Inflammasome Antagonism by Human Parainfluenza Virus Type 3 C Protein.

Human parainfluenza virus type 3 (HPIV3) is a negative-sense single-stranded RNA virus belonging to the Paramyxoviridae family. HPIV3 is a lung-tropic virus causing airway diseases, including pneumonia, croup, and bronchiolitis, during infancy and childhood. The activation of the inflammasome by pathogens results in the production of proinflammatory cytokines such as interleukin-1ß (IL-1ß) during infection. Thus, the inflammasome-mediated proinflammatory response plays a critical role in regulating the immune response and virus clearance. The inflammasome is a multimeric protein complex triggering caspase-1 activation. Activated caspase-1 cleaves pro-IL-1ß into its mature (and active) secretory form. Our study revealed inflammasome activation in macrophages following HPIV3 infection. Specifically, the activation of the NLRP3/ASC inflammasome resulted in the production of mature IL-1ß from HPIV3-infected cells. Furthermore, Toll-like receptor 2 (TLR2) activation (first signal) and potassium efflux (second signal) constituted two cellular events mediating inflammasome activation following HPIV3 infection. During our studies, we surprisingly identified the HPIV3 C protein as an antagonist of inflammasome activation. The HPIV3 C protein is an accessory protein encoded by the open reading frame of the viral phosphoprotein (P) gene. The HPIV3 C protein interacted with the NLRP3 protein and blocked inflammasome activation by promoting the proteasomal degradation of the NLRP3 protein. Thus, our studies report NLRP3/ASC inflammasome activation by HPIV3 via TLR2 signaling and potassium efflux. Furthermore, we have identified HPIV3 C as a viral component involved in antagonizing inflammasome activation.IMPORTANCE Human parainfluenza virus type 3 (HPIV3) is a paramyxovirus that causes respiratory tract diseases during infancy and childhood. Currently, there is no effective vaccine or antiviral therapy for HPIV3. Therefore, in order to develop anti-HPIV3 agents (therapeutics and vaccines), it is important to study the HPIV3-host interaction during the immune response. Inflammasomes play an important role in the immune response. Inflammasome activation by HPIV3 has not been previously reported. Our studies demonstrated inflammasome activation by HPIV3 in macrophages. Specifically, HPIV3 activated the NLRP3/ASC inflammasome by TLR2 activation and potassium efflux. C proteins of paramyxoviruses are accessory proteins encoded by the viral phosphoprotein gene. The role of the C protein in inflammasome regulation was unknown. Surprisingly, our studies revealed that the HPIV3 C protein antagonizes inflammasome activation. In addition, we highlighted for the first time a mechanism utilized by paramyxovirus accessory proteins to block inflammasome activation. The HPIV3 C protein interacted with the NLRP3 protein to trigger the proteasomal degradation of the NLRP3 protein.

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.

N-terminal phosphorylation of phosphoprotein of vesicular stomatitis virus is required for preventing nucleoprotein from binding to cellular RNAs and for functional template formation.

The phosphoprotein (P) of vesicular stomatitis virus (VSV) plays essential roles in viral RNA synthesis. It associates with nascent nucleoprotein (N) to form N(0)-P (free of RNAs), thereby preventing the N from binding to cellular RNAs and maintaining the N in a viral genomic RNA encapsidation-competent form for transcription and replication. The contributions of phosphorylation of P to transcription and replication have been studied intensively, but a concrete mechanism of action still remains unclear. In this study, using a VSV minigenome system, we demonstrated that a mutant of P lacking N-terminal phosphorylation (P3A), in which the N-terminal phosphate acceptor sites are replaced with alanines (S60/A, T62/A, and S64/A), does not support transcription and replication. However, results from protein interaction assays showed that P3A self-associates and interacts with N and the large protein (L) as efficiently as P does. Furthermore, purified recombinant P3A from Sf21 cells supported transcription in an in vitro transcription reconstitution assay. We also proved that P3A is not distributed intranuclearly in vivo. CsCl gradient centrifugation showed that P3A is incapable of preventing N from binding to cellular RNAs and therefore prevents functional template formation. Taken together, our results demonstrate that N-terminal phosphorylation is indispensable for P to prevent N from binding to nonviral RNAs and to maintain the N-specific encapsidation of viral genomic RNA for functional template formation.

An unconventional pathway of mRNA cap formation by vesiculoviruses.

mRNAs of vesicular stomatitis virus (VSV), a prototype of nonsegmented negative strand (NNS) RNA viruses (e.g., rabies, measles, mumps, Ebola, and Borna disease viruses), possess the 5'-terminal cap structure identical to that of eukaryotic mRNAs, but the mechanism of mRNA cap formation is distinctly different from the latter. The elucidation of the unconventional capping of VSV mRNA remained elusive for three decades since the discovery of the cap structure in some viral and eukaryotic mRNAs in 1975. Only recently our biochemical studies revealed an unexpected strategy employed by vesiculoviruses (VSV and Chandipura virus, an emerging arbovirus) to generate the cap structure. This article summarizes the historical and current research that led to the discovery of the novel vesiculoviral mRNA capping reaction.

Domain within the C protein of human parainfluenza virus type 3 that regulates interferon signaling.

Human parainfluenza virus type 3 (HPIV3), one of the paramyxoviruses, uses its accessory C protein as an antagonist against interferon (IFN)-mediated host innate immunity. We have previously shown that the C protein significantly decreased the IFN-induced phosphorylation of signal transducer and activator of transcription (Stat) 1 and the formation of gamma IFN activation factor (GAF) complex, thus abrogating the antiviral activity of the IFNs against vesicular stomatitis virus (VSV) replication. Here, by mutational analyses we demonstrated that the N-terminal truncation of the C protein (CNdelta25 and CNdelta50) substantially (approximately 50%) recovers the IFN-induced responses, suggesting the critical role of the N-terminal region of the C protein in IFN signaling. Furthermore, our results indicate that the charged amino acid residues within the N-terminal region of the C protein regulate the antagonistic effect of the C protein on IFN signaling.

Structural and functional properties of the vesicular stomatitis virus nucleoprotein-RNA complex as revealed by proteolytic digestion.

To gain insight into the structural and functional properties of the vesicular stomatitis virus nucleocapsid-RNA complex (vN-RNA), we analyzed it by treatment with proteolytic enzymes. Chymotrypsin treatment to the vN-RNA results in complete digestion of the C-terminal 86 amino acids of the N protein. The residual chymotrypsin resistant vN-RNA complex (vDeltaN-RNA) carrying N-terminal 336 amino acids of the N protein (DeltaN) was inactive in transcription. The DeltaN protein retained its capability to protect the genomic RNA from nuclease digestion but failed to interact to the P protein. Interestingly, addition of excess amount of P protein rendered the vN-RNA complex resistant to the chymotrypsin digestion. Finally, our data revealed that the recombinant N-RNA complex purified from bacteria (bN-RNA) is resistant to chymotrypsin digestion, suggesting that the C-terminal unstructured domain (C-loop) remains inaccessible to protease digestion. Detailed comparative analyses of the vN-RNA and vDeltaN-RNA are discussed.

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.

The HR motif in the RNA-dependent RNA polymerase L protein of Chandipura virus is required for unconventional mRNA-capping activity.

Chandipura virus (CHPV) is an emerging human pathogen associated with acute encephalitis and is related closely to vesicular stomatitis virus (VSV), a prototype rhabdovirus. Here, we demonstrate that the RNA polymerase L protein of CHPV exhibits a VSV-like RNA:GDP polyribonucleotidyltransferase (PRNTase) activity, which transfers the 5'-monophosphorylated (p-) viral mRNA start sequence to GDP to produce a capped RNA, and that the conserved HR motif in the CHPV L protein is essential for the PRNTase activity. Interestingly, the CHPV L protein was found to form two distinct SDS-resistant complexes with the CHPV mRNA and leader RNA start sequences; mutations in the HR motif significantly reduced the formation of the former complex (a putative covalent enzyme-pRNA intermediate in the PRNTase reaction), but not the latter complex. These results suggest that the rhabdoviral L proteins universally use the active-site HR motif for the PRNTase reaction at the step of the enzyme-pRNA intermediate formation.

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.

N-terminally truncated C protein, CNDelta25, of human parainfluenza virus type 3 is a potent inhibitor of viral replication.

The C protein of human parainfluenza virus type 3 (HPIV3) is a multifunctional accessory protein that inhibits viral transcription and interferon (IFN) signaling. In the present study, we found that removal of N-terminal 25 or 50 amino acid residues from the C protein (CNDelta25 or CNDelta50) totally abolished viral RNA synthesis in the HPIV3 minigenome system. Further N-terminal or C-terminal deletion impaired the inhibitory ability of CNDelta25 and CNDelta50. Subsequent mutagenesis analysis suggested that the N-terminal-charged amino acid residues (K3, K6, K12, E16, and R24) contribute to the higher inhibition caused by CNDelta25 than the C protein. Consistent with viral RNA synthesis inhibition, the growth of HPIV3 was significantly decreased by 5 logs in HeLa-derived cell line expressing CNDelta25. Interestingly, replication of respiratory syncytial virus (RSV), another important respiratory tract pathogen, was also strongly inhibited in the presence of CNDelta25. These findings provide a promising potential to use CNDelta25 as an antiviral agent against the clinically important respiratory tract diseases caused by HPIV3 and RSV.

Evidence for phosphorylation of human parainfluenza virus type 3 C protein: mutant C proteins exhibit variable inhibitory activities in vitro.

The P mRNA of human parainfluenza virus type 3, like other members of the subfamily Paramyxovirinae, gives rise to several polypeptides, one amongst them, the C protein, which is involved in inhibition of viral RNA synthesis as well as counteracting the host interferon signaling pathway. As a further step towards characterizing the function of C protein we present evidence to demonstrate the phosphorylation of C protein. Evidence for this observation emerged from deletion mapping studies coupled with mass spectroscopy analysis confirming residues S7, S22, S47T48 and S81 residues as the phosphorylation sites within the NH(2)-terminus of C protein. Here, we utilized a HPIV 3 minigenome replication assay and real time RT-PCR analysis to measure the relative RNA levels synthesized in the presence of mutant C proteins. Mutants S7A and S81A displayed low levels of RNA while mutant 5A that was devoid of all these phosphorylation sites exhibited high RNA level in comparison to wild type C during transcription. Interestingly, high levels of RNA were observed in the presence of S81A and mutant 5A during replication. Taken together, our results indicate that phosphorylation may differentially affect the inhibitory activity of C protein thereby regulating viral RNA synthesis.

Phosphoprotein, P of human parainfluenza virus type 3 prevents self-association of RNA-dependent RNA polymerase, L.

The RNA-dependent RNA-polymerase (RdRp) of human parainfluenza virus type 3 (HPIV3) is a large protein (L, 2233 amino acids), and along with the phosphoprotein (P, 603 amino acids) forms a heterocomplex that transcribes the genome RNA into mRNAs in vitro and in vivo that are 5'-capped and methylated and 3'-polyadenylated. The interaction of the P protein, an obligatory cofactor, imparts the RdRp activity of the L protein, which is otherwise inactive. The precise mechanism underlying this activation process remains unknown. Several recent reports suggested that the L proteins of paramyxoviruses, when expressed alone, self-associate to form an oligomeric structure. The presumptive oligomerization domain lies in the N-terminal part of the L protein (for HPIV3, 889 amino acids). Here, we demonstrate that a series of N-terminally deleted L proteins as well as several truncated proteins that span different regions of the L protein can also efficiently co-immunoprecipitate the full length L protein. In addition, by several biochemical parameters, the L-L interaction was shown to form aggregates rather than oligomers. In contrast, when the P protein was co-expressed with the L protein, the former bound to a domain spanning the N-terminal 1060 amino acids of the latter, which prevented L-L self-association, resulting in the formation of structurally competent and functionally active RdRp.

Formation of guanosine(5')tetraphospho(5')adenosine cap structure by an unconventional mRNA capping enzyme of vesicular stomatitis virus.

The RNA-dependent RNA polymerase L protein of vesicular stomatitis virus (VSV) elicits GTPase and RNA:GDP polyribonucleotidyltransferase (PRNTase) activities to produce a 5'-cap core structure, guanosine(5')triphospho(5')adenosine (GpppA), on viral mRNAs. Here, we report that the L protein produces an unusual cap structure, guanosine(5')tetraphospho(5')adenosine (GppppA), that is formed by the transfer of the 5'-monophosphorylated viral mRNA start sequence to GTP by the PRNTase activity before the removal of the gamma-phosphate from GTP by GTPase. Interestingly, GppppA-capped and polyadenylated full-length mRNAs were also found to be synthesized by an in vitro transcription system with the native VSV RNP.

Different effect of proteasome inhibition on vesicular stomatitis virus and poliovirus replication.

Proteasome activity is an important part of viral replication. In this study, we examined the effect of proteasome inhibitors on the replication of vesicular stomatitis virus (VSV) and poliovirus. We found that the proteasome inhibitors significantly suppressed VSV protein synthesis, virus accumulation, and protected infected cells from toxic effect of VSV replication. In contrast, poliovirus replication was delayed, but not diminished in the presence of the proteasome inhibitors MG132 and Bortezomib. We also found that inhibition of proteasomes stimulated stress-related processes, such as accumulation of chaperone hsp70, phosphorylation of eIF2alpha, and overall inhibition of translation. VSV replication was sensitive to this stress with significant decline in replication process. Poliovirus growth was less sensitive with only delay in replication. Inhibition of proteasome activity suppressed cellular and VSV protein synthesis, but did not reduce poliovirus protein synthesis. Protein kinase GCN2 supported the ability of proteasome inhibitors to attenuate general translation and to suppress VSV replication. We propose that different mechanisms of translational initiation by VSV and poliovirus determine their sensitivity to stress induced by the inhibition of proteasomes. To our knowledge, this is the first study that connects the effect of stress induced by proteasome inhibition with the efficiency of viral infection.

Quercetinase pirin makes poliovirus replication resistant to flavonoid quercetin.

Flavonoid quercetin and its derivative, methylquercetin, inhibit the replication of poliovirus in several cell lines. Here, we show that replication of poliovirus is inhibited by quercetin and that the extent of this inhibition depends on the intracellular content of pirin, a quercetinase. HeLa cells contain higher content of pirin protein than normal kidney human epithelial (NKE) or 293 cells do. Poliovirus replication in HeLa cells is significantly more resistant to quercetin than its replication in NKE and 293 cells. Overexpression of pirin reduced antiviral inhibitory effect of quercetin, while siRNA-induced suppression of pirin level made poliovirus replication more sensitive to the flavonoid. The results suggest that quercetinase activity of pirin determines the resistance of poliovirus infection to quercetin.

Inhibition of human parainfluenza virus type 3 infection by novel small molecules.

Human parainfluenza virus type 3 (HPIV3) is an important respiratory tract pathogen of infants and children. There are no vaccines or antivirals currently approved for prevention or treatment of HPIV3 infection. Towards developing an antiviral therapy to combat HPIV3 infection, we have established a green fluorescent protein (GFP)-tagged HPIV3 infected-cell assay and used it for screening of a small molecule library obtained from ChemBridge Diver. Two novel small molecules (C5 and C7) which shared structural similarities were identified and their inhibitory effects on HPIV3 were confirmed in CV-1 and human lung epithelium A549 cells by plaque assay, Western blot and Northern blot analyses. C5 and C7 effectively prevented the cytopathic effect in cells infected with HPIV3, achieving IC(50) values of 2.36 microM and 0.08 microM, respectively, for infectious virus production. The inhibition appears to be at the primary transcriptional level of HPIV3 life cycle based on sequential time course test, binding and internalization assays, and finally by a minigenome transcription assay in cells as well as measuring viral transcripts in cells in the presence of anisomycin. Interestingly, vesicular stomatitis virus (VSV), another member of mononegavirales order, was also inhibited by these compounds, whereas poliovirus-a picornavirus was not. Use of these inhibitors has a strong potential to develop novel antiviral agents against this important human pathogen.

Interaction of vesicular stomatitis virus P and N proteins: identification of two overlapping domains at the N terminus of P that are involved in N0-P complex formation and encapsidation of viral genome RNA.

The nucleocapsid (N) protein of nonsegmented negative-strand (NNS) RNA viruses, when expressed in eukaryotic cells, aggregates and forms nucleocapsid-like complexes with cellular RNAs. The phosphoprotein (P) has been shown to prevent such aggregation by forming a soluble complex with the N protein free from cellular RNAs (designated N(0)). The N(0)-P complex presumably mediates specific encapsidation of the viral genome RNA. The precise mechanism by which the P protein carries out this function remains unclear. Here, by using a series of deleted and truncated mutant forms of the P protein of vesicular stomatitis virus (VSV), Indiana serotype, we present evidence that the N-terminal 11 to 30 amino acids (aa) of the P protein are essential in keeping the N protein soluble. Furthermore, glutathione S-transferase fused to the N-terminal 40 aa by itself is able to form the N(0)-P complex. Interestingly, the N-terminal 40-aa stretch failed to interact with the viral genome N-RNA template whereas the C-terminal 72 aa of the P protein interacted specifically with the latter. With an in vivo VSV minigenome transcription system, we further show that a deletion mutant form of P (PDelta1-10) lacking the N-terminal 10 aa which is capable of forming the N(0)-P complex was unable to support VSV minigenome transcription, although it efficiently supported transcription in vitro in a transcription-reconstitution reaction when used as purified protein. However, the same mutant protein complemented minigenome transcription when expressed together with a transcription-defective P deletion mutant protein containing N-terminal aa 1 to 210 (PDeltaII+III). Since the minigenome RNA needs to be encapsidated before transcription ensues, it seems that the entire N-terminal 210 aa are required for efficient genome RNA encapsidation. Taking these results together, we conclude that the N-terminal 11 to 30 aa are required for N(0)-P complex formation but the N-terminal 210 aa are required for genome RNA encapsidation.

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.

Unconventional mechanism of mRNA capping by the RNA-dependent RNA polymerase of vesicular stomatitis virus.

All known eukaryotic and some viral mRNA capping enzymes (CEs) transfer a GMP moiety of GTP to the 5'-diphosphate end of the acceptor RNA via a covalent enzyme-GMP intermediate to generate the cap structure. In striking contrast, the putative CE of vesicular stomatitis virus (VSV), a prototype of nonsegmented negative-strand (NNS) RNA viruses including rabies, measles, and Ebola, incorporates the GDP moiety of GTP into the cap structure of transcribing mRNAs. Here, we report that the RNA-dependent RNA polymerase L protein of VSV catalyzes the capping reaction by an RNA:GDP polyribonucleotidyltransferase activity, in which a 5'-monophosphorylated viral mRNA-start sequence is transferred to GDP generated from GTP via a covalent enzyme-RNA intermediate. Thus, the L proteins of VSV and, by extension, other NNS RNA viruses represent a new class of viral CEs, which have evolved independently from known eukaryotic CEs.