency but had dramatic effects on Vif processing. In general, mutations that markedly increased or decreased the sensitivity of Vif to proteolytic processing severely impaired or completely abolished Vif function. In contrast, mutations at the same site that had little or no effect on processing efficiency also did not influence Vif function. None of the mutants affected the ability of the virus to replicate in permissive cell lines. Our data suggest that mutations in Vif that cause a profound change in the sensitivity to Pr-dependent processing also severely impaired Vif function, suggesting that intravirion processing of Vif is important for the production of infectious viruses.

Research paper thumbnail of Activity of Human Immunodeficiency Virus Type 1 Protease Inhibitors against the Initial Autocleavage in Gag-Pol Polyprotein Processing

Antimicrobial Agents and Chemotherapy, 2012

Inhibitors of HIV protease have proven to be important drugs in combination anti-HIV therapy. The... more Inhibitors of HIV protease have proven to be important drugs in combination anti-HIV therapy. These inhibitors were designed to target mature protease and prevent viral particle maturation by blocking Gag and Gag-Pol processing by mature protease. Currently there are few data assessing the ability of these protease inhibitors to block the initial step in autoproteolytic processing of Gag-Pol. This unique step involves the dimerization of two Gag-Pol polyproteins and autocleavage of the Gag-Pol polyprotein by the embedded dimeric protease. We developed a plasmid encoding a modified form of Gag-Pol that can undergo autoprocessing only at the initial cleavage site between p2 and nucleocapsid. Using an in vitro transcription/translation system, we assessed the ability of six different approved protease inhibitors (darunavir, indinavir, nelfinavir, ritonavir, saquinavir, and tipranavir) to block this initial autocleavage step. Of these inhibitors, darunavir and saquinavir were the most effective. Darunavir and saquinavir were also the most effective at blocking the initial autoprocessing of full-length Gag-Pol in HIV-1-infected T cells. Thus, we have identified at least two HIV-1 protease inhibitors that have activity against the primary autocatalytic step of the embedded HIV-1 protease in Gag-Pol at concentrations that may be attained in HIV-1-infected patients. Due to unique aspects of the initial processing step, it may be possible to develop inhibitors with greater potency against this step, thus halting viral maturation at the earliest stages. The transcription/translation assay could be used to develop more potent inhibitors of this essential first step in viral maturation.

Research paper thumbnail of Identification of Caspase Cleavage Sites in KSHV Latency-Associated Nuclear Antigen and Their Effects on Caspase-Related Host Defense Responses

PLoS pathogens, 2015

Kaposi's sarcoma-associated herpesvirus (KSHV), also known as human herpesvirus-8, is the cau... more Kaposi's sarcoma-associated herpesvirus (KSHV), also known as human herpesvirus-8, is the causative agent of three hyperproliferative disorders: Kaposi's sarcoma, primary effusion lymphoma (PEL) and multicentric Castleman's disease. During viral latency a small subset of viral genes are produced, including KSHV latency-associated nuclear antigen (LANA), which help the virus thwart cellular defense responses. We found that exposure of KSHV-infected cells to oxidative stress, or other inducers of apoptosis and caspase activation, led to processing of LANA and that this processing could be inhibited with the pan-caspase inhibitor Z-VAD-FMK. Using sequence, peptide, and mutational analysis, two caspase cleavage sites within LANA were identified: a site for caspase-3 type caspases at the N-terminus and a site for caspase-1 and-3 type caspases at the C-terminus. Using LANA expression plasmids, we demonstrated that mutation of these cleavage sites prevents caspase-1 and caspase...

Research paper thumbnail of Next-Generation Sequencing Analysis Reveals Differential Expression Profiles of MiRNA-mRNA Target Pairs in KSHV-Infected Cells

PLOS ONE, 2015

Kaposi&am... more Kaposi's sarcoma associated herpesvirus (KSHV) causes several tumors, including primary effusion lymphoma (PEL) and Kaposi's sarcoma (KS). Cellular and viral microRNAs (miRNAs) have been shown to play important roles in regulating gene expression. A better knowledge of the miRNA-mediated pathways affected by KSHV infection is therefore important for understanding viral infection and tumor pathogenesis. In this study, we used deep sequencing to analyze miRNA and cellular mRNA expression in a cell line with latent KSHV infection (SLKK) as compared to the uninfected SLK line. This approach revealed 153 differentially expressed human miRNAs, eight of which were independently confirmed by qRT-PCR. KSHV infection led to the dysregulation of ~15% of the human miRNA pool and most of these cellular miRNAs were down-regulated, including nearly all members of the 14q32 miRNA cluster, a genomic locus linked to cancer and that is deleted in a number of PEL cell lines. Furthermore, we identified 48 miRNAs that were associated with a total of 1,117 predicted or experimentally validated target mRNAs; of these mRNAs, a majority (73%) were inversely correlated to expression changes of their respective miRNAs, suggesting miRNA-mediated silencing mechanisms were involved in a number of these alterations. Several dysregulated miRNA-mRNA pairs may facilitate KSHV infection or tumor formation, such as up-regulated miR-708-5p, associated with a decrease in pro-apoptotic caspase-2 and leukemia inhibitory factor LIF, or down-regulated miR-409-5p, associated with an increase in the p53-inhibitor MDM2. Transfection of miRNA mimics provided further evidence that changes in miRNAs are driving some observed mRNA changes. Using filtered datasets, we also identified several canonical pathways that were significantly enriched in differentially expressed miRNA-mRNA pairs, such as the epithelial-to-mesenchymal transition and the interleukin-8 signaling pathways. Overall, our data provide a more detailed understanding of KSHV latency and guide further studies of the biological significance of these changes.

Research paper thumbnail of A conserved hydrogen-bonding network of P2 bis-tetrahydrofuran-containing HIV-1 protease inhibitors (PIs) with a protease active-site amino acid backbone aids in their activity against PI-resistant HIV

Antimicrobial agents and chemotherapy, 2014

In the present study, GRL008, a novel nonpeptidic human immunodeficiency virus type 1 (HIV-1) pro... more In the present study, GRL008, a novel nonpeptidic human immunodeficiency virus type 1 (HIV-1) protease inhibitor (PI), and darunavir (DRV), both of which contain a P2-bis-tetrahydrofuranyl urethane (bis-THF) moiety, were found to exert potent antiviral activity (50% effective concentrations [EC50s], 0.029 and 0.002 μM, respectively) against a multidrug-resistant clinical isolate of HIV-1 (HIVA02) compared to ritonavir (RTV; EC50, >1.0 μM) and tipranavir (TPV; EC50, 0.364 μM). Additionally, GRL008 showed potent antiviral activity against an HIV-1 variant selected in the presence of DRV over 20 passages (HIVDRV(R)P20), with a 2.6-fold increase in its EC50 (0.097 μM) compared to its corresponding EC50 (0.038 μM) against wild-type HIV-1NL4-3 (HIVWT). Based on X-ray crystallographic analysis, both GRL008 and DRV showed strong hydrogen bonds (H-bonds) with the backbone-amide nitrogen/carbonyl oxygen atoms of conserved active-site amino acids G27, D29, D30, and…

Research paper thumbnail of Systemic neutralizing antibodies induced by long interval mucosally primed systemically boosted immunization correlate with protection from mucosal SHIV challenge

This article appeared in a journal published by Elsevier. The attached copy is furnished to the a... more This article appeared in a journal published by Elsevier. The attached copy is furnished to the author for internal non-commercial research and education use, including for instruction at the authors institution and sharing with colleagues. Other uses, including reproduction and distribution, or selling or licensing copies, or posting to personal, institutional or third party websites are prohibited. In most cases authors are permitted to post their version of the article (e.g. in Word or Tex form) to their personal website or institutional repository. Authors requiring further information regarding Elsevier's archiving and manuscript policies are encouraged to visit: http://www.elsevier.com/copyright Immune correlates of vaccine protection from HIV-1 infection would provide important milestones to guide HIV-1 vaccine development. In a proof of concept study using mucosal priming and systemic boosting, the titer of neutralizing antibodies in sera was found to correlate with protection of mucosally exposed rhesus macaques from SHIV infection. Mucosal priming consisted of two sequential immunizations at 12-week intervals with replicating host range mutants of adenovirus type 5 (Ad5hr) expressing the HIV-1 89.6p env gene. Following boosting with either heterologous recombinant protein or alphavirus replicons at 12-week intervals animals were intrarectally exposed to infectious doses of the CCR5 tropic SHIV SF162p4. Heterologous mucosal prime systemic boost immunization elicited neutralizing antibodies (Nabs), antibody-dependent cytotoxicity (ADCC), and specific patterns of antibody binding to envelope peptides. Vaccine induced protection did not correlate with the type of boost nor T-cell responses, but rather with the Nab titer prior to exposure.

Research paper thumbnail of Analysis of Memory B Cell Responses and Isolation of Novel Monoclonal Antibodies with Neutralizing Breadth from HIV-1-Infected Individuals

PLoS ONE, 2010

The isolation of human monoclonal antibodies (mAbs) that neutralize a broad spectrum of primary H... more The isolation of human monoclonal antibodies (mAbs) that neutralize a broad spectrum of primary HIV-1 isolates and the characterization of the human neutralizing antibody B cell response to HIV-1 infection are important goals that are central to the design of an effective antibody-based vaccine. We immortalized IgG(+) memory B cells from individuals infected with diverse clades of HIV-1 and selected on the basis of plasma neutralization profiles that were cross-clade and relatively potent. Culture supernatants were screened using various recombinant forms of the envelope glycoproteins (Env) in multiple parallel assays. We isolated 58 mAbs that were mapped to different Env surfaces, most of which showed neutralizing activity. One mAb in particular (HJ16) specific for a novel epitope proximal to the CD4 binding site on gp120 selectively neutralized a multi-clade panel of Tier-2 HIV-1 pseudoviruses, and demonstrated reactivity that was comparable in breadth, but distinct in neutralization specificity, to that of the other CD4 binding site-specific neutralizing mAb b12. A second mAb (HGN194) bound a conserved epitope in the V3 crown and neutralized all Tier-1 and a proportion of Tier-2 pseudoviruses tested, irrespective of clade. A third mAb (HK20) with broad neutralizing activity, particularly as a Fab fragment, recognized a highly conserved epitope in the HR-1 region of gp41, but showed striking assay-dependent selectivity in its activity. This study reveals that by using appropriate screening methods, a large proportion of memory B cells can be isolated that produce mAbs with HIV-1 neutralizing activity. Three of these mAbs show unusual breadth of neutralization and therefore add to the current panel of HIV-1 neutralizing antibodies with potential for passive protection and template-based vaccine design.

Research paper thumbnail of The Initial Step in Human Immunodeficiency Virus Type 1 GagProPol Processing Can Be Regulated by Reversible Oxidation

PLoS ONE, 2010

Background: Maturation of human immunodeficiency virus type 1 (HIV-1) occurs upon activation of H... more Background: Maturation of human immunodeficiency virus type 1 (HIV-1) occurs upon activation of HIV-1 protease embedded within GagProPol precursors and cleavage of Gag and GagProPol polyproteins. Although reversible oxidation can regulate mature protease activity as well as retrovirus maturation, it is possible that the effects of oxidation on viral maturation are mediated in whole, or part, through effects on the initial intramolecular cleavage event of GagProPol. In order assess the effect of reversible oxidation on this event, we developed a system to isolate the first step in protease activation involving GagProPol.

Research paper thumbnail of Reversible Oxidative Modification as a Mechanism for Regulating Retroviral Protease Dimerization and Activation

Journal of Virology, 2003

Human immunodeficiency virus protease activity can be regulated by reversible oxidation of a sulf... more Human immunodeficiency virus protease activity can be regulated by reversible oxidation of a sulfurcontaining amino acid at the dimer interface. We show here that oxidation of this amino acid in human immunodeficiency virus type 1 protease prevents dimer formation. Moreover, we show that human T-cell leukemia virus type 1 protease can be similarly regulated through reversible glutathionylation of its two conserved cysteine residues. Based on the known three-dimensional structures and multiple sequence alignments of retroviral proteases, it is predicted that the majority of retroviral proteases have sulfur-containing amino acids at the dimer interface. The regulation of protease activity by the modification of a sulfurcontaining amino acid at the dimer interface may be a conserved mechanism among the majority of retroviruses.

Research paper thumbnail of Transmission of Simian Immunodeficiency Virus Carrying Multiple Cytotoxic T-Lymphocyte Escape Mutations with Diminished Replicative Ability Can Result in AIDS Progression in Rhesus Macaques

Journal of Virology, 2008

Cytotoxic T-lymphocyte (CTL) responses frequently select for immunodeficiency virus mutations tha... more Cytotoxic T-lymphocyte (CTL) responses frequently select for immunodeficiency virus mutations that result in escape from CTL recognition with viral fitness costs. The replication in vivo of such viruses carrying not single but multiple escape mutations in the absence of the CTL pressure has remained undetermined. Here, we have examined the replication of simian immunodeficiency virus (SIV) with five gag mutations selected in a macaque possessing the major histocompatibility complex haplotype 90-120-Ia after its transmission into 90-120-Ia-negative macaques. Our results showed that even such a "crippled" SIV infection can result in persistent viral replication, multiple reversions, and AIDS progression.

Research paper thumbnail of Design and evaluation of an in-house HIV-1 (group M and O), SIVmnd and SIVcpz antigen capture assay

Journal of Virological Methods, 1998

... To design an in-house antigen capture ... positive sample signals were obtained when using pu... more ... To design an in-house antigen capture ... positive sample signals were obtained when using purified IgG 1 for coating at a concentration of 4 μg/well and purified and conjugated IgG 2 for detection at a concentration of 5 μg ml −1 . The anti-p24 titer for IgG 1 was 1/10 000 and 1 ...

Research paper thumbnail of The HIV-2 genotype and the HIV-1 syncytium-inducing phenotype are associated with a lower virus replication in dendritic cells

Journal of Medical Virology, 2000

During sexual transmission, HIV infects the mucosal dendritic cells and is transferred to CD4 T c... more During sexual transmission, HIV infects the mucosal dendritic cells and is transferred to CD4 T cells. Whether HIV variants of a particular genetic (sub)type or phenotype selectively infect dendritic cells (DC) or are preferentially transferred to T cells remains highly controversial. To avoid the cumbersome use of primary dendritic cells, in vitro dendritic cell models were generated from precursors, either hematopoietic progenitor cells (HPC) or monocytes (MO). Productive infection in the dendritic cells and transfer of the virus to T cells was assessed for a range of HIV variants. HPC-derived dendritic cells (HPC-DC) were more susceptible to HIV-1 than to HIV-2 isolates. The HIV-1 group O strains were more productive in HPC-DC than group M, but amongst the latter, no subtype-related difference was observed. Both non-syncytium-inducing (NSI) and SI HIV isolates and lab strains could productively infect HPC-DC, albeit with a different efficiency. Adding blocking antibodies confirmed that both CCR-5 and CXCR-4 co-receptors were functional. Biological HIV-1 clones of the NSI/R5 phenotype infected more readily HPC-DC than SI/X4 clones. MO-derived dendritic cells were, however, more exclusive in their preference for NSI/R5 clones. Some HIV variants, that did not grow readily in HPC-DC alone, could be rescued by adding resting or pre-activated T cells. The present data show that HIV-2 isolates and SI clones replicate less in model-DC, but no preference for a particular HIV-1 subtype was evident. Co-culture with T cells could "correct" a limited growth in dendritic cells. Clearly, both intrinsic dendritic cell susceptibility and enhancement by T cells are explained only partly by HIV genotype and phenotype. The in vitro dendritic cell models seem useful tools to further unravel interactions between HIV, DC, and T cells.

Research paper thumbnail of Serum inactivation contributes to the failure of stromal-derived factor-1 to block HIV-I infection in vivo

Journal of Leukocyte Biology, 2003

The chemokine stromal-derived factor-1 (SDF-1) can block human immunodeficiency virus type 1 (HIV... more The chemokine stromal-derived factor-1 (SDF-1) can block human immunodeficiency virus type 1 (HIV-1) infection in vitro by binding to the CXC chemokine receptor, CXCR-4, which serves as a coreceptor for T cell tropic HIV-1. In spite of being constitutively expressed in vivo, SDF-1 does not appear to block HIV-1 infection and spread in vivo. We report that SDF-1 is consistently measured in normal serum (15.4؎3.0 ng/ ml; mean؎SD) and in serum from AIDS patients (16.6؎3.7 ng/ml). However, we find that circulating SDF-1 is modified to an inactive form. When exposed to serum, recombinant SDF-1 is specifically and rapidly altered to yield an apparently smaller chemokine that does not bind to SDF-1 receptor-expressing cells, does not have chemoattractive or pre-B cell stimulatory activity, and does not block HIV-1 infection. Thus, serum modification and inactivation contribute to the failure of SDF-1 to block HIV-1 infection and spread in man. The inactivation of circulating SDF-1 may be critical in permitting local gradients to develop and direct cell trafficking. J. Leukoc. Biol. 74: 000 -000; 2003.

Research paper thumbnail of Detection of Serum Antibodies to a Kaposi's Sarcoma‐Associated Herpesvirus‐Specific Peptide

The Journal of Infectious Diseases, 1997

Kaposi's sarcoma (KS) -associated herpesvirus/human herpesvirus type 8 (KSHV/HHV-8) may play an e... more Kaposi's sarcoma (KS) -associated herpesvirus/human herpesvirus type 8 (KSHV/HHV-8) may play an etiologic role in the pathogenesis of KS. In an attempt to assess KSHV/HHV-8 infection, an ELISA was developed using an 18 -amino acid peptide from a putative minor capsid protein of KSHV/HHV-8 conjugated to bovine serum albumin. Overall, sera from human immunodeficiency virus type 1 (HIV-1) -positive patients with KS had a higher reactivity in the assay than did sera from HIV-1 -positive patients without KS (P Å .018). Of 35 HIV-1 -positive patients with KS, 60% were antibody positive, compared with 27% of 33 HIV-1 -positive patients without KS. Of 30 healthy blood donors, 20% were antibody positive. The ELISA responses did not correlate with antibody titers to Epstein-Barr virus.

Research paper thumbnail of Kaposi's sarcoma-associated herpesvirus (human herpesvirus 8): epidemiology and pathogenesis

Journal of Antimicrobial Chemotherapy, 2000

The complete ORF54 of the Kaposi's sarcoma-associated herpesvirus (KSHV) (human herp... more The complete ORF54 of the Kaposi's sarcoma-associated herpesvirus (KSHV) (human herpes-virus-8; HHV-8) was cloned and expressed in E. coli. The results show that KSHV/HHV-8 ORF54 encodes a functional dUTPase which specifically hydrolyses dUTP ...

Research paper thumbnail of 130 Inhibition of HIV-1 Release by Cell Permeable Peptides

JAIDS Journal of Acquired Immune Deficiency Syndromes, 2009

Research paper thumbnail of Identification of carboxypeptidase N as an enzyme responsible for C-terminal cleavage of stromal cell-derived factor-1  in the circulation

Blood, 2005

The chemokine stromal-derived factor-1␣ (SDF-1␣) is an essential regulator of hematopoiesis, lymp... more The chemokine stromal-derived factor-1␣ (SDF-1␣) is an essential regulator of hematopoiesis, lymphocyte homing, pre-Bcell growth, and angiogenesis. As SDF-1␣ is constitutively expressed in many tissues, chemokine function is mostly regulated by proteolytic degradation. Human serum cleaves the 68-amino acid chemokine, SDF-1␣, at both termini. The enzyme or enzymes responsible for the removal of the carboxy-terminal lysine from SDF-1␣, leading to significant reduction in biologic activity, have not been identified.

Research paper thumbnail of Regulation of HIV-1 Protease Activity through Cysteine Modification

Biochemistry, 1996

The homodimeric protease of the human immunodeficiency virus 1 contains two cysteine residues per... more The homodimeric protease of the human immunodeficiency virus 1 contains two cysteine residues per monomer which are highly conserved among viral isolates. However, these cysteine residues are not essential for catalytic activity which raises the question of why they are conserved. We have found previously that these cysteine residues are unusually susceptible to oxidation by metal ions, and this results in inhibition of protease activity. Recombinant protease mutants (C67A, C95A, and the double mutant C67A,C95A) were prepared to assess the possible role of these cysteines in redox regulation of the enzyme. Mixed disulfides were formed between the cysteine residues of the enzymes and low molecular weight thiols. Enzyme activity was lost when a mixed disulfide was formed between 5,5′-dithiobis(2nitrobenzoic acid) and cysteine 95, while the same mixed disulfide at cysteine 67 reduced activity by 50%. This effect was reversible as normal activity could be restored when the enzyme was treated with dithiothreitol. The cysteines could also be modified with the common cellular thiol glutathione. Modification with glutathione was verified by mass spectrometry of the protein peaks obtained from HPLC separation. Glutathiolation of cysteine 95 abolished activity whereas modification at cysteine 67 increased the k cat by more than 2-fold with no effect on K m . In addition, glutathiolation at cysteine 67 markedly stabilized the enzyme activity presumably by reducing autoproteolysis. These results demonstrate one possible mechanism for regulation of the HIV-1 protease through cysteine modification and identify additional targets for affecting protease activity other than the active site.

Research paper thumbnail of HIV-2 protease is inactivated after oxidation at the dimer interface and activity can be partly restored with methionine sulphoxide reductase

Biochemical Journal, 2000

Human immunodeficiency viruses encode a homodimeric protease that is essential for the production... more Human immunodeficiency viruses encode a homodimeric protease that is essential for the production of infectious virus. Previous studies have shown that HIV-1 protease is susceptible to oxidative inactivation at the dimer interface at Cys-95, a process that can be reversed both chemically and enzymically.

Research paper thumbnail of Inhibition of HIV-1 replication by a peptide dimerization inhibitor of HIV-1 protease

Antiviral Research, 2006

Peptides based on the amino (N) and carboxy (C)-terminal regions of human immunodeficiency virus ... more Peptides based on the amino (N) and carboxy (C)-terminal regions of human immunodeficiency virus type-1 (HIV-1) protease and on the Cterminus of p6 * can inhibit HIV-1 protease activity by preventing dimerization. We developed a peptide dimerization inhibitor, P27, that included these domains and a cell permeable domain derived from HIV-1 Tat. P27 inhibited wild type (WT) and protease inhibitor (PI)-resistant HIV-1 protease (IC 50 : 0.23-0.32 M). Kinetic and biochemical assays confirmed that P27 inhibits protease dimerization. Fluorescein-labeled peptide accumulated in MT-2 cells and protected acutely infected MT-2 cells from HIV-1-induced cytotoxicity (IC 50 : 5.1 M). P27 also inhibited p24 accumulation from H9 and U937 cells chronically infected with WT or PI-resistant HIV-1. Immunoblot analysis on the supernatants and infected cells revealed a block in virus release by P27 rather than an inhibition of polyprotein processing. However, inhibition of p55 Gag processing by active-site inhibitors was enhanced when combined with P27, suggesting that P27 can affect protease function in maturing virions. Although P27 was rationally designed to block dimerization of the mature HIV-1 protease, the effects of P27 on HIV-1 replication may be related to partial inhibition of Gag-Pol processing leading to a disruption in virus release. Published by Elsevier B.V.

Research paper thumbnail of Intravirion Processing of the Human Immunodeficiency Virus Type 1 Vif Protein by the Viral Protease May Be Correlated with Vif Function

Journal of Virology, 2002

The human immunodeficiency virus type 1 (HIV-1) Vif protein is specifically packaged into virus p... more The human immunodeficiency virus type 1 (HIV-1) Vif protein is specifically packaged into virus particles through an interaction with viral genomic RNA in which it associates with the viral nucleoprotein complex. We now demonstrate for the first time that virus-associated Vif is subject to proteolytic processing by the viral protease (Pr). Pr-dependent processing of Vif was observed both in vivo and in vitro. In vivo processing of Vif was cell type independent and evident by the appearance of a 7-kDa processing product, which was restricted to cell-free virus preparations. Processing of Vif required an active viral Pr and was sensitive to Pr inhibitors such as ritonavir. The processing site in Vif was characterized both in vivo and in vitro and mapped to Ala 150 . Interestingly, the Vif processing site is located in a domain that is highly conserved among HIV-1, HIV-2, and simian immunodeficiency virus Vif isolates. Mutations at or near the processing site did not affect protein stability or packaging efficiency but had dramatic effects on Vif processing. In general, mutations that markedly increased or decreased the sensitivity of Vif to proteolytic processing severely impaired or completely abolished Vif function. In contrast, mutations at the same site that had little or no effect on processing efficiency also did not influence Vif function. None of the mutants affected the ability of the virus to replicate in permissive cell lines. Our data suggest that mutations in Vif that cause a profound change in the sensitivity to Pr-dependent processing also severely impaired Vif function, suggesting that intravirion processing of Vif is important for the production of infectious viruses.