Ratan K. Maitra

Temporal activation of NF-κB regulates an interferon-independent innate antiviral response against cytoplasmic RNA viruses

NF-κB is known to exert its antiviral innate immune response via the IFN-β-induced Janus kinase/signal transducers and activators of transcription pathway. However, our current studies have demonstrated that activated NF-κB is capable of directly establishing an antiviral state independent of IFN or secreted soluble factor(s) against two highly pathogenic respiratory RNA viruses. Human parainfluenza virus type 3, a mildly cytopathic virus that induced NF-κB very early during infection was converted to a virulent virus after NF-κB inhibition. In contrast, a highly cytopathic virus, human respiratory syncytial virus that induced NF-κB late during infection, was converted to a mildly cytopathic virus after NF-κB induction before virus replication. This interconversion of cytopathic phenotypes of viruses after NF-κB modulation was further shown to be independent of IFN and soluble secreted factors(s). Moreover, tumor necrosis factor α (TNF-α) and IL-1β elicited an antiviral response, which was NF-κB-dependent. Thus, NF-κB induction directly confers an essential innate antiviral response against human parainfluenza virus type 3 and respiratory syncytial virus, which is independent of IFN-inducible factor(s).

Inhibition of STAT 1 phosphorylation by human parainfluenza virus type 3 C protein

ABSTRACT The P mRNA of the viruses belonging to the subfamily Paramyxovirinae possesses a unique property of giving rise to several accessory proteins by a process that involves the utilization of overlapping open reading frames (the C proteins) and by an “RNA-editing” mechanism (the V proteins). Although these proteins are considered accessory, numerous studies have highlighted the importance of these proteins in virus transcription and interferon signaling, including our previous observation on the role of human parainfluenza virus type 3 (HPIV 3) C protein in the transcription of viral genome (Malur et al., Virus Res. 99:199-204, 2004). In this report, we have addressed its role in interferon signaling by generating a stable cell line, L-C6, by using the lentiviral expression system which expresses HPIV 3 C protein. The L-C6 cells were efficient in abrogating both alpha and gamma interferon-induced antiviral states and demonstrated a drastic reduction in the formation of gamma-activated factor complexes in the cell extracts. Western blot analysis subsequently revealed a defect in the phosphorylation of STAT 1 in these cells. Taken together, our results indicate that HPIV 3 C protein is capable of counteracting the interferon signaling pathway by specifically inhibiting the activation of STAT 1.

Human immunodeficiency virus type 1 (HIV-1) provirus expression and LTR transcription are repressed in NEF-expressing cell lines

Human immunodeficiency virus type 1 (HIV-1) NEF protein has been demonstrated to be a negative regulator of HIV-1 replication and HIV-1 LTR transcription under transient expression conditions. The difficulty of several laboratories to reproduce these findings led us to reexamine the role of NEF in HIV-1 provirus expression and HIV-1 LTR transcription. Basal transcription from the HIV-1 LTR in the presence of a NEF expression vector was compared to that in the presence of a mutated NEF vector. NEF expression led to a greater than 10-fold repression of LTR transcription under these conditions. HeLa and Jurkat cell lines carrying the nef gene linked to the CMV promoter or the HIV-1 LTR were isolated by coselection for neomycin resistance. Single cell isolates were further selected for the expression of nef transcripts. With the exception of the anti-sense nef cell lines, all the nef cell lines expressed the 27-kDa NEF protein, detectable by immunoprecipitation. NEF+ HeLa cell lines were at least 5-fold less efficient than NEF- HeLa cell lines in transient proviral expression. Provirus expression was also repressed in the NEF+ Jurkat cell lines. TAT-activated LTR transcription from an HIV-1 LTR-linked CAT expression vector was repressed 10-fold in the NEF+ HeLa and NEF+ Jurkat cell lines. When infected with HIV-1, NEF expressing T lymphoid cell lines showed moderate delays in onset and peak of reverse transcriptase production. However, none of these cell lines completely arrested virus replication. Our data confirm a negative regulatory effect of NEF on both virus production and LTR driven CAT expression in the cell lines tested. It is possible that cell specific factors may influence NEF activity.

TARGETING RNA FOR DEGRADATION WITH A (2',5')-OLIGOADENYLATE ANTISENSE CHIMERA

Abstract A novel method is described to selectively cleave RNA by harnessing the 2-5A-dependent ribonuclease.

Silencing the myotrophin gene by RNA interference leads to the regression of cardiac hypertrophy

Myotrophin-induced activation of NF-kappaB has been shown to be associated with cardiac hypertrophy (CH) that progresses to heart failure (HF). In the present study, we examined the cause-and-effect relationship between myotrophin and NF-kappaB activation using small hairpin RNA (shRNA) against myotrophin both in vitro (using neonatal rat myocytes) and in vivo [using myotrophin transgenic (Myo-Tg) mice, which overexpress myotrophin in the heart, develop CH, and gradually progress to HF]. Among several lentiviral vectors expressing myotrophin shRNAs, L-sh-109 showed the best silencing effect at both the mRNA (155.3 +/- 5.9 vs. 32.5 +/- 5.5, P < 0.001) and protein levels associated with a significant reduction of atrial natriuretic factor (ANF) and NF-kappaB. In vivo, when L-sh-109 was delivered directly into the hearts of 10-wk-old Myo-Tg mice, we observed a significant regression of cardiac mass (8.0 vs. 5.7 mg/g, P < 0.001) and myotrophin gene expression (54.5% over untreated Myo-Tg mice, P < 0.001) associated with a reduction in ANF and NF-kappaB signaling components. Our data suggest that using RNA interference to silence the myotrophin gene prevents NF-kappaB activation, associated with an attenuation of CH. This strategy could be an excellent therapeutic means for the treatment of CH and HF.

Targeting HIV mRNA for Degradation: 2,5-A Antisense Chimeras as Potential Chemotherapeutic Agents for AIDS

Abstract We have identified a region within a 1 kb HIV gag RNA which can be ablated in vitro using a 2,5-A antisense chimera. The cleavage was specific and almost complete at a concentration of 100 nM chimera.

2-5A-Antisense: A Novel Approach to Cancer Therapy

In the past two decades, the identification and cloning of genes responsible for human malignancies has led to the first concerted efforts aimed at controlling cancer by blocking the expression of both oncogenes and genes which suppress the immune system’s response to cancer. Attempts at inhibiting the expression of harmful genes is approached in a selective and versatile manner through the use of antisense nucleic acids (reviewed in refs. 1 – 5). “Antisense” refers to the concept that nucleic acids which are complementary in sequence may anneal by Watson-CIick base pairing in cells and inhibit gene expression. Because mRNA is considered to be in the “sense” orientation, complementary sequence is called antisense. Antisense is highly selective in its mode of action because most genes have at least a stretch of sequence which is unique in nature. Therefore, one can use antisense which will bind with a much higher affinity to the targeted nucleic acid than to any other sequence. This highly selective mode of action makes antisense much more specific than conventional chemotherapeutic agents. Any gene, the sequence of which is known, can be targeted by synthesizing a strand of antisense. Binding of antisense to RNA produces stretches of duplex or double-stranded nucleic acid that can interfere with RNA processing and function at any one of several different levels. Possible mechanisms of antisense action include, but are not limited to, inhibition of transcription through formation of triplex structures with DNA, interference with mRNA splicing and transport to cytoplasm, induction mRNA decay, and inhibition of translation.

Molecular Functional Studies of HIV-1 REV and NEF Proteins

Human immunodeficiency virus type 1 (HIV-1), the etiological agent of AIDS, preferentially infects the CD4+ helper subset of human T lymphocytes. Clinically, HIV-1 infection is characterized by a chronic phase lasting several years with a paucity of infected lymphocytes in the circulation. Notwithstanding, HIV-1 infection of primary human T lymphocytes or CD4+ cell lines in vitro leads to massive acute infection and cell death (20). This dichotomy between the natural history of virus infection and its behavior in tissue culture implies the existence of viral and cellular determinants of viral latency and reactivation.