C Krishna Prasad

Publications of C Krishna Prasad

• Mos 3' UTR regulatory differences underlie species-specific temporal patterns of Mos mRNA cytoplasmic polyadenylation and translational recruitment during oocyte maturation.

  Authors: C Krishna Prasad, Mahendran Mahadevan, Melanie C MacNicol, Angus M MacNicol

  Molecular reproduction and development. 09/2008; 75(8):1258-68.

  The Mos proto-oncogene is a critical regulator of vertebrate oocyte maturation. The maturation-dependent translation of Mos protein correlates with the cytoplasmic polyadenylation of the maternal MosThe Mos proto-oncogene is a critical regulator of vertebrate oocyte maturation. The maturation-dependent translation of Mos protein correlates with the cytoplasmic polyadenylation of the maternal Mos mRNA. However, the precise temporal requirements for Mos protein function differ between oocytes of model mammalian species and oocytes of the frog Xenopus laevis. Despite the advances in model organisms, it is not known if the translation of the human Mos mRNA is also regulated by cytoplasmic polyadenylation or what regulatory elements may be involved. We report that the human Mos 3' untranslated region (3' UTR) contains a functional cytoplasmic polyadenylation element (CPE) and demonstrate that the endogenous Mos mRNA undergoes maturation-dependent cytoplasmic polyadenylation in human oocytes. The human Mos 3' UTR interacts with the human CPE-binding protein and exerts translational control on a reporter mRNA in the heterologous Xenopus oocyte system. Unlike the Xenopus Mos mRNA, which is translationally activated by an early acting Musashi/polyadenylation response element (PRE)-directed control mechanism, the translational activation of the human Mos 3' UTR is dependent on a late acting CPE-dependent process. Taken together, our findings suggest a fundamental difference in the 3' UTR regulatory mechanisms controlling the temporal induction of maternal Mos mRNA polyadenylation and translational activation during Xenopus and mammalian oocyte maturation.

• Multiple human papillomavirus genes affect the adeno-associated virus life cycle.

  Authors: Hong You, Yong Liu, C Krishna Prasad, Nalini Agrawal, Dazhi Zhang, Sarmistha Bandyopadhyay, Hongmei Liu, Helen H Kay, Jawahar L Mehta, Paul L Hermonat

  Virology. 02/2006; 344(2):532-40.

  The risk of cervical cancer, one of the most prevalent cancers in the world, is determined by two viruses. Human papillomavirus (HPV) is the main risk factor for developing cervical cancer. However,The risk of cervical cancer, one of the most prevalent cancers in the world, is determined by two viruses. Human papillomavirus (HPV) is the main risk factor for developing cervical cancer. However, although little known, it is well substantiated that the human Parvovirus adeno-associated virus type 2 (AAV), and its encoded Rep78 protein, interacts with HPV and lowers the risk of cervical cancer. HPV also contributes to AAV inhibition by serving as a helper virus for AAV and stimulating higher AAV replication levels. Here we surveyed four HPV-16 early genes, E1, E2, E6 and E7, for their ability to increase/decrease the basal level of AAV replication in stratifying squamous epithelium (the epithelial raft culture system). It was found that the HPV-16 E1, E2 and E6 genes were able to help/enhance AAV-2 replication in epithelial raft cultures. Under these conditions, with all the HPV genes being expressed from the AAV p5 promoter, E1 appeared to have the strongest enhancing effect on AAV DNA replication (Southern blot), RNA expression (RT-PCR), protein expression (Western blot) and AAV virion production (2 plate-Southern blot). Further study of E1 mutants showed that the carboxy-half of E1, the putative helicase/ATPase domain, was the main contributor of helper activity. These data are important for understanding the HPV-AAV interaction and its effect on modifying cervical cancer risk. These data also suggest the possibility that the identified HPV helper genes may be useful in the generation of recombinant (r)AAV virions for gene therapy, as rAAV is increasing in popularity for such purposes.

• Use and specificity of breast cancer antigen/milk protein BA46 for generating anti-self-cytotoxic T lymphocytes by recombinant adeno-associated virus-based gene loading of dendritic cells.

  Authors: Yong Liu, Maurizio Chiriva-Internati, Changxuan You, Rongcheng Luo, Hong You, C Krishna Prasad, Fabio Grizzi, Everardo Cobos, V Suzanne Klimberg, Helen Kay, Jawahar L Mehta, Paul L Hermonat

  Cancer gene therapy. 04/2005; 12(3):304-12.

  Antigen-targeted immunotherapy is an emerging treatment for breast cancer. However, useful breast cancer antigens are only found in a subset of cancer patients. BA46, also known as lactadherin, is aAntigen-targeted immunotherapy is an emerging treatment for breast cancer. However, useful breast cancer antigens are only found in a subset of cancer patients. BA46, also known as lactadherin, is a membrane-associated glycoprotein that is expressed in most breast cancer cells but not in general hematopoietic cell populations. Moreover, it is much more difficult to generate CTLs against self-antigens. We wished to determine if the use of recombinant adeno-associated virus (rAAV) type 2 vectors for gene-loading of dendritic cells (DCs) could generate rapid, effective cytotoxic T lymphocytes (CTLs) against BA46. We were able to demonstrate that AAV/BA46/Neo-loading of DCs resulted in: (1) BA46 expression in DCs, (2) chromosomal integration of the AAV/BA46/Neo vector within DCs, (3) strong, rapid BA46-specific, MHC class I-restricted CTLs in only 1 week, (4) T-cell populations with significant interferon-gamma (IFN-gamma) expression but low IL-4 expression, (5) high CD80 and CD86 expression in DCs, and (6) high CD8:CD4 and CD8:CD56 T cell ratios. These data suggest that rAAV-loading of DCs may be useful for immunotherapeutic protocols against self-antigens in addition to viral antigens and that the BA46 antigen is potentially appropriate for cell-mediated immunotherapeutic protocols addressing ductal breast cancer.

• Infection, replication, and cytopathology of human papillomavirus type 31 in trophoblasts.

  Authors: Hong You, Yong Liu, Nalini Agrawal, C Krishna Prasad, Maurizio Chiriva-Internati, Curtis L Lowery, Helen H Kay, Paul L Hermonat

  Virology. 12/2003; 316(2):281-9.

  Human papillomavirus (HPV) DNA is preferentially found in spontaneous abortions, specifically residing in trophoblasts, and transfected HPV-16 DNA replicates and produces progeny in 3A trophoblastsHuman papillomavirus (HPV) DNA is preferentially found in spontaneous abortions, specifically residing in trophoblasts, and transfected HPV-16 DNA replicates and produces progeny in 3A trophoblasts in culture. In this study 3A trophoblasts were shown to display both HPV receptors and infection by HPV-31b and HPV-6 virus resulted in de novo (increasing) HPV DNA replication in these cells (inhibited by neutralizing anti-HPV31b antibodies). Reverse transcription-polymerase chain reaction analysis revealed that E1;E4, E6, and L1 were significantly expressed at days 5 (early) and 10 (late), respectively, and in situ immunocytochemistry verified L1 protein expression. Perhaps most important, HPV 31b virus infection caused both a decrease in 3A trophoblast cell numbers in a dose-dependent manner and a low trophoblast-endometrial cell adhesion (both inhibited by neutralizing anti-HPV-31 antibodies). These data further support the hypothesis that HPVs are fully active in trophoblasts and may cause some spontaneous abortions.

• The adeno-associated virus major regulatory protein Rep78-c-Jun-DNA motif complex modulates AP-1 activity.

  Authors: C Krishna Prasad, Craig Meyers, De-Jin Zhan, Hong You, Maurizio Chiriva-Internati, Jawahar L Mehta, Yong Liu, Paul L Hermonat

  Virology. 10/2003; 314(1):423-31.

  Multiple epidemiologic studies show that adeno-associated virus (AAV) is negatively associated with cervical cancer (CX CA), a cancer which is positively associated with human papillomavirus (HPV)Multiple epidemiologic studies show that adeno-associated virus (AAV) is negatively associated with cervical cancer (CX CA), a cancer which is positively associated with human papillomavirus (HPV) infection. Mechanisms for this correlation may be by Rep78's (AAV's major regulatory protein) ability to bind the HPV-16 p97 promoter DNA and inhibit transcription, to bind and interfere with the functions of the E7 oncoprotein of HPV-16, and to bind a variety of HPV-important cellular transcription factors such as Sp1 and TBP. c-Jun is another important cellular factor intimately linked to the HPV life cycle, as well as keratinocyte differentiation and skin development. Skin is the natural host tissue for both HPV and AAV. In this article it is demonstrated that Rep78 directly interacts with c-Jun, both in vitro and in vivo, as analyzed by Western blot, yeast two-hybrid cDNA, and electrophoretic mobility shift-supershift assay (EMSA supershift). Addition of anti-Rep78 antibodies inhibited the EMSA supershift. Investigating the biological implications of this interaction, Rep78 inhibited the c-Jun-dependent c-jun promoter in transient and stable chloramphenicol acetyl-transferase (CAT) assays. Rep78 also inhibited c-Jun-augmented c-jun promoter as well as the HPV-16 p97 promoter activity (also c-Jun regulated) in in vitro transcription assays in T47D nuclear extracts. Finally, the Rep78-c-Jun interaction mapped to the amino-half of Rep78. The ability of Rep78 to interact with c-Jun and down-regulate AP-1-dependent transcription suggests one more mechanism by which AAV may modulate the HPV life cycle and the carcinogenesis process.

• Generation of recombinant skin in vitro by adeno-associated virus type 2 vector transduction.

  Authors: Nalini Agrawal, Hong You, Yong Liu, Maurizio Chiriva-Internati, John Bremner, Tarun Garg, Fabio Grizzi, C Krishna Prasad, Jawahar L Mehta, Paul L Hermonat

  Tissue engineering. 10(11-12):1707-15.

  It has long been recognized that skin may be a particularly good target for pharmacologic gene therapy and as a platform for the secretion of systemically distributed molecules. Adeno-associatedIt has long been recognized that skin may be a particularly good target for pharmacologic gene therapy and as a platform for the secretion of systemically distributed molecules. Adeno-associated virus type 2 (AAV) is a useful vector for skin gene therapy because skin is the natural host tissue for AAV, in which it functions as an autonomous parvovirus. We demonstrate here that recombinant (r) AAV vectors carrying the granulocyte-macrophage colony-stimulating factor (GM-CSF), human papillomavirus E6, or green fluorescent protein (GFP) transgene could transduce primary human keratinocytes in ex vivo culture. We further demonstrate that these transduced cells could be used to form a transgene-positive recombinant skin (r-skin), using the organotypic epithelial raft culture system. Transduction of keratinocytes was demonstrated by reverse transcriptase-polymerase chain reaction (RT-PCR) for RNA expression, enzyme-linked immunosorbent assay (ELISA) for product secretion, intracellular staining for protein expression, vector-chromosomal junction PCR and Southern blot analysis of proviral sequences, in situ immunohistochemistry analysis of protein expression, and ultraviolet light fluorescence for GFP expression. AAV/GM-CSF/Neo-infected keratinocyte/raft skins secreted GM-CSF at levels as high as 25 ng/cm(2) of skin and maintained expression to 60 days postinfection. These data support the utility and efficiency of AAV-based gene delivery to produce genetically altered keratinocytes and r-skin.