Nagadenahalli B. Siddappa

Curcumin, a novel p300/CREB-binding protein-specific inhibitor of acetyltransferase, represses the acetylation of histone/nonhistone proteins and histone acetyltransferase-dependent chromatin transcription.

Acetylation of histones and non-histone proteins is an important post-translational modification involved in the regulation of gene expression in eukaryotes and all viral DNA that integrates into the human genome (e.g. the human immunodeficiency virus). Dysfunction of histone acetyltransferases (HATs) is often associated with the manifestation of several diseases. In this respect, HATs are the new potential targets for the design of therapeutics. In this study, we report that curcumin (diferuloylmethane), a major curcumanoid in the spice turmeric, is a specific inhibitor of the p300/CREB-binding protein (CBP) HAT activity but not of p300/CBP-associated factor, in vitro and in vivo. Furthermore, curcumin could also inhibit the p300-mediated acetylation of p53 in vivo. It specifically represses the p300/CBP HAT activity-dependent transcriptional activation from chromatin but not a DNA template. It is significant that curcumin could inhibit the acetylation of HIV-Tat protein in vitro by p300 as well as proliferation of the virus, as revealed by the repression in syncytia formation upon curcumin treatment in SupT1 cells. Thus, non-toxic curcumin, which targets p300/CBP, may serve as a lead compound in combinatorial HIV therapeutics.

Tat Protein of Human Immunodeficiency Virus Type 1 Subtype C Strains Is a Defective Chemokine

ABSTRACT Human immunodeficiency virus type 1 (HIV-1)-associated dementia (HAD) is correlated with increased monocyte migration to the brain, and the incidence of HAD among otherwise asymptomatic subjects appears to be lower in India than in the United States and Europe (1 to 2% versus 15 to 30%). Because of the genetic differences between HIV-1 strains circulating in these regions, we sought to identify viral determinants associated with this difference. We targeted Tat protein for these studies in view of its association with monocyte chemotactic function. Analyses of Tat sequences representing nine subtypes revealed that at least six amino acid residues are differentially conserved in subtype C Tat (C-Tat). Of these, cysteine (at position 31) was highly (>99%) conserved in non-subtype C viruses and more than 90% of subtype C viruses encoded a serine. We hypothesized a compromised chemotactic function of C-Tat due to the disruption of CC motif and tested it with the wild type C-Tat (CS) and its two isogenic variants (CC and SC) derived by site-directed mutagenesis. We found that the CS natural variant was defective for monocyte chemotactic activity without a loss in the transactivation property. While the CC mutant is functionally competent for both the functions, in contrast, the SC mutant was defective in both. Therefore, the loss of the C-Tat chemotactic property may underlie the reduced incidence of HAD; although not presenting conclusive evidence, this study provides the first evidence for a potential epidemiologic phenomenon associated with biological differences in the subtype C viruses.

Clade-specific differences in neurotoxicity of human immunodeficiency virus-1 B and C Tat of human neurons: significance of dicysteine C30C31 motif

Human immunodeficiency virus‐1 (HIV‐1) causes mild to severe cognitive impairment and dementia. The transactivator viral protein, Tat, is implicated in neuronal death responsible for neurological deficits. Several clades of HIV‐1 are unequally distributed globally, of which HIV‐1 B and C together account for the majority of the viral infections. HIV‐1–related neurological deficits appear to be most common in clade B, but not clade C prevalent areas. Whether clade‐specific differences translate to varied neuropathogenesis is not known, and this uncertainty warrants an immediate investigation into neurotoxicity on human neurons of Tat derived from different viral clades

HIV-1 clade-specific differences in the induction of neuropathogenesis

Human immunodeficiency virus (HIV)-associated dementia (HAD) is common among clade B HIV-infected individuals, but less common and less severe among individuals infected with clade C HIV-1, suggesting clade-specific differences in neuropathogenicity. Although differences in neuropathogenicity have been investigated in vitro using viral proteins responsible for HAD, to date there are no virological studies using animal models to address this issue. Therefore, we investigated neuropathogenesis induced by HIV-1 clades using the severe combined immune deficiency (SCID) mouse HIV encephalitis model, which involves intracranial injection of macrophages infected with representative clade B (HIV-1ADA) or clade C (HIV-1Indie-C1) HIV-1 isolates into SCID mice. In cognitive tests, mice exposed to similar inputs of HIV-1 clade C made fewer memory errors than those exposed to HIV-1 clade B. Histopathological analysis of mice exposed to clade B exhibited greater astrogliosis and increased loss of neuronal network integrity. In vitro experiments revealed differences in a key characteristic of HIV-1 that influences HAD, increased monocyte infiltration. HIV-1Indie-C1-infected macrophages recruited monocytes poorly in vitro compared with HIV-1ADA-infected macrophages. Monocyte recruitment was HIV-1 Tat and CCL2 dependent. This is the first demonstration, ever since HIV neuropathogenesis was first recognized, that viral genetic differences between clades can affect disease severity and that such studies help identify key players in neuropathogenesis by HIV-1.

Anti-HIV IgA isotypes: differential virion capture and inhibition of transcytosis are linked to prevention of mucosal R5 SHIV transmission.

Objective:Although passive immunization with anti-HIV-1 Env IgG1 neutralizing monoclonal antibodies (nmAbs) prevented simian–human immunodeficiency virus (SHIV) infection in rhesus monkeys, IgA nmAbs have not been tested. Here, we sought to determine whether human anti-HIV-1 dimeric (d)IgA1, dIgA2, and IgG1 differ in their ability to prevent mucosal R5 SHIV acquisition in rhesus monkeys. Design:DIgA1, dIgA2, and IgG1 versions of nmAb HGN194 were applied intrarectally in three rhesus monkey groups 30 min before intrarectal SHIV challenge. Methods:After a control pharmacokinetic study confirmed that nmAb concentrations in rectal fluids over time were similar for all HGN194 isotypes, control and nmAb-treated animals were challenged intrarectally with an R5 SHIV, and viral loads were monitored. Results:Unexpectedly, dIgA1 provided the best protection in vivo – although all nmAbs showed similar neutralizing activity in vitro. Five out of the six dIgA1-treated rhesus monkeys remained virus-free compared to only one out of six animals given dIgA2 (P = 0.045 by log-rank test) and two out of six rhesus monkeys treated with IgG1 forms of the nmAb (P = 0.12). Protection correlated significantly with virion capture activity by a given nmAb form, as well as inhibition of transcytosis of cell-free virus across an epithelial cell layer in vitro. Conclusions:Our data imply that dIgA1-mediated capturing of virions in mucosal secretions and inhibition of transcytosis can provide significant prevention of lentiviral acquisition – over and above direct virus neutralization. Vaccine strategies that induce mucosal IgA, especially IgA1, should be developed as a first line of defense against HIV-1, a virus predominantly transmitted mucosally.

An Anti-HIV-1 V3 Loop Antibody Fully Protects Cross-Clade and Elicits T-Cell Immunity in Macaques Mucosally Challenged with an R5 Clade C SHIV

Neutralizing antibodies have been shown to protect macaques against SHIV challenge. However, genetically diverse HIV-1 clades have evolved, and a key question left unanswered is whether neutralizing antibodies can confer cross-clade protection in vivo. The novel human monoclonal antibody HGN194 was isolated from an individual infected with an HIV-1 clade AG recombinant circulating recombinant form (CRF). HGN194 targets an epitope in the third hypervariable loop (V3) of HIV-1 gp120 and neutralizes a range of relatively neutralization-sensitive and resistant viruses. We evaluated the potential of HGN194 to protect infant rhesus monkeys against a SHIV encoding a primary CCR5-tropic HIV-1 clade C envelope. After high-dose mucosal challenge, all untreated controls became highly viremic while all HGN194-treated animals (50 mg/kg) were completely protected. When HGN194 was given at 1 mg/kg, one out of two monkeys remained aviremic, whereas the other had delayed, lower peak viremia. Interestingly, all protected monkeys given high-dose HGN194 developed Gag-specific proliferative responses of both CD4+ and CD8+ T cells. To test whether generation of the latter involved cryptic infection, we ablated CD8+ cells after HGN194 clearance. No viremia was detected in any protected monkeys, thus ruling out virus reservoirs. Thus, induction of CD8 T-cell immunity may have resulted from transient “Hit and Run” infection or cross priming via Ag-Ab-mediated cross-presentation. Together, our data identified the HGN194 epitope as protective and provide proof-of-concept that this anti-V3 loop mAb can prevent infection with sterilizing immunity after challenge with virus of a different clade, implying that V3 is a potential vaccine target.

SHIV-1157i and passaged progeny viruses encoding R5 HIV-1 clade C env cause AIDS in rhesus monkeys

BackgroundInfection of nonhuman primates with simian immunodeficiency virus (SIV) or chimeric simian-human immunodeficiency virus (SHIV) strains is widely used to study lentiviral pathogenesis, antiviral immunity and the efficacy of AIDS vaccine candidates. SHIV challenges allow assessment of anti-HIV-1 envelope responses in primates. As such, SHIVs should mimic natural HIV-1 infection in humans and, to address the pandemic, encode HIV-1 Env components representing major viral subtypes worldwide.ResultsWe have developed a panel of clade C R5-tropic SHIVs based upon env of a Zambian pediatric isolate of HIV-1 clade C, the worlds most prevalent HIV-1 subtype. The parental infectious proviral clone, SHIV-1157i, was rapidly passaged through five rhesus monkeys. After AIDS developed in the first animal at week 123 post-inoculation, infected blood was infused into a sixth monkey. Virus reisolated at this late stage was still exclusively R5 tropic and mucosally transmissible. Here we describe the long-term follow-up of this initial cohort of six monkeys. Two have remained non-progressors, whereas the other four gradually progressed to AIDS within 123–270 weeks post-exposure. Two progressors succumbed to opportunistic infections, including a case of SV40 encephalitis.ConclusionThese data document the disease progression induced by the first mucosally transmissible, pathogenic R5 non-clade B SHIV and suggest that SHIV-1157i-derived viruses, including the late-stage, highly replication-competent SHIV-1157ipd3N4 previously described (Song et al., 2006), display biological characteristics that mirror those of HIV-1 clade C and support their expanded use for AIDS vaccine studies in nonhuman primates.

Identification of Subtype C Human Immunodeficiency Virus Type 1 by Subtype-Specific PCR and Its Use in the Characterization of Viruses Circulating in the Southern Parts of India

ABSTRACT Human immunodeficiency virus type 1 (HIV-1) subtype C viruses are associated with nearly half of worldwide HIV-1 infections and are most predominant in India and the southern and eastern parts of Africa. Earlier reports from India identified the preponderance of subtype C and a small proportion of subtype A viruses. Subsequent reports identifying multiple subtypes suggest new introductions and/or their detection due to extended screening. The southern parts of India constitute emerging areas of the epidemic, but it is not known whether HIV-1 infection in these areas is associated with subtype C viruses or is due to the potential new introduction of non-subtype C viruses. Here, we describe the development of a specific and sensitive PCR-based strategy to identify subtype C-viruses (C-PCR). The strategy is based on amplifying a region encompassing a long terminal repeat and gag in the first round, followed by two sets of nested primers; one amplifies multiple subtypes, while the other is specific to subtype C. The common HIV and subtype C-specific fragments are distinguishable by length differences in agarose gels and by the difference in the numbers of NF-κB sites encoded in the subtype C-specific fragment. We implemented this method to screen 256 HIV-1-infected individuals from 35 towns and cities in four states in the south and a city in the east. With the exception of single samples of subtypes A and B and a B/C recombinant, we found all to be infected with subtype C viruses, and the subtype assignments were confirmed in a subset by using heteroduplex mobility assays and phylogenetic analysis of sequences. We propose the use of C-PCR to facilitate rapid molecular epidemiologic characterization to aid vaccine and therapeutic strategies.

Transactivation and signaling functions of Tat are not correlated: biological and immunological characterization of HIV-1 subtype-C Tat protein

BackgroundOf the diverse subtypes of Human Immunodeficiency Virus Type-1 (HIV-1), subtype-C strains cause a large majority of infections worldwide. The reasons for the global dominance of HIV-1 subtype-C infections are not completely understood. Tat, being critical for viral infectivity and pathogenesis, may differentially modulate pathogenic properties of the viral subtypes. Biochemical studies on Tat are hampered by the limitations of the current purification protocols. Tat purified using standard protocols often is competent for transactivation activity but defective for a variety of other biological functions. Keeping this limitation in view, we developed an efficient protein purification strategy for Tat.ResultsTat proteins obtained using the novel strategy described here were free of contaminants and retained biological functions as evaluated in a range of assays including the induction of cytokines, upregulation of chemokine coreceptor, transactivation of the viral promoter and rescue of a Tat-defective virus. Given the highly unstable nature of Tat, we evaluated the effect of the storage conditions on the biological function of Tat following purification. Tat stored in a lyophilized form retained complete biological activity regardless of the storage temperature. To understand if variations in the primary structure of Tat could influence the secondary structure of the protein and consequently its biological functions, we determined the CD spectra of subtype-C and -B Tat proteins. We demonstrate that subtype-C Tat may have a relatively higher ordered structure and be less flexible than subtype-B Tat. We show that subtype-C Tat as a protein, but not as a DNA expression vector, was consistently inferior to subtype-B Tat in a variety of biological assays. Furthermore, using ELISA, we evaluated the anti-Tat antibody titers in a large number of primary clinical samples (n = 200) collected from all four southern Indian states. Our analysis of the Indian populations demonstrated that Tat is non-immunodominant and that a large variation exists in the antigen-specific antibody titers.ConclusionOur report not only describes a simple protein purification strategy for Tat but also demonstrates important structural and functional differences between subtype-B and -C Tat proteins. Furthermore, this is the first report of protein purification and characterization of subtype-C Tat.

Relative Transmissibility of an R5 Clade C Simian- Human Immunodeficiency Virus Across Different Mucosae in Macaques Parallels the Relative Risks of Sexual HIV-1 Transmission in Humans via Different Routes

BACKGROUND Worldwide, approximately 90% of all human immunodeficiency virus (HIV) transmissions occur mucosally; almost all involve R5 strains. Risks of sexual HIV acquisition are highest for rectal, then vaginal, and finally oral exposures. METHODS Mucosal lacerations may affect the rank order of susceptibility to HIV but cannot be assessed in humans. We measured relative virus transmissibility across intact mucosae in macaques using a single stock of SHIV-1157ipd3N4, a simian-human immunodeficiency virus encoding a primary R5 HIV clade C env (SHIV-C). RESULTS The penetrability of rhesus macaque mucosae differed significantly, with rectal challenge requiring the least virus, followed by vaginal and then oral routes (P = .031, oral vs vaginal; P < .001 rectal vs vaginal). These findings imply that intrinsic mucosal properties are responsible for the differential mucosal permeability. The latter paralleled the rank order reported for humans, with relative risk estimates within the range of epidemiological human studies. To test whether inflammation facilitates virus transmission--as predicted from human studies--we established a macaque model of localized buccal inflammation. Systemic infection occurred across inflamed but not normal buccal mucosa. CONCLUSION Our primate data recapitulate virus transmission risks observed in humans, thus establishing R5 SHIV-1157ipd3N4 in macaques as a robust model system to study cofactors involved in human mucosal HIV transmission and its prevention.