Bishnu P. De

Specific Interaction in Vitro and in Vivo of Glyceraldehyde-3-phosphate Dehydrogenase and LA Protein with Cis-acting RNAs of Human Parainfluenza Virus Type 3

Human parainfluenza virus type 3 (HPIV3) genome RNA is transcribed and replicated by the virus-encoded RNA-dependent RNA polymerase, and specific cellular proteins play a regulatory role in these processes. To search for cellular proteins potentially interacting with HPIV3 cis-acting regulatory RNAs, a gel mobility shift assay was used. Two cellular proteins specifically interacted with the viral cis-acting RNAs containing the genomic 3′-noncoding region and the plus-sense leader sequence region. Surprisingly, by biochemical and immunological analyses, one of the cellular proteins was identified as the key glycolytic enzyme, glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The other protein was characterized as the autoantigen, LA protein. Both GAPDH and LA protein also interacted with the same cis-acting RNA sequences in vivo and were found to be associated with the HPIV3 ribonucleoprotein complex in the infected cells. By double immunofluorescent labeling, GAPDH was found to be co-localized with viral ribonucleoprotein in the perinuclear region. These observations strongly suggest that cellular GAPDH and LA Protein participate in the regulation of HPIV3 gene expression.

Systemic transforming growth factor-β1 gene therapy induces Foxp3+ regulatory cells, restores self-tolerance, and facilitates regeneration of beta cell function in overtly diabetic nonobese diabetic mice

Background. Type 1 diabetes results from auto-aggressive T–cell-mediated destruction of beta cells of the pancreas. Recent data suggest that restoration of self-tolerance may facilitate islet-cell regeneration/recovery. In view of the immunoregulatory activity of transforming growth factor (TGF)-&bgr;1, we investigated whether systemic TGF-&bgr;1 gene therapy blocks islet destructive autoimmunity and facilitates regeneration of beta-cell function in overtly diabetic nonobese diabetic (NOD) mice. Methods. We used site-directed mutagenesis to create cysteine to serine mutation at sites 224 and 226 and constructed a replication deficient adenovirus (Ad) vector encoding active form of human TGF-&bgr;1 (Ad-hTGF-&bgr;1). Overtly diabetic NOD mice received intravenous injection of Ad-hTGF-&bgr;1. Seven to 14 days after the injection, the mice received transplants with 500 syngeneic islets under the kidney capsule. Islet-graft survival and regeneration of endogenous beta-cell function were examined. Results. Syngeneic islet grafts failed by day 17 in all untreated mice, whereas Ad-hTGF-&bgr;1 therapy prolonged survival of islet grafts. Islet grafts from treated mice showed well-preserved islets with a peri-islet infiltrate primarily of CD4+ T cells and expression of CD25 and Foxp3. Systemic TGF-&bgr;1 gene therapy was associated with islet regeneration in the native pancreas. Native pancreas of treated mice revealed islets staining strongly for insulin. Similar to what was found in the syngeneic islet graft, there were well-demarcated peri-islet infiltrates that were positive for CD4, TGF-&bgr;1, and Foxp3. Conclusions. Our data demonstrate that systemic TGF-&bgr;1 gene therapy blocks islet destructive autoimmunity, facilitates islet regeneration, and cures diabetes in diabetic NOD mice.

Gene expression of nonsegmented negative strand RNA viruses

Nonsegmented negative strand RNA viruses comprise major human and animal pathogens in nature. This class of viruses is ubiquitous and infects vertebrates, invertebrates, and plants. Our laboratory has been working on the gene expression of two prototype nonsegmented negative strand RNA viruses, vesicular stomatitis virus (a rhabdovirus) and human parainfluenza virus 3 (a paramyxovirus). An RNA-dependent RNA polymerase (L and P protein) is packaged within the virion which faithfully copies the genome RNA in vitro and in vivo; this enzyme complex, in association with the nucleocapsid protein (N), is also involved in the replication process. In this review, we have presented up-to-date information of the structure and function of the RNA polymerases of these two viruses, the mechanisms of transcription and replication, and the role of host proteins in the life-cycle of the viruses. These detailed studies have led us to a better understanding of the roles of viral and cellular proteins in the viral gene expression.

AAV2-mediated CLN2 gene transfer to rodent and non-human primate brain results in long-term TPP-I expression compatible with therapy for LINCL

Late infantile neuronal ceroid lipofuscinosis (LINCL) is a fatal, autosomal recessive disease resulting from mutations in the CLN2 gene with consequent deficiency in its product tripeptidyl peptidase I (TPP-I). In the central nervous system (CNS), the deficiency of TPP-I results in the accumulation of proteins in lysosomes leading to a loss of neurons causing progressive neurological decline, and death by ages 10–12 years. To establish the feasibility of treating the CNS manifestations of LINCL by gene transfer, an adeno-associated virus 2 (AAV2) vector encoding the human CLN2 cDNA (AAV2CUhCLN2) was assessed for its ability to establish therapeutic levels of TPP-I in the brain. In vitro studies demonstrated that AAV2CUhCLN2 expressed CLN2 and produced biologically active TPP-I protein of which a fraction was secreted as the pro-TPP-I precursor and was taken up by nontransduced cells (ie, cross-correction). Following AAV2-mediated CLN2 delivery to the rat striatum, enzymatically active TPP-I protein was detected. By immunohistochemistry TPP-I protein was detected in striatal neurons (encompassing nearly half of the target structure) for up to 18 months. At the longer time points following striatal administration, TPP-I-positive cell bodies were also observed in the substantia nigra, frontal cerebral cortex and thalamus of the injected hemisphere, and the frontal cerebral cortex of the noninjected hemisphere. These areas of the brain contain neurons that extend axons into the striatum, suggesting that CNS circuitry may aid the distribution of the gene product. To assess the feasibility of human CNS delivery, a total of 3.6 × 1011 particle units of AAV2CUhCLN2 was administered to the CNS of African green monkeys in 12 distributed doses. Assessment at 5 and 13 weeks demonstrated widespread detection of TPP-I in neurons, but not glial cells, at all regions of injection. The distribution of TPP-I-positive cells was similar between the two time points at all injection sites. Together, these data support the development of direct CNS gene transfer using an AAV2 vector expressing the CLN2 cDNA for the CNS manifestations of LINCL.

Cocaine Analog Coupled to Disrupted Adenovirus: A Vaccine Strategy to Evoke High-titer Immunity Against Addictive Drugs

Based on the concept that anticocaine antibodies could prevent inhaled cocaine from reaching its target receptors in the brain, an effective anticocaine vaccine could help reverse cocaine addiction. Leveraging the knowledge that E1(-)E3(-) adenovirus (Ad) gene transfer vectors are potent immunogens, we have developed a novel vaccine platform for addictive drugs by covalently linking a cocaine analog to the capsid proteins of noninfectious, disrupted Ad vector. The Ad-based anticocaine vaccine evokes high-titer anticocaine antibodies in mice sufficient to completely reverse, on a persistent basis, the hyperlocomotor activity induced by intravenous administration of cocaine.

A common mechanism for cytoplasmic dynein-dependent microtubule binding shared among adeno-associated virus and adenovirus serotypes

ABSTRACT During infection, adenovirus-associated virus (AAV) undergoes microtubule-dependent retrograde transport as part of a program of vectorial transport of viral genome to the nucleus. A microtubule binding assay was used to evaluate the hypothesis that cytoplasmic dynein mediates AAV interaction with microtubules. Binding of AAV serotype 2 (AAV2) was enhanced in a nucleotide-dependent manner by the presence of total cellular microtubule-associated proteins (MAPs) but not cytoplasmic dynein-depleted MAPs. Excess AAV2 capsid protein prevented microtubule binding by AAV serotypes 2, 5, and rh.10, as well as adenovirus serotype 5, indicating that similar binding sites are used by these viruses.

Novel Cocaine Vaccine Linked to a Disrupted Adenovirus Gene Transfer Vector Blocks Cocaine Psychostimulant and Reinforcing Effects

Immunotherapy is a promising treatment for drug addiction. However, insufficient immune responses to vaccines in most subjects pose a challenge. In this study, we tested the efficacy of a new cocaine vaccine (dAd5GNE) in antagonizing cocaine addiction-related behaviors in rats. This vaccine used a disrupted serotype 5 adenovirus (Ad) gene transfer vector coupled to a third-generation cocaine hapten, termed GNE (6-(2R,3S)-3-(benzoyloxy)-8-methyl-8-azabicyclo [3.2.1] octane-2-carboxamido-hexanoic acid). Three groups of rats were immunized with dAd5GNE. One group was injected with 3H-cocaine, and radioactivity in the blood and brain was determined. A second group was tested for cocaine-induced locomotor sensitization. A third group was examined for cocaine self-administration, extinction, and reinstatement of responding for cocaine. Antibody titers were determined at various time-points. In each experiment, we added a control group that was immunized with dAd5 without a hapten. The vaccination with dAd5GNE produced long-lasting high titers (>105) of anti-cocaine antibodies in all of the rats. The vaccination inhibited cocaine-induced hyperlocomotor activity and sensitization. Vaccinated rats acquired cocaine self-administration, but they showed less motivation to self-administer cocaine under a progressive-ratio schedule than control rats. When cocaine was not available in a session, control rats exhibited ‘extinction burst’ responding, whereas vaccinated rats did not. Moreover, when primed with cocaine, vaccinated rats did not reinstate responding, suggesting a blockade of cocaine-seeking behavior. These data strongly suggest that our dAd5GNE vector-based vaccine may be effective in treating cocaine abuse and addiction.

Phosphorylation of Sendai Virus Phosphoprotein by Cellular Protein Kinase C ζ

The phosphoproteins (P) of nonsegmented negative strand RNA viruses are viral RNA polymerase subunits involved in both transcription and replication during the virus life cycle. Phosphorylation of P proteins in several negative strand RNA viruses by specific cellular kinases was found to be required for P protein function. In the present study, using bacterially expressed unphosphorylated P protein of Sendai virus, a mouse parainfluenza virus, we have shown that the major cellular kinase that phosphorylates P protein in vitro is biochemically and immunologically indistinguishable from protein kinase C (PKC) ζ isoform. PKC ζ was packaged into the Sendai virion and remained associated with purified viral ribonucleoprotein, where it phosphorylated both the P and the nucleocapsid protein in vitro. When PKC ζ-specific inhibitory pseudosubstrate peptide was introduced into LLC-MK2 cells prior to Sendai virus infection, production of progeny virus was dramatically attenuated, and kinetic analysis revealed that primary transcription was repressed. These data indicate that phosphorylation of the Sendai virus P protein by PKC ζ plays a critical role in the virus life cycle.

Role of NH2- and COOH-Terminal Domains of the P Protein of Human Parainfluenza Virus Type 3 in Transcription and Replication

ABSTRACT The phosphoproteins (P proteins) of paramyxoviruses play a central role in transcription and replication of the viruses by forming the RNA polymerase complex L-P and encapsidation complex (N-P) with nucleocapsid protein (N) and binding to N protein-encapsidated genome RNA template (N-RNA template). We have analyzed the human parainfluenza virus type 3 (HPIV3) P protein and deletion mutants thereof in an in vitro transcription and in vivo replication system. The in vitro system utilizes purified N-RNA template and cell extract containing L and P proteins coexpressed via plasmids using a recombinant vaccinia virus expression system. The in vivo system takes advantage of minigenome replication, which measures luciferase reporter gene expression from HPIV3 minigenomes by viral proteins in a recombinant vaccinia virus expression system. These studies revealed that the C-terminal 20-amino-acid region of P is absolutely required for transcription in vitro and luciferase expression in vivo, suggesting its critical role in viral RNA synthesis. The N-terminal 40-amino-acid region, on the other hand, is essential for luciferase expression but dispensable for transcription in vitro. Consistent with these findings, the C-terminal domain is required for binding of P protein to the N-RNA template involved in both transcription and replication, whereas the N-terminal domain is required for the formation of soluble N-P complex involved in encapsidation of nascent RNA chains during replication. Coimmunoprecipitation analysis showed that the P protein forms a stable homooligomer (perhaps a trimer) that is present in L-P and N-P complexes in the higher oligomeric forms (at least a pentamer). Interestingly, coexpression of a large excess of N- or C-terminally deleted P with wild-type P had no effect on minigenome replication in vivo, notwithstanding the formation of heterooligomeric complexes. These data indicate that P protein with a deleted terminal domain can function normally within the P heterooligomeric complex to carry out transcription and replication in vivo.

Aav-directed persistent expression of an anti-nicotine antibody gene for smoking cessation

Gene therapy with an anti-nicotine monoclonal antibody limits nicotine access to the brain in mice and may be a potential therapy for cigarette addiction. Extinguishing Addiction Those who smoke and try to quit have variously described their feelings as a never-ending hunger, terror, immense panic, rage, and the loss of a loved one—reactions that stem from nicotine addiction and withdrawal. Given cigarette smoke’s destructive effects on health and cost to society, a means of overcoming such addiction would be enormously beneficial. For most smokers, current antismoking therapies fail to work. One newer idea is an anti-nicotine vaccine; in this approach, nicotine (coupled to a larger molecule) is administered, generating an immune response. The resulting anti-nicotine antibodies bind the nicotine from cigarette smoke in the blood, intercepting the drug before it affects reward centers in the brain. In disappointing clinical trials, however, such vaccines result in variable antibody titers and have only limited success in halting smoking, with best results seen in people with the strongest antibody response. Hicks et al. now describe a different tactic to get to the same end; they use gene transfer to attain persistent, high levels of anti-nicotine antibodies in smokers’ blood. The researchers constructed an adeno-associated virus–based vector that expressed a high-affinity anti-nicotine monoclonal antibody. Mice injected with a single dose of this vector made high antibody titers (which were in 40-fold molar excess over the nicotine concentrations seen in people who smoke continuously), which remained high for at least 18 weeks. When these mice were injected with nicotine, the antibodies effectively sequestered this compound in the blood: nicotine concentrations in the brain were only 15% of those in brains of mice that did not express the antibodies. Furthermore, the usual nicotine-induced changes in blood pressure, heart rate, and locomotor activity were abolished or greatly reduced in mice that expressed the anti-nicotine antibodies. Further work will be needed to test this vector in a rodent model trained to self-administer nicotine, because the mice in this study were not addicted to the drug. Successful results from such a test would then support investigating this approach in clinical trials. Current strategies to help tobacco smokers quit have limited success as a result of the addictive properties of the nicotine in cigarette smoke. We hypothesized that a single administration of an adeno-associated virus (AAV) gene transfer vector expressing high levels of an anti-nicotine antibody would persistently prevent nicotine from reaching its receptors in the brain. To test this hypothesis, we constructed an AAVrh.10 vector that expressed a full-length, high-affinity, anti-nicotine antibody derived from the Fab fragment of the anti-nicotine monoclonal antibody NIC9D9 (AAVantiNic). In mice treated with this vector, blood concentrations of the anti-nicotine antibody were dose-dependent, and the antibody showed high specificity and affinity for nicotine. The antibody shielded the brain from systemically administered nicotine, reducing brain nicotine concentrations to 15% of those in naïve mice. The amount of nicotine sequestered in the serum of vector-treated mice was more than seven times greater than that in untreated mice, with 83% of serum nicotine bound to immunoglobulin G. Treatment with the AAVantiNic vector blocked nicotine-mediated alterations in arterial blood pressure, heart rate, and locomotor activity. In summary, a single administration of a gene transfer vector expressing a high-affinity anti-nicotine monoclonal antibody elicited persistent (18 weeks), high titers of an anti-nicotine antibody that obviated the physiologic effects of nicotine. If this degree of efficacy translates to humans, AAVantiNic could be an effective preventative therapy for nicotine addiction.