Amiya K. Banerjee

Gene expression of vesicular stomatitis virus genome RNA

It is just over 20 years since the landmark discovery was made by Baltimore et al. (1970) that vesicular stomatitis virus (VSV) packages within the mature virion the vital RNA-dependent RNA polymerase that transcribes the negative sense (anti-message sense) genome RNA into messenger RNAs. This unique observation provided the window of opportunity to study the regulation of VSV gene expression in the test tube and made VSV an early paradigm for RNA viral gene expression, a position that it has maintained ever since. In the past two decades, availability of information pertaining to the mechanism of gene expression of VSV has burgeoned, providing a better understanding of the structure and function of not only the VSV genome and its products but also those of negative-strand RNA viruses in general. The complete nucleotide sequence of the genome is now known (Rose and Schubert, 1987) and hence the sequence of the mRNAs and the deduced amino acid sequences of the viral proteins. All virus-coded proteins have been identified and their possible functions in the viral life cycle assigned. Notwithstanding this progress, the precise mechanisms by which the linear, single-strand RNA genome is transcribed into mRNAs and eventually replicated still remain a challenge to molecular virologists. VSV, a rhabdovirus, is thus a prototype model of all nonsegmented negative-strand RNA viruses, such as rabies, measles, mumps, parainfluenza, respiratory syncytial, and Sendai, to mention a few. While rabies is a rhabdovirus, all the other examples belong to the Paramyxoviridae family. The gene order and overall mechanism of gene expression in these two families of viruses are generally expected to be very similar. Available evidence has in fact demonstrated a commonality in their modes of transcription and replication. However, there are specific differences between VSV and the paramyxoviruses (as there are among the individual members of the Paramyxoviridae family) that warrant

Role of Nucleolin in Human Parainfluenza Virus Type 3 Infection of Human Lung Epithelial Cells

ABSTRACT Human parainfluenza virus type 3 (HPIV-3) is an airborne pathogen that infects human lung epithelial cells from the apical (luminal) plasma membrane domain. In the present study, we have identified cell surface-expressed nucleolin as a cellular cofactor required for the efficient cellular entry of HPIV-3 into human lung epithelial A549 cells. Nucleolin was enriched on the apical cell surface domain of A549 cells, and HPIV-3 interacted with nucleolin during entry. The importance of nucleolin during HPIV-3 replication was borne out by the observation that HPIV-3 replication was significantly inhibited following (i) pretreatment of cells with antinucleolin antibodies and (ii) preincubation of HPIV-3 with purified nucleolin prior to its addition to the cells. Moreover, HPIV-3 cellular internalization and attachment assays performed in the presence of antinucleolin antibodies and purified nucleolin revealed the requirement of nucleolin during HPIV-3 internalization but not during attachment. Thus, these results suggest that nucleolin expressed on the surfaces of human lung epithelial A549 cells plays an important role during HPIV-3 cellular entry.

Phospholipid Scramblase 1 Potentiates the Antiviral Activity of Interferon

ABSTRACT Phospholipid scramblase 1 (PLSCR1) is an interferon (IFN)- and growth factor-inducible, calcium-binding protein that either inserts into the plasma membrane or binds DNA in the nucleus depending on its state of palmyitoylation. In certain hematopoietic cells, PLSCR1 is required for normal maturation and terminal differentiation from progenitor cells as regulated by select growth factors, where it promotes recruitment and activation of Src kinases. PLSCR1 is a substrate of Src (and Abl) kinases, and transcription of the PLSCR1 gene is regulated by the same growth factor receptor pathways in which PLSCR1 potentiates afferent signaling. The marked transcriptional upregulation of PLSCR1 by IFNs led us to explore whether PLSCR1 plays an analogous role in cellular responses to IFN, with specific focus on antiviral activities. Accordingly, human cells in which PLSCR1 expression was decreased with short interfering RNA were rendered relatively insensitive to the antiviral activity of IFNs, resulting in higher titers of vesicular stomatitis virus (VSV) and encephalomyocarditis virus. Similarly, VSV replicated to higher titers in mouse PLSCR1−/− embryonic fibroblasts than in identical cells transduced to express PLSCR1. PLSCR1 inhibited accumulation of primary VSV transcripts, similar to the effects of IFN against VSV. The antiviral effect of PLSCR1 correlated with increased expression of a subset of IFN-stimulated genes (ISGs), including ISG15, ISG54, p56, and guanylate binding proteins. Our results suggest that PLSCR1, which is itself an ISG-encoded protein, provides a mechanism for amplifying and enhancing the IFN response through increased expression of a select subset of potent antiviral genes.

RNA editing in the phosphoprotein gene of the human parainfluenza virus type 3

n Abstractn n RNA editing of the human parainfluenza virus type 3 (HPIV3) phosphoprotein (P) gene was found to occur for the accession of an alternate discontinuous cistron. Editing occurred within a purine-rich sequence (AAUUAAAAAAGGGGG) found at the mRNA nucleotides 791–805. This sequence resembles an HPIV3 consensus transcription termination sequence and is located at the 5′-end of the putative D protein coding sequences. Editing at an alternate site (AAUUGGAAAGGAAAGG), mRNA nucleotides 1121–1136, for accession of a conserved V cistron, which is present in a number of paramyxovirus P genes, was not found to occur in HPIV3. In contrast with many other paramyxoviruses, editing was indiscriminate with the insertion of 1–12 additional G residues not present in the gene template. RNA editing was found to occur in both in vivo (HPIV3 infected cells) and in vitro (purified nucleocapsid complexes) synthesized mRNAs. Further, the in vitro prepared mRNA was edited regardless of whether the nucleocapsid complexes were transcribed in the presence or absence of uninfected human lung carcinoma (HLC) cell lysates. These results support the notion that RNA editing appears to be exclusively a function of viral proteins.n n

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.

Histidine-mediated RNA transfer to GDP for unique mRNA capping by vesicular stomatitis virus RNA polymerase

The RNA-dependent RNA polymerase L protein of vesicular stomatitis virus, a prototype of nonsegmented negative-strand (NNS) RNA viruses, forms a covalent complex with a 5′-phosphorylated viral mRNA-start sequence (L-pRNA), a putative intermediate in the unconventional mRNA capping reaction catalyzed by the RNA:GDP polyribonucleotidyltransferase (PRNTase) activity. Here, we directly demonstrate that the purified L-pRNA complex transfers pRNA to GDP to produce the capped RNA (Gpp-pRNA), indicating that the complex is a bona fide intermediate in the RNA transfer reaction. To locate the active site of the PRNTase domain in the L protein, the covalent RNA attachment site was mapped. We found that the 5′-monophosphate end of the RNA is linked to the histidine residue at position 1,227 (H1227) of the L protein through a phosphoamide bond. Interestingly, H1227 is part of the histidine-arginine (HR) motif, which is conserved within the L proteins of the NNS RNA viruses including rabies, measles, Ebola, and Borna disease viruses. Mutagenesis analyses revealed that the HR motif is required for the PRNTase activity at the step of the enzyme-pRNA intermediate formation. Thus, our findings suggest that an ancient NNS RNA viral polymerase has acquired the PRNTase domain independently of the eukaryotic mRNA capping enzyme during evolution and PRNTase becomes a rational target for designing antiviral agents.

Phosphoprotein of Human Parainfluenza Virus Type 3 Blocks Autophagosome-Lysosome Fusion to Increase Virus Production

Autophagy is a multistep process in which cytoplasmic components, including invading pathogens, are captured by autophagosomes that subsequently fuse with degradative lysosomes. Negative-strand RNA viruses, including paramyxoviruses, have been shown to alter autophagy, but the molecular mechanisms remain largely unknown. We demonstrate that human parainfluenza virus type 3 (HPIV3) induces incomplete autophagy by blocking autophagosome-lysosome fusion, resulting in increased virus production. The viral phosphoprotein (P) is necessary and sufficient to inhibition autophagosome degradation. P binds to SNAP29 and inhibits its interaction with syntaxin17, thereby preventing these two host SNARE proteins from mediating autophagosome-lysome fusion. Incomplete autophagy and resultant autophagosome accumulation increase extracellular viral production but do not affect viral protein synthesis. These findings highlight how viruses can block autophagosome degradation by disrupting the function of SNARE proteins.

Small-molecule activators of RNase L with broad-spectrum antiviral activity

RNase L, a principal mediator of innate immunity to viral infections in higher vertebrates, is required for a complete IFN antiviral response against certain RNA stranded viruses. dsRNA produced during viral infections activates IFN-inducible synthetases that produce 5′-phosphorylated, 2′,5′-oligoadenylates (2-5A) from ATP. 2-5A activates RNase L in a wide range of different mammalian cell types, thus blocking viral replication. However, 2-5A has unfavorable pharmacologic properties; it is rapidly degraded, does not transit cell membranes, and leads to apoptosis. To obtain activators of RNase L with improved drug-like properties, high-throughput screening was performed on chemical libraries by using fluorescence resonance energy transfer. Seven compounds were obtained that activated RNase L at micromolar concentrations, and structure–activity relationship studies resulted in identification of an additional four active compounds. Two lead compounds were shown to have a similar mechanistic path toward RNase L activation as the natural activator 2-5A. The compounds bound to the 2-5A-binding domain of RNase L (as determined by surface plasmon resonance and confirmed by computational docking), and the compounds induced RNase L dimerization and activation. Interestingly, the low-molecular-weight activators of RNase L had broad-spectrum antiviral activity against diverse types of RNA viruses, including the human pathogen human parainfluenza virus type 3, yet these compounds by themselves were not cytotoxic at the effective concentrations. Therefore, these RNase L activators are prototypes for a previously uncharacterized class of broad-spectrum antiviral agents.

Role of cellular casein kinase II in the function of the phosphoprotein (P) subunit of RNA polymerase of vesicular stomatitis virus.

The phosphorylation of the P protein of vesicular stomatitis virus by cellular casein kinase II (CKII) is essential for its activity in viral transcription. Recent in vitro studies have demonstrated that CKII converts the inactive unphosphorylated form of P (P0) to an active phosphorylated form P1, after phosphorylation at two serine residues, Ser-59 and Ser-61. To gain insight into the role of CKII-mediated phosphorylation in the structure and function of the P protein, we have carried out circular dichroism (CD) and biochemical analyses of both P0 and P1. The results of CD analyses reveal that phosphorylation of P0 to P1 significantly increases the predicted α-helical structure of the P1 protein from 27 to 48%. The phosphorylation defective double serine mutant (P59/61), which is transcriptionally inactive, possesses a secondary structure similar to that of P0. P1, at a protein concentration of 50 μg/ml, elutes from a gel filtration column apparently as a dimer, whereas both P0 and the double serine mutant elute as a monomer at the same concentration. Interestingly, unlike wild-type P1 protein, the P mutants in which either Ser-59 or Ser-61 is altered to alanine required a high concentration of CKII for optimal phosphorylation. We demonstrate here that phosphorylation of either Ser-59 or Ser-61 is necessary and sufficient to transactivate L polymerase although alteration of one serine residue significantly decreases its affinity for CKII. We have also shown that P1 binds to the N-RNA template more efficiently than P0 and the formation of P1 is a prerequisite for the subsequent phosphorylation by L protein-associated kinase. In addition, mutant P59/61 acts as a transdominant negative mutant when used in a transcription reconstitution assay in the presence of wild-type P protein.

Anti-malaria drug blocks proteotoxic stress response: Anti-cancer implications

The number of physical conditions and chemical agents induce accumulation of misfolded proteins creating proteotoxic stress. This leads to activation of adaptive pro-survival pathway, known as heat shock response (HSR), resulting in expression of additional chaperones. Several cancer treatment approaches, such as proteasome inhibitor Bortezomib and hsp90 inhibitor geldanamycin, involve activation of proteotoxic stress. Low efficacy of these therapies is likely due to the protective effects of HSR induced in treated cells, making this pathway an attractive target for pharmacological suppression. We found that the anti-malaria drugs quinacrine (QC) and emetine prevented HSR in cancer cells, as judged by induction of hsp70 expression. As opposed to emetine, which inhibited general translation, QC did not affect protein synthesis, but rather suppressed inducible HSF1-dependent transcription of the hsp70 gene in a relatively selective manner. The treatment of tumor cells in vitro with a combination of non-toxic concentrations of QC and proteotoxic stress inducers resulted in rapid induction of apoptosis. The effect was similar if QC was substituted by siRNA against hsp70, suggesting that the HSR inhibitory activity of QC was responsible for cell sensitization to proteotoxic stress inducers. QC was also found to enhance the antitumor efficacy of proteotoxic stress inducers in vivo: combinatorial treatment with 17-DMAG+QC resulted in suppression of tumor growth in two mouse syngeneic models. These results reveal that QC is an inhibitor of HSF1-mediated HSR. As such, this compound has significant clinical potential as an adjuvant in therapeutic strategies aimed at exploiting the cytotoxic potential of proteotoxic stress.