Virus multiplication and induction of apoptosis by Sendai virus: role of the C proteins

Sendai virus (SeV) P gene encodes a nested set of carboxyl-coterminal proteins (C', C, Y1 and Y2), which are referred to collectively as the C proteins. Characterization of the virus multiplication and cellular responses in HEp-2 cells infected with the recombinant SeV which lacks two (C' and C), three (C', C and Y1) or all the four C proteins revealed that all the recombinant viruses can grow in the cells to various extents, depending, apparently, on the number of species expressing C protein. In reverse proportion to the viral growth ability, these viruses induced apoptosis in the infected cells. These results indicate that Y2 protein has an antiapoptotic activity, and suggest that this activity works in an additive manner with the longer C protein(s) (C' and/or C) of SeV in order to suppress virus-induced apoptosis in the SeV-infected cells. Apparently, the antiapoptotic activity of the C proteins supports virus multiplication in the infected cells.     

Knockout of the Sendai virus C gene eliminates the viral ability to prevent the interferon-alpha/beta-mediated responses.

Sendai virus (SeV) renders cells unresponsive to interferon (IFN)-alpha. To identify viral factors involved in this process, we examined whether recombinant SeVs, which could not express V protein, subsets of C proteins (C, C', Y1 and Y2) or any of four C proteins, retained the capability of impeding IFN-alpha-mediated responses. Among these viruses, only the 4C knockout virus completely lost the ability to suppress the induction of IFN-alpha-stimulated gene products and the subsequent establishment of an anti-viral state. These findings reveal crucial roles of the SeV C proteins in blocking IFN-alpha-mediated responses.     

Versatility of the accessory C proteins of Sendai virus: contribution to virus assembly as an additional role

The P/C mRNA of Sendai virus (SeV) encodes a nested set of accessory proteins, C', C, Y1, and Y2, referred to collectively as C proteins, using the +1 frame relative to the open reading frame of phospho (P) protein and initiation codons at different positions. The C proteins appear to be basically nonstructural proteins as they are found abundantly in infected cells but greatly underrepresented in the virions. We previously created a 4C(-) SeV, which expresses none of the four C proteins, and concluded that the C proteins are categorically nonessential gene products but greatly contribute to viral full replication and infectivity (A. Kurotani et al., Genes Cells 3:111-124, 1998). Here, we further characterized the 4C(-) virus multiplication in cultured cells. The viral protein and mRNA synthesis was enhanced with the mutant virus relative to the parental wild-type (WT) SeV. However, the viral yields were greatly reduced. In addition, the 4C(-) virions appeared to be highly anomalous in size, shape, and sedimentation profile in a sucrose gradient and exhibited the ratios of infectivity to hemagglutination units significantly lower than those of the WT. In the WT infected cells, C proteins appeared to colocalize almost perfectly with the matrix (M) proteins, pretty well with an external envelope glycoprotein (hemagglutinin-neuraminidase [HN]), and very poorly with the internal P protein. In the absence of C proteins, there was a significant delay of the incorporation of M protein and both of the envelope proteins, HN and fusion (F) proteins, into progeny virions. These results strongly suggest that the accessory and basically nonstructural C proteins are critically required in the SeV assembly process. This role of C proteins was further found to be independent of their recently discovered function to counteract the antiviral action of interferon-alpha/beta. SeV C proteins thus appear to be quite versatile.     

Sendai virus C proteins must interact directly with cellular components to interfere with interferon action

Sendai virus (SeV) infection of interferon (IFN)-competent cells is one of the most efficient ways of inducing IFN production. Virus replication is nevertheless largely unaffected, since SeV infection also interfers with IFN action, a prerequisite for the establishment of an antiviral state. This property has been mapped by reverse genetics to the viral C gene, which is also known to act as a promoter-specific inhibitor of viral RNA synthesis. Using luciferase reporter plasmids containing IFN-responsive promoters, we have found that all four C proteins effectively interdict IFN signaling when expressed independently of SeV infection. The C proteins must therefore interact directly with cellular components to carry this out. The C gene in the context of an SeV infection was also found to induce STAT1 instability in some cells, whereas in other cells it apparently acts to prevent the synthesis of STAT1 in response to the virus infection or IFN treatment. The SeV C proteins appear to act in at least two ways to counteract the IFN induced by SeV infection.     

The Sendai paramyxovirus accessory C proteins inhibit viral genome amplification in a promoter-specific fashion.

Many paramyxoviruses express small basic C proteins, from an alternate, overlapping open reading frame of the P gene mRNA, which were previously found to inhibit mRNA synthesis. During recent experiments in which infectious Sendai virus (SeV) was recovered from cDNA via the initial expression of the viral N, P, and L genes from plasmids, the abrogation of C protein expression from the plasmid P gene was found to be necessary for virus recovery. We have investigated the effect of C coexpression on the amplification of an internally deleted defective interfering (DI) genome directly in the transfected cell, for which, in contrast to virus recovery experiments, genome amplification is independent of mRNA synthesis carried out by the SeV polymerase. We find that C protein coexpression also strongly inhibits the amplification of this DI genome but has little or no effect on that of a copy-back DI genome (DI-H4). We have also characterized the C protein from a mutant SeV and found that (i) it had lost most of its inhibitory activity on internally deleted DI genome amplification and (ii) its coexpression no longer prevented the recovery of SeV from DNA. However, consistent with the insensitivity of copy-back DI genomes to C protein inhibition, C coexpression did not prevent the recovery of copy-back nondefective viruses from DNA. The inhibitory effects of C coexpression thus appear to be promoter specific.     

Sendai Virus C Proteins Counteract the Interferon-Mediated Induction of an Antiviral State

We have studied the relationship between the Sendai virus (SeV) C proteins (a nested set of four proteins initiated at different start codons) and the interferon (IFN)-mediated antiviral response in IFN-competent cells in culture. SeV strains containing wild-type or various mutant C proteins were examined for their ability (i) to induce an antiviral state (i.e., to prevent the growth of vesicular stomatitis virus [VSV] following a period of SeV infection), (ii) to induce the elevation of Stat1 protein levels, and (iii) to prevent IFN added concomitant with the SeV infection from inducing an antiviral state. We find that expression of the wild-type C gene and, specifically, the AUG114-initiated C protein prevents the establishment of an antiviral state: i.e., cells infected with wild-type SeV exhibited little or no increase in Stat1 levels and were permissive for VSV replication, even in the presence of exogenous IFN. In contrast, in cells infected with SeV lacking the AUG114-initiated C protein or containing a single amino acid substitution in the C protein, the level of Stat1 increased and VSV replication was inhibited. The prevention of the cellular IFN-mediated antiviral response appears to be a key determinant of SeV pathogenicity.     

Construction and characterization of a fluorescent sendai virus carrying the gene for envelope fusion protein fused with enhanced green fluorescent protein

Sendai virus (SeV) is an enveloped virus with a non-segmented negative-strand RNA genome. SeV envelope fusion (F) glycoproteins play crucial roles in the viral life cycle in processes such as viral binding, assembly, and budding. In this study, we developed a viable recombinant SeV designated F-EGFP SeV/ΔF, in which the F protein was replaced by an F protein fused to EGFP at the carboxyl terminus. Living infected cells of the recombinant virus were directly visualized by green fluorescence. The addition of EGFP to the F protein maintained the activities of the F protein in terms of intracellular transport to the plasma membrane via the ER and the Golgi apparatus and fusion activity in the infected cells. These results suggest that this fluorescent SeV is a useful tool for studying the viral binding, assembly, and budding mechanisms of F proteins and the SeV life cycle in living infected cells.     

Coxsackievirus B Exits the Host Cell in Shed Microvesicles Displaying Autophagosomal Markers

Coxsackievirus B3 (CVB3), a member of the picornavirus family and enterovirus genus, causes viral myocarditis, aseptic meningitis, and pancreatitis in humans. We genetically engineered a unique molecular marker, “fluorescent timer” protein, within our infectious CVB3 clone and isolated a high-titer recombinant viral stock (Timer-CVB3) following transfection in HeLa cells. “Fluorescent timer” protein undergoes slow conversion of fluorescence from green to red over time, and Timer-CVB3 can be utilized to track virus infection and dissemination in real time. Upon infection with Timer-CVB3, HeLa cells, neural progenitor and stem cells (NPSCs), and C2C12 myoblast cells slowly changed fluorescence from green to red over 72 hours as determined by fluorescence microscopy or flow cytometric analysis. The conversion of “fluorescent timer” protein in HeLa cells infected with Timer-CVB3 could be interrupted by fixation, suggesting that the fluorophore was stabilized by formaldehyde cross-linking reactions. Induction of a type I interferon response or ribavirin treatment reduced the progression of cell-to-cell virus spread in HeLa cells or NPSCs infected with Timer-CVB3. Time lapse photography of partially differentiated NPSCs infected with Timer-CVB3 revealed substantial intracellular membrane remodeling and the assembly of discrete virus replication organelles which changed fluorescence color in an asynchronous fashion within the cell. “Fluorescent timer” protein colocalized closely with viral 3A protein within virus replication organelles. Intriguingly, infection of partially differentiated NPSCs or C2C12 myoblast cells induced the release of abundant extracellular microvesicles (EMVs) containing matured “fluorescent timer” protein and infectious virus representing a novel route of virus dissemination. CVB3 virions were readily observed within purified EMVs by transmission electron microscopy, and infectious virus was identified within low-density isopycnic iodixanol gradient fractions consistent with membrane association. The preferential detection of the lipidated form of LC3 protein (LC3 II) in released EMVs harboring infectious virus suggests that the autophagy pathway plays a crucial role in microvesicle shedding and virus release, similar to a process previously described as autophagosome-mediated exit without lysis (AWOL) observed during poliovirus replication. Through the use of this novel recombinant virus which provides more dynamic information from static fluorescent images, we hope to gain a better understanding of CVB3 tropism, intracellular membrane reorganization, and virus-associated microvesicle dissemination within the host.     

Isolation of 3-(4,8,12-Trimethyltridecyl)-furan (“Phytofuran”) from Burley Tobacco

Sendai virus (SeV) is an enveloped virus with a non-segmented negative-strand RNA genome. SeV envelope fusion (F) glycoproteins play crucial roles in the viral life cycle in processes such as viral binding, assembly, and budding. In this study, we developed a viable recombinant SeV designated F-EGFP SeV/ΔF, in which the F protein was replaced by an F protein fused to EGFP at the carboxyl terminus. Living infected cells of the recombinant virus were directly visualized by green fluorescence. The addition of EGFP to the F protein maintained the activities of the F protein in terms of intracellular transport to the plasma membrane via the ER and the Golgi apparatus and fusion activity in the infected cells. These results suggest that this fluorescent SeV is a useful tool for studying the viral binding, assembly, and budding mechanisms of F proteins and the SeV life cycle in living infected cells.     

Sequential Formation of Vaccinia Virus Proteins and Viral Deoxyribonucleic Acid Replication

In vaccinia virus-infected cell cultures, cellular protein synthesis was inhibited 50% at 2 hr postinfection (PI) and 80 to 90% by 4 hr PI. Input virus was responsible for this inhibition. Five early proteins, coded for by the viral genome, could be detected at 2 to 3 hr PI. Normally, their synthesis did not continue beyond 6 hr PI, at which time synthesis of a different set of proteins began. When DNA replication was blocked, synthesis of these early proteins continued until 9 to 12 hr PI. The bulk of the proteins which were incorporated into mature virus were synthesized at 8 hr PI and thereafter. The time of their formation was close to the time at which virus maturation occurred. However, 15% of the protein found in mature virus was synthesized early in the infectious cycle. The quantity of “early viral protein” which was not incorporated into mature virus was almost as large as the quantity of viral protein which did appear in mature virus. The “early” and “late” proteins could be shown to have separate and distinct immunological properties. The role of this large quantity of “early” protein is discussed.     

Sendai Virus C Proteins Regulate Viral Genome and Antigenome Synthesis To Dictate the Negative Genome Polarity

The order Mononegavirales comprises a large number of nonsegmented negative-strand RNA viruses (NNSVs). How the genome polarity is determined is a central issue in RNA virus biology. Using a prototypic species, vesicular stomatitis virus (VSV), it has been established that the negative polarity of the viral genome is defined solely by different strengths of the cis-acting replication promoters located at the 3′ ends of the genome and antigenome, resulting in the predominance of the genome over the antigenome. This VSV paradigm has long been applied for the Mononegavirales in general without concrete proof. We now found that another prototypic species, Sendai virus (SeV), undergoes a marked shift from the early antigenome-dominant to the late genome-dominant phase during the course of infection. This shift appeared to be governed primarily by the expression of the accessory C protein, because no such shift occurred in a recombinant SeV with the C gene deleted, and antigenomes were dominant throughout infection, generating antigenome-dominant and noninfectious progeny virions. Therefore, we proposed for the first time a trans-regulatory mechanism, the SeV paradigm, to dictate the genome polarity of an NNSV. A series of promoter-swapped SeV recombinants suggested the importance of the primary as well as secondary structures of the promoters in this trans-regulation.     

A highly recombinogenic system for the recovery of infectious Sendai paramyxovirus from cDNA: generation of a novel copy-back nondefective interfering virus.

We have recovered infectious Sendai virus (SeV) from full-length cDNA (FL-3) by transfecting this cDNA and pGEM plasmids expressing the nucleocapsid protein (NP), phosphoprotein and large proteins into cells infected with a vaccinia virus which expresses T7 RNA polymerase. These cells were then injected into chicken eggs, in which SeV grows to very high titers. FL-3 was marked with a BglII site in the leader region and an NsiI site (ATGCAT) in the 5' nontranslated region of the NP gene, creating a new, out-of-frame, 5' proximal AUG. All the virus stocks generated eventually removed this impediment to NP expression, by either point mutation or recombination between FL-3 and pGEM-NP. The recovery system was found to be highly recombinogenic. Even in the absence of selective pressure, one in 20 of the recombinant SeV generated had exchanged the NP gene of FL-3 with that of pGEM-NP. When a fifth plasmid containing a new genomic 3' end without the presumably deleterious BglII site was included as another target for recombination, the new genomic 3' end was found in the recombinant SeV in 12 out of 12 recoveries. Using this approach, a novel copy-back nondefective virus was generated which interferes with wild-type virus replication.     

Longer and Shorter Forms of Sendai Virus C Proteins Play Different Roles in Modulating the Cellular Antiviral Response

The Sendai virus (SeV) C gene codes for a nested set of four C proteins that carry out several functions, including the modulation of viral RNA synthesis and countering of the cellular antiviral response. Using mutant C genes (and in particular a C gene with a deletion of six amino acids present only in the larger pair of C proteins) and recombinant SeV carrying these mutant C genes, we find that the nested set of C proteins carry out a nested set of functions. All of the C proteins interdict interferon (IFN) signaling to IFN-stimulated genes (ISGs) and prevent pY701-Stat1 formation. However, only the larger C proteins can induce STAT1 instability, prevent IFN from inducing an antiviral state, or prevent programmed cell death. Remarkably, interdiction of IFN signaling to ISGs and the absence of pY701-Stat1 formation did not prevent IFN-alpha from inducing an anti-Vesicular stomatitis virus (VSV) state. It is possible that IFN-alpha signaling to induce an anti-VSV state can occur independently of the well-established Jak/Stat/ISGF3 pathway and that it is this parallel pathway that is targeted by the longer C proteins.     

Mouse hepatitis virus liver pathology is dependent on ADP-ribose-1'-phosphatase, a viral function conserved in the alpha-like supergroup

Viral infection of the liver can lead to severe tissue damage when high levels of viral replication and spread in the organ are coupled with strong induction of inflammatory responses. Here we report an unexpected correlation between the expression of a functional X domain encoded by the hepatotropic mouse hepatitis virus strain A59 (MHV-A59), the high-level production of inflammatory cytokines, and the induction of acute viral hepatitis in mice. X-domain (also called macro domain) proteins possess poly-ADP-ribose binding and/or ADP-ribose-1′′-phosphatase (ADRP) activity. They are conserved in coronaviruses and in members of the “alpha-like supergroup” of phylogenetically related positive-strand RNA viruses that includes viruses of medical importance, such as rubella virus and hepatitis E virus. By using reverse genetics, we constructed a recombinant murine coronavirus MHV-A59 mutant encoding a single-amino-acid substitution of a strictly conserved residue that is essential for coronaviral ADRP activity. We found that the mutant virus replicated to slightly reduced titers in livers but, strikingly, did not induce liver disease. In vitro, the mutant virus induced only low levels of the inflammatory cytokines tumor necrosis factor alpha and interleukin-6 (IL-6). In vivo, we found that IL-6 production, in particular, was reduced in the spleens and livers of mutant virus-infected mice. Collectively, our data demonstrate that the MHV X domain exacerbates MHV-induced liver pathology, most likely through the induction of excessive inflammatory cytokine expression.     

Identification of a cis-acting element required for shunt-mediated translational initiation of the Sendai virus Y proteins

Shunting is a mechanism that permits translational initiation at internal codons positioned in proximity to a ribosome acceptor sequence. Sendai virus exploits shunting to express a series of proteins that initiate at the fourth and fifth start sites on the P/C mRNA (namely, the Y1 and Y2 proteins, respectively). Shunt-mediated initiation at these sites is codon independent. In an attempt to characterise the acceptor site, an extensive deletion analysis was performed spanning the entire C ORF. Only mutants flanking the Y1/Y2 start sites exhibited altered shunt phenotypes. Some of these significantly enhanced shunting efficiency to the point where the Y1/Y2 proteins were the major translational products of the mRNA. Additionally, removal of a short region just downstream of the Y2 start codon (referred to as Δ10) ablated all Y protein initiation via shunting but had no effect on Y expression when the AUG codons were viewed by a scanning ribosome. Point mutations introduced into this Δ10 sequence severely perturbed shunt-mediated initiation. We also provide evidence that changes in this region of the P/C mRNA may be used to modulate Y protein expression levels in different viral strains.