Inhibition Of Viral RNA Synthesis Research Articles

In vitro antiviral activity of abietane diterpenoids isolated from Torreya nucifera against rotavirus infection

Although vaccines have been developed for rotavirus infections, there is currently no effective therapeutic treatment. Therefore, this study aimed to evaluate the efficacy of 18-hydroxyferruginol (1) and 18-oxoferruginol (2) isolated from Torreya nucifera (T. nucifera) against bovine G8P[7] and porcine G5P[7] rotaviruses using two different assay strategies: 1) reduction of viral infectivity by neutralizing the virus (virucidal assay) and 2) inhibition of viral replication after infection (post-treatment assay). In the post-treatment assay, compounds 1 and 2 exhibited strong anti-rotavirus activity, with 50% effective concentration values of 24.7 μM (selectivity index; SI= 2.52) and 23.2 μM (SI= 1.75) against bovine G8P[7], 21.1 μM (SI= 2.95) and 22.6 μM (SI= 1.80) against porcine G5P[7], respectively. During viral replication, the two compounds demonstrated stronger inhibition of viral RNA synthesis in the late stages (18 h) than in the early stages (6 h). Compounds 1 and 2 also inhibited the synthesis of viral proteins such as VP6, as determined by immunofluorescence assay. Thus, it appears that compounds 1 and 2 isolated from T. nucifera possess strong antiviral activity against rotaviruses, inhibit viral replication, and may be developed into potential plant-derived therapeutic candidates against rotavirus infection.

Heterogeneous Nuclear Ribonucleoprotein L Negatively Regulates Foot-and-Mouth Disease Virus Replication through Inhibition of Viral RNA Synthesis by Interacting with the Internal Ribosome Entry Site in the 5′ Untranslated Region

Upon infection, the highly structured 5' untranslated region (5' UTR) of picornavirus is involved in viral protein translation and RNA synthesis. As a critical element in the 5' UTR, the internal ribosome entry site (IRES) binds to various cellular proteins to function in the processes of picornavirus replication. Foot-and-mouth disease virus (FMDV) is an important member in the family Picornaviridae, and its 5' UTR contains a functional IRES element. In this study, the cellular heterogeneous nuclear ribonucleoprotein L (hnRNP L) was identified as an IRES-binding protein for FMDV by biotinylated RNA pulldown assays, mass spectrometry (MS) analysis, and determination of hnRNP L-IRES interaction regions. Further, we found that hnRNP L inhibited the growth of FMDV through binding to the viral IRES and that the inhibitory effect of hnRNP L on FMDV growth was not due to FMDV IRES-mediated translation, but to influence on viral RNA synthesis. Finally, hnRNP L was demonstrated to coimmunoprecipitate with RNA-dependent RNA polymerase (3Dpol) in an FMDV RNA-dependent manner in the infected cells. Thus, our results suggest that hnRNP L, as a critical IRES-binding protein, negatively regulates FMDV replication by inhibiting viral RNA synthesis, possibly by remaining in the replication complex.IMPORTANCE Picornaviruses, as a large family of human and animal pathogens, cause a bewildering array of disease syndromes. Many host factors are implicated in the pathogenesis of these viruses, and some proteins interact with the viral IRES elements to affect function. Here, we report for the first time that cellular hnRNP L specifically interacts with the IRES of the picornavirus FMDV and negatively regulates FMDV replication through inhibiting viral RNA synthesis. Further, our results showed that hnRNP L coimmunoprecipitates with FMDV 3Dpol in a viral RNA-dependent manner, suggesting that it may remain in the replication complex to function. The data presented here would facilitate further understanding of virus-host interactions and the pathogenesis of picornavirus infections.

Benzamidine ML336 inhibits plus and minus strand RNA synthesis of Venezuelan equine encephalitis virus without affecting host RNA production

Venezuelan equine encephalitis virus (VEEV) is an alphavirus that is endemic to the Americas. VEEV outbreaks occur periodically and cause encephalitis in both humans and equids. There are currently no therapeutics or vaccines for treatment of VEEV in humans. Our group has previously reported on the development of a benzamidine VEEV inhibitor, ML336, which shows potent antiviral activity in both in vitro and in vivo models of infection. In cell culture experiments, ML336 inhibits viral RNA synthesis when added 2–4 h post-infection, and mutations conferring resistance occur within the viral nonstructural proteins (nsP2 and nsP4). We hypothesized that ML336 targets an activity of the viral replicase complex and inhibits viral RNA synthesis. To test this hypothesis, we employed various biochemical and cellular assays. Using structural analogues of ML336, we demonstrate that the cellular antiviral activity of these compounds correlates with their inhibition of viral RNA synthesis. For instance, the IC50 of ML336 for VEEV RNA synthesis inhibition was determined as 1.1 nM, indicating potent anti-RNA synthesis activity in the low nanomolar range. While ML336 efficiently inhibited VEEV RNA synthesis, a much weaker effect was observed against the Old World alphavirus Chikungunya virus (IC50 > 4 μM), agreeing with previous data from a cell based assay. Using a tritium incorporation assay, we demonstrated that there was no significant inhibition of cellular transcription. With a combination of fluorography, strand-specific qRT-PCR, and tritium incorporation, we demonstrated that ML336 inhibits the synthesis of the positive sense genomic, negative sense template, and subgenomic RNAs of VEEV. Based on these results, we propose that the mechanism of action for this class of antiviral compounds is inhibition of viral RNA synthesis through interaction with the viral replicase complex.

Anti-influenza virus activity of a salcomine derivative mediated by inhibition of viral RNA synthesis.

Influenza virus infection is a major threat to global health. Although vaccines and anti-influenza virus drugs are available, annual influenza virus epidemics result in severe illness, and an influenza pandemic occurs every 20-30years. To identify candidate anti-influenza virus compounds, we screened approximately 5,000 compounds in an in-house library. We identified MZ7465, a salcomine derivative, as a potent inhibitor of influenza virus propagation. We analyzed the antiviral propagation mechanism of the hit compound by determining the amounts of viral proteins and RNA in infected cells treated with or without the hit compound. Treatment of infected cells with MZ7465 decreased both viral protein and RNA synthesis. In addition, an in vitro assay showed that viral RNA synthesis was directly inhibited by MZ7465. These results suggest that salcomine and its derivatives are potential candidates for the treatment of influenza virus infections.

Antiviral activity of A771726, the active metabolite of leflunomide, against Junín virus.

The aim of this study was to investigate the effect of A771726, the active metabolite of leflunomide, (CONICET-UBA), Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Ciudad against the infection with Junín virus (JUNV), agent of Argentine hemorrhagic fever (AHF). The treatment with non-cytotoxic concentrations of A771726 of Vero and A549 cells infected with JUNV inhibited virus replication in a dose-dependent manner, as determined by virus yield reduction assay. The antiviral effectiveness of A771726 was not importantly affected by the multiplicity of infection and the virus strain. Moreover, the combination of A771726 and ribavirin had a significantly more potent antiviral activity than each single drug treatment. Mechanistic studies showed that the main action of A771726 is exerted before 6 h of JUNV infection. Accordingly, inhibition of viral RNA synthesis was detected in treated infected cells by real time RT-PCR. The exogenous addition of uridine or orotic acid produced a partial reversal of the inhibitory effect of A771726 on infective virus production whereas a total reversion was detected on JUNV RNA synthesis, probably by restoration of the enzymatic activity of dihydroorotate dehydrogenase (DHODH) and the intracellular pyrimidine pools. In conclusion, these results suggest that the antiviral target would be viral RNA synthesis through pyrimidine depletion, but any other effect of the compound on JUNV infection cannot be excluded. This study opens the possibility of the therapeutic application of a wide spectrum host-targeted compound alone or in combination with ribavirin to combat AHF as well as other human pathogenic arenaviruses.

Nuclear TRIM25 Specifically Targets Influenza Virus Ribonucleoproteins to Block the Onset of RNA Chain Elongation

TRIM25 is an E3 ubiquitin ligase that activates RIG-I to promote the antiviral interferon response. The NS1 protein from all strains of influenza A virus binds TRIM25, although not all virus strains block the interferon response, suggesting alternative mechanisms for TRIM25 action. Here we present a nuclear role for TRIM25 in specifically restricting influenza A virus replication. TRIM25 inhibits viral RNA synthesis through a direct mechanism that is independent of its ubiquitin ligase activity and the interferon pathway. This activity can be inhibited by the viral NS1 protein. TRIM25 inhibition of viral RNA synthesis results from its binding to viral ribonucleoproteins (vRNPs), the structures containing individual viral RNA segments, the viral polymerase, and multiple viral nucleoproteins. TRIM25 binding does not inhibit initiation of capped-RNA-primed viral mRNA synthesis by the viral polymerase. Rather, the onset of RNA chain elongation is inhibited because TRIM25 prohibits the movement of RNA into the polymerase complex.

Influenza B virus non-structural protein 1 counteracts ISG15 antiviral activity by sequestering ISGylated viral proteins

The ubiquitin-like protein ISG15 and its conjugation to proteins (ISGylation) are strongly induced by type I interferon. Influenza B virus encodes non-structural protein 1 (NS1B) that binds human ISG15 and provides an appropriate model for determining how ISGylation affects virus replication in human cells. Here using a recombinant virus encoding a NS1B protein defective in ISG15 binding, we show that NS1B counteracts ISGylation-mediated antiviral activity by binding and sequestering ISGylated viral proteins, primarily ISGylated viral nucleoprotein (NP), in infected cells. ISGylated NP that is not sequestered by mutant NS1B acts as a dominant-negative inhibitor of oligomerization of the more abundant unconjugated NP. Consequently formation of viral ribonucleoproteins that catalyse viral RNA synthesis is inhibited, causing decreased viral protein synthesis and virus replication. We verify that ISGylated NP is largely responsible for inhibition of viral RNA synthesis by generating recombinant viruses that lack known ISGylation sites in NP.

Regulation of Viral RNA Synthesis by the V Protein of Parainfluenza Virus 5

Paramyxoviruses include many important animal and human pathogens. The genome of parainfluenza virus 5 (PIV5), a prototypical paramyxovirus, encodes a V protein that inhibits viral RNA synthesis. In this work, the mechanism of inhibition was investigated. Using mutational analysis and a minigenome system, we identified regions in the N and C termini of the V protein that inhibit viral RNA synthesis: one at the very N terminus of V and the second at the C terminus of V. Furthermore, we determined that residues L16 and I17 are critical for the inhibitory function of the N-terminal region of the V protein. Both regions interact with the nucleocapsid protein (NP), an essential component of the viral RNA genome complex (RNP). Mutations at L16 and I17 abolished the interaction between NP and the N-terminal domain of V. This suggests that the interaction between NP and the N-terminal domain plays a critical role in V inhibition of viral RNA synthesis by the N-terminal domain. Both the N- and C-terminal regions inhibited viral RNA replication. The C terminus inhibited viral RNA transcription, while the N-terminal domain enhanced viral RNA transcription, suggesting that the two domains affect viral RNA through different mechanisms. Interestingly, V also inhibited the synthesis of the RNA of other paramyxoviruses, such as Nipah virus (NiV), human parainfluenza virus 3 (HPIV3), measles virus (MeV), mumps virus (MuV), and respiratory syncytial virus (RSV). This suggests that a common host factor may be involved in the replication of these paramyxoviruses. We identified two regions of the V protein that interact with NP and determined that one of these regions enhances viral RNA transcription via its interaction with NP. Our data suggest that a common host factor may be involved in the regulation of paramyxovirus replication and could be a target for broad antiviral drug development. Understanding the regulation of paramyxovirus replication will enable the rational design of vaccines and potential antiviral drugs.

Solar and temperature treatments affect the ability of human rotavirus wa to bind to host cells and synthesize viral RNA.

Rotavirus, the leading cause of diarrheal diseases in children under the age of five, is often resistant to conventional wastewater treatment and thus can remain infectious once released into the aquatic environment. Solar and heat treatments can inactivate rotavirus, but it is unknown how these treatments inactivate the virus on a molecular level. To answer this question, our approach was to correlate rotavirus inactivation with the inhibition of portions of the virus life cycle as a means to identify the mechanisms of solar or heat inactivation. Specifically, the integrity of the rotavirus NSP3 gene, virus-host cell interaction, and viral RNA synthesis were examined after heat (57°C) or solar treatment of rotavirus. Only the inhibition of viral RNA synthesis positively correlated with a loss of rotavirus infectivity; 57°C treatment of rotavirus resulted in a decrease of rotavirus RNA synthesis at the same rate as rotavirus infectivity. These data suggest that heat treatment neutralized rotaviruses primarily by targeting viral transcription functions. In contrast, when using solar disinfection, the decrease in RNA synthesis was responsible for approximately one-half of the decrease in infectivity, suggesting that other mechanisms, including posttranslational, contribute to inactivation. Nevertheless, both solar and heat inactivation of rotaviruses disrupted viral RNA synthesis as a mechanism for inactivation.

Mechanism of action for respiratory syncytial virus inhibitor RSV604.

Respiratory syncytial virus (RSV) is the leading cause of acute lower respiratory tract infections in young children and other high-risk populations. RSV nucleoprotein (N) is essential for virus assembly and replication as part of the viral ribonucleoprotein (RNP) complex. RSV604 was a putative N inhibitor in phase 2 clinical trials whose molecular mechanism of action (MoA) was not well understood. This study investigated the cell line-dependent potency of RSV604 and demonstrated its direct binding to the N protein in vitro, providing the first evidence of direct target engagement for this class of inhibitors reported to date. The affinity of RSV604 N binding was not affected by RSV604 resistance mutations in the N protein. RSV604 engaged in two different MoAs in HeLa cells, inhibiting both RSV RNA synthesis and the infectivity of released virus. The lack of inhibition of viral RNA synthesis in some cell lines explained the cell-type-dependent potency of the inhibitor. RSV604 did not inhibit viral RNA synthesis in the RSV subgenomic replicon cells or in the cell-free RNP assay, suggesting that it might act prior to viral replication complex formation. RSV604 did not alter N protein localization in the infected cells. Taken together, these results provide new insights leading to an understanding of the MoAs of RSV604 and other similar N inhibitors.

Targeting membrane-bound viral RNA synthesis reveals potent inhibition of diverse coronaviruses including the middle East respiratory syndrome virus.

Coronaviruses raise serious concerns as emerging zoonotic viruses without specific antiviral drugs available. Here we screened a collection of 16671 diverse compounds for anti-human coronavirus 229E activity and identified an inhibitor, designated K22, that specifically targets membrane-bound coronaviral RNA synthesis. K22 exerts most potent antiviral activity after virus entry during an early step of the viral life cycle. Specifically, the formation of double membrane vesicles (DMVs), a hallmark of coronavirus replication, was greatly impaired upon K22 treatment accompanied by near-complete inhibition of viral RNA synthesis. K22-resistant viruses contained substitutions in non-structural protein 6 (nsp6), a membrane-spanning integral component of the viral replication complex implicated in DMV formation, corroborating that K22 targets membrane bound viral RNA synthesis. Besides K22 resistance, the nsp6 mutants induced a reduced number of DMVs, displayed decreased specific infectivity, while RNA synthesis was not affected. Importantly, K22 inhibits a broad range of coronaviruses, including Middle East respiratory syndrome coronavirus (MERS–CoV), and efficient inhibition was achieved in primary human epithelia cultures representing the entry port of human coronavirus infection. Collectively, this study proposes an evolutionary conserved step in the life cycle of positive-stranded RNA viruses, the recruitment of cellular membranes for viral replication, as vulnerable and, most importantly, druggable target for antiviral intervention. We expect this mode of action to serve as a paradigm for the development of potent antiviral drugs to combat many animal and human virus infections.

Role of Human Hypoxanthine Guanine Phosphoribosyltransferase in Activation of the Antiviral Agent T-705 (Favipiravir)

6-Fluoro-3-hydroxy-2-pyrazinecarboxamide (T-705) is a novel antiviral compound with broad activity against influenza virus and diverse RNA viruses. Its active metabolite, T-705-ribose-5'-triphosphate (T-705-RTP), is recognized by influenza virus RNA polymerase as a substrate competing with GTP, giving inhibition of viral RNA synthesis and lethal virus mutagenesis. Which enzymes perform the activation of T-705 is unknown. We here demonstrate that human hypoxanthine guanine phosphoribosyltransferase (HGPRT) converts T-705 into its ribose-5'-monophosphate (RMP) prior to formation of T-705-RTP. The anti-influenza virus activity of T-705 and T-1105 (3-hydroxy-2-pyrazinecarboxamide; the analog lacking the 6-fluoro atom) was lost in HGPRT-deficient Madin-Darby canine kidney cells. This HGPRT dependency was confirmed in human embryonic kidney 293T cells undergoing HGPRT-specific gene knockdown followed by influenza virus ribonucleoprotein reconstitution. Knockdown for adenine phosphoribosyltransferase (APRT) or nicotinamide phosphoribosyltransferase did not change the antiviral activity of T-705 and T-1105. Enzymatic assays showed that T-705 and T-1105 are poor substrates for human HGPRT having Km(app) values of 6.4 and 4.1 mM, respectively. Formation of the RMP metabolites by APRT was negligible, and so was the formation of the ribosylated metabolites by human purine nucleoside phosphorylase. Phosphoribosylation and antiviral activity of the 2-pyrazinecarboxamide derivatives was shown to require the presence of the 3-hydroxyl but not the 6-fluoro substituent. The crystal structure of T-705-RMP in complex with human HGPRT showed how this compound binds in the active site. Since conversion of T-705 by HGPRT appears to be inefficient, T-705-RMP prodrugs may be designed to increase the antiviral potency of this new antiviral agent.

Identification of a Broad-Spectrum Inhibitor of Viral RNA Synthesis: Validation of a Prototype Virus-Based Approach

There are no approved therapeutics for the most deadly nonsegmented negative-strand (NNS) RNA viruses, including Ebola (EBOV). To identify chemical scaffolds for the development of broad-spectrum antivirals, we undertook a prototype-based lead identification screen. Using the prototype NNS virus, vesicular stomatitis virus (VSV), multiple inhibitory compounds were identified. Three compounds wereinvestigated for broad-spectrum activity andinhibited EBOV infection. The most potent, CMLDBU3402, was selected for further study. CMLDBU3402 did not show significant activity against segmented negative-strand RNA viruses, suggesting proscribed broad-spectrum activity. Mechanistic analysis indicated that CMLDBU3402 blocked VSV viral RNA synthesis and inhibited EBOV RNA transcription, demonstrating a consistent mechanism of action against genetically distinct viruses. The identification of this chemical backbone as a broad-spectrum inhibitor of viral RNA synthesis offers significant potential for the development of new therapies for highly pathogenic viruses.

Hepatitis C Virus Translation Preferentially Depends on Active RNA Replication

Hepatitis C virus (HCV) RNA initiates its replication on a detergent-resistant membrane structure derived from the endoplasmic reticulum (ER) in the HCV replicon cells. By performing a pulse-chase study of BrU-labeled HCV RNA, we found that the newly-synthesized HCV RNA traveled along the anterograde-membrane traffic and moved away from the ER. Presumably, the RNA moved to the site of translation or virion assembly in the later steps of viral life cycle. In this study, we further addressed how HCV RNA translation was regulated by HCV RNA trafficking. When the movement of HCV RNA from the site of RNA synthesis to the Golgi complex was blocked by nocodazole, an inhibitor of ER-Golgi transport, HCV protein translation was surprisingly enhanced, suggesting that the translation of viral proteins occurred near the site of RNA synthesis. We also found that the translation of HCV proteins was dependent on active RNA synthesis: inhibition of viral RNA synthesis by an NS5B inhibitor resulted in decreased HCV viral protein synthesis even when the total amount of intracellular HCV RNA remained unchanged. Furthermore, the translation activity of the replication-defective HCV replicons or viral RNA with an NS5B mutation was greatly reduced as compared to that of the corresponding wildtype RNA. By performing live cell labeling of newly synthesized HCV RNA and proteins, we further showed that the newly synthesized HCV proteins colocalized with the newly synthesized viral RNA, suggesting that HCV RNA replication and protein translation take place at or near the same site. Our findings together indicate that the translation of HCV RNA is coupled to RNA replication and that the both processes may occur at the same subcellular membrane compartments, which we term the replicasome.

Inhibition of viral RNA synthesis in canine distemper virus infection by proanthocyanidin A2

Canine distemper virus (CDV) is a contagious and multisystemic viral disease that affects domestic and wild canines as well as other terrestrial and aquatic carnivores. The disease in dogs is often fatal and no specific antiviral therapy is currently available.In this study, we evaluated the in vitro antiviral activity against CDV of proanthocyanidin A2 (PA2), a phenolic dimer belonging to the class of condensed tannins present in plants. Our results showed that PA2 exerted in vitro antiviral activity against CDV with a higher selectivity index compared to ribavirin, included in our study for the previously tested anti-CDV activity. The time of addition assay led us to observe that PA2 was able to decrease the viral RNA synthesis and to reduce progeny virus liberation, at different times post infection suggesting multiple mechanisms of action including inhibition of viral replicative complex and modulation of the redox milieu. These data suggest that PA2, isolated from the bark of Aesculus hippocastanum, has potential usefulness as an anti-CDV compound inhibiting viral replication.

Inhibition of viral RNA synthesis in canine distemper virus infection by proanhocyanidin A2

Inhibition of viral RNA synthesis in canine distemper virus infection by proanhocyanidin A2

Anti-viral mechanism of barramundi Mx against betanodavirus involves the inhibition of viral RNA synthesis through the interference of RdRp

Nervous necrosis virus (NNV) belongs to the betanodavirus of the Nodaviridae family. It is the causative agent of viral nervous necrosis (VNN) disease, and has inflicted devastating damage on the world-wide aquaculture industry. The fish that survived after the outbreak of VNN become persistently NNV-infected carriers. NNV-persistent infection has been demonstrated in a barramundi brain (BB) cell line, and it involves the type I interferon (IFN) response with the expression of Mx gene. However, little of the defense mechanism in fish cells against NNV infection is understood. In this study, the anti-NNV mechanism of barramundi Mx protein (BMx) was elucidated in cBB cells which were derived from BB cell line after serial treatments by NNV-specific antiserum and then became an NNV-free cell line. After NNV infection of cBB cells, the level of viral RNA-dependent RNA polymerase (RdRp) increased with time over a period of 24 h post-infection (hpi), but decreased when the BMx expression increased 48 and 72 hpi. When the expression of BMx was down-regulated by BMx-specific siRNA, the expression levels of viral RNA, proteins and progeny viral titers were restored. The BMx was found to colocalize with viral RdRp at the perinuclear area 24 hpi and coprecipitate with viral RdRp, indicating that they could bind with each other. Viral RdRp was also revealed to colocalize with lysosomes 48 hpi as the NNV RdRp level started to decline. Therefore, it is suggested that BMx inhibited the viral RNA synthesis by interaction with viral RdRp, and redistributed RdRp to perinuclear area for degradation.

Anti-Bovine Viral Diarrhoea Virus and Hepatitis C Virus Activity of the Cyclooxygenase Inhibitor SC-560

A number of compounds were examined for their inhibitory effect on bovine viral diarrhoea virus (BVDV) replication in cell cultures and found that some cyclooxygenase (COX) inhibitors had antiviral activity against the virus. Determination of compounds for their anti-BVDV activity was on the basis of the inhibition of virus-induced cytopathogenicity in Mardin-Darby bovine kidney (MDBK) cells. Anti-hepatitis C virus (HCV) activity was assessed by the inhibition of viral RNA synthesis in the subgenomic HCV RNA replicon cells. Among the test compounds, 5-(4-chlorophenyl)-1-(4-methoxyphenyl)-3-(trifluoromethyl)-1H-pyrazole (SC-560) was the most active against BVDV, and its 50% effective and cytotoxic concentrations were 10.9 +/-2.8 and 93.9 +/-24.5 microM in virus and mock-infected MDBK cells, respectively. The compound also suppressed BVDV RNA synthesis in a dose-dependent fashion. Studies on the mechanism of action revealed that SC-560 did not interfere with viral entry to the host cells. Furthermore, it was assumed that the antiviral activity of SC-560 was not associated with its inhibitory effect on COX. The combination of SC-560 and interferon-alpha was additive to synergistic in inhibiting BVDV replication. More importantly, the compound proved to be a selective inhibitor of HCV replication. SC-560 and its derivative might have potential as novel antiviral agents against HCV.

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.

Paramyxovirus Sendai virus C proteins are essential for maintenance of negative-sense RNA genome in virus particles

The Sendai virus (SeV) C proteins are a nested set of four accessory proteins, C′, C, Y1, and Y2, encoded on the P mRNA from an alternate reading frame. The C proteins are multifunctional proteins involved in viral pathogenesis, inhibition of viral RNA synthesis, counteracting the innate immune response of the host cell, inhibition of virus-induced apoptosis, and facilitating virus-like particle (VLP)/virus budding. Among these functions, the roles for pathogenesis and counteracting host cell interferon (IFN) responses have been studied extensively, but the others are less well understood. In this paper, we found that the C proteins contributed in many ways to the efficient production of infectious virus particles by using a series of SeV recombinants without one or more C protein expression. Knockout of both C′ and C protein expression resulted in reduced virus release despite higher viral protein synthesis in the cells. Interestingly, for the viruses without C′ and C, or all four C protein expression, non-infectious virions containing antigenomic RNAs were produced predominantly compared to genomic RNA-containing infectious virions, due to aberrant viral RNA synthesis. Our results demonstrate for the first time that the C proteins regulate balance of viral genome and antigenome RNA synthesis for efficient production of infectious virus particles in the course of virus infection.