Generation of a recombinant Saffold Virus expressing UnaG as a marker for the visualization of viral infection.

BACKGROUND: Saffold virus (SAFV), which belongs to the genus Cardiovirus of the family Picornaviridae, is associated with acute respiratory or gastrointestinal illnesses in children; it is also suspected to cause severe diseases, such as acute flaccid paralysis and aseptic meningitis. However, the understanding of the mechanism of its pathogenicity is still limited due to the many unknowns about its lifecycle; for example, the cellular receptor for its infection remains to be determined. A system to monitor SAFV infection in vitro and in vivo is required in order to accelerate research on SAFV. RESULTS: We generated a recombinant SAFV expressing green fluorescent protein (GFP) or UnaG, a novel fluorescent protein derived from Japanese eel. HeLa cells infected by either GFP or UnaG-expressing SAFV showed a bright green fluorescent signal, enabling convenient monitoring of SAFV infection. However, the expression of GFP but not UnaG was quickly lost during virus passaging due to the difference in genetic stability in the SAFV virus genome; the UnaG gene was stably maintained in the virus genome after at least five passages. CONCLUSIONS: SAFV infection of cultured cells can easily be monitored using UnaG-expressing SAFV, which is superior to GFP in terms of genetic stability in the virus genome. This virus could be a useful tool for SAFV research, such as comparing the susceptibility of various cells to SAFV infection and evaluating the effects of antivirals on SAFV infection in high-throughput screening.

Theilovirus 3C Protease Cleaves the C-Terminal Domain of the Innate Immune RNA Sensor, Melanoma Differentiation-Associated Gene 5, and Impairs Double-Stranded RNA-Mediated IFN Response.

Melanoma differentiation-associated gene 5 (MDA5), a member of the retinoic acid-inducible gene I (RIG-I)-like receptors (RLRs), has pivotal roles in innate immune responses against many positive-stranded RNA viruses, including picornavirus and coronavirus. Upon engagement with dsRNA derived from viral infection, MDA5 initiates coordinated signal transduction leading to type I IFN induction to restrict viral replication. In this study, we describe a targeted cleavage events of MDA5 by the 3C protease from Theilovirus. Upon ectopic expression of theilovirus 3C protease from Saffold virus or Theiler's murine encephalomyelitis virus but not encephalomyocarditis virus, fragments of cleaved MDA5 were observed in a dose-dependent manner. When enzymatically inactive Theilovirus 3C protease was expressed, MDA5 cleavage was completely abrogated. Mass spectrometric analysis identified two cleavage sites at the C terminus of MDA5, cleaving off one of the RNA-binding domains. The same cleavage pattern was observed during Theilovirus infection. The cleavage of MDA5 by Theilovirus protease impaired ATP hydrolysis, RNA binding, and filament assembly on RNA, resulting in dysfunction of MDA5 as an innate immune RNA sensor for IFN induction. Furthermore, the cleavage-resistant MDA5 mutant against the 3C protease showed an enhanced IFN response during Saffold virus infection, indicating that Theilovirus has a strategy to circumvent the antiviral immune response by cleaving MDA5 using 3C protease. In summary, these data suggest MDA5 cleavage by 3C protease as a novel immune evasive strategy of Theilovirus.

A mouse monoclonal antibody against influenza C virus attenuates acetaminophen-induced liver injury in mice.

Molecular mimicry is one of the main processes for producing autoantibodies during infections. Although some autoantibodies are associated with autoimmune diseases, the functions of many autoantibodies remain unknown. Previously, we reported that S16, a mouse (BALB/c) monoclonal antibody against the hemagglutinin-esterase fusion glycoprotein of influenza C virus, recognizes host proteins in some species of animals, but we could not succeed in identifying the proteins. In the present study, we found that S16 cross-reacted with acetyl-CoA acyltransferase 2 (ACAA2), which is expressed in the livers of BALB/c mice. ACAA2 was released into the serum after acetaminophen (APAP) administration, and its serum level correlated with serum alanine aminotransferase (ALT) activity. Furthermore, we observed that S16 injected into mice with APAP-induced hepatic injury prompted the formation of an immune complex between S16 and ACAA2 in the serum. The levels of serum ALT (p < 0.01) and necrotic areas in the liver (p < 0.01) were reduced in the S16-injected mice. These results suggest that S16 may have a mitigation function in response to APAP-induced hepatotoxicity. This study shows the therapeutic function of an autoantibody and suggests that an antibody against extracellular ACAA2 might be a candidate for treating APAP-induced hepatic injury.

Saffold Cardiovirus Infection in a 2-Year-Old Boy with Acute Pancreatitis.

Saffold cardiovirus (SAFV), first identified in a stool sample in 2007, is thought to be associated with respiratory disease and gastroenteritis. On the other hand, animal experiments suggested that the major viral load, following intraperitoneal inoculation of SAFV in mice, may be detected in the pancreas. However, until now, no cases of SAFV in patients with pancreatitis have been reported. This report presents a unique case in a patient who developed relapsing acute pancreatitis (AP) after hand, foot, and mouth disease, and was suspected to have SAFV-1 infection. A 2-year-old boy was admitted to the hospital because of severe abdominal pain. His serum amylase and lipase levels were elevated. Enhanced computed tomography showed pancreatic swelling and dilation of the main pancreatic duct, leading to a diagnosis of severe AP. The viral genome of SAFV-1 was detected by reverse transcription polymerase chain reaction from fecal samples. Furthermore, the serum neutralization titer for SAFV was elevated during AP, but decreased after 1 year. These findings strongly suggest the patient developed SAFV-1 infection concurrent with AP. Therefore, we propose that a cohort study is required to clarify the relationship between SAFV and AP.

Effect of cysteine mutations in the extracellular domain of CM2 on the influenza C virus replication.

CM2 is the second membrane protein of influenza C virus and possesses three conserved cysteines at residue 1, 6 and 20 in its extracellular domain, all of which are involved in the formation of disulfide-linked oligomers of the molecule. In the present study, to examine the effect of CM2 oligomerization on virus replication, we generated a mutant recombinant virus, rC1620A, in which all three cysteines on CM2 were substituted to alanines. The rC1620A virus was more attenuated than the recombinant wild-type (rWT) virus in cultured cells. The CM2 protein synthesized in rC1620A-infected cells could not apparently be detected as a tetramer and was transported to the cell surface less efficiently than was authentic CM2. The amount of CM2 protein incorporated into the rC1620A virions was comparable to that into the rWT virions, although the main CM2 species in the rC1620A virions was in the form of a dimer. Analyses of one-step grown virions and virus-infected cells could not provide evidence for any difference in growth between rC1620A and rWT. On the other hand, the amount of genome present in VLPs possessing the mutant CM2 (C1620A-VLPs) was approximately 31% of that in VLPs possessing wild-type CM2 (WT-VLPs). The incoming genome from VLPs was less efficiently transported to the nucleus in the C1620A-VLP-infected cells than in WT-VLP-infected cells, leading to reduced reporter gene expression in the C1620A-VLP-infected cells. Taken together, these findings demonstrate that CM2 oligomerization affects the packaging and uncoating processes. Thus, we concluded that disulfide-linked CM2 oligomers facilitate virus growth by affecting the replication processes.

Saffold virus type 3 (SAFV-3) persists in HeLa cells.

Saffold virus (SAFV) was identified as a human cardiovirus in 2007. Although several epidemiological studies have been reported, they have failed to provide a clear picture of the relationship between SAFV and human diseases. SAFV genotype 3 has been isolated from the cerebrospinal fluid specimen of patient with aseptic meningitis. This finding is of interest since Theiler's murine encephalomyelitis virus (TMEV), which is the closely related virus, is known to cause a multiple sclerosis-like syndrome in mice. TMEV persistently infects in mouse macrophage cells in vivo and in vitro, and the viral persistence is essential in TMEV-induced demyelinating disease. The precise mechanism(s) of SAFV infection still remain unclear. In order to clarify the SAFV pathogenicity, in the present study, we studied the possibilities of the in vitro persistent infection of SAFV. The two distinct phenotypes of HeLa cells, HeLa-N and HeLa-R, were identified. In these cells, the type of SAFV-3 infection was clearly different. HeLa-N cells were lyticly infected with SAFV-3 and the host suitable for the efficient growth. On the other hand, HeLa-R cells were persistently infected with SAFV-3. In addition, the SAFV persistence in HeLa-R cells is independent of type I IFN response of host cells although the TMEV persistence in mouse macrophage cells depends on the response. Furthermore, it was suggested that SAFV persistence may be influenced by the expression of receptor(s) for SAFV infection on the host cells. The present findings on SAFV persistence will provide the important information to encourage the research of SAFV pathogenicity.

Glycosylation of CM2 is important for efficient replication of influenza C virus.

CM2 is the second membrane protein of influenza C virus and possesses a conserved motif for N-glycosylation. To investigate the role(s) of CM2 glycosylation in the virus replication, we generated rN11A, a recombinant influenza C virus lacking the glycosylation site. The rN11A virus grew less efficiently than the wild-type (WT) virus, although the biochemical characteristics of the mutant CM2 were similar to those of authentic CM2. The amount of the genome (GFP-vRNA) in the CM2-N11A-virus-like particles (VLPs) was 13% of that found in WT-VLPs. The incoming GFP-vRNA was less efficiently transported to the nucleus in CM2-N11A-VLP-infected cells than WT-VLP-infected cells, leading to the reduced reporter gene expression in CM2-N11A-VLP-infected cells. Thus the glycosylation of CM2 is required for efficient replication of influenza C virus, and the obtained findings confirmed and extended the previous observation that CM2 is involved in the genome packaging and uncoating processes.

The preparation of an infectious full-length cDNA clone of Saffold virus.

The pathogenicity of Saffold virus (SAFV) among humans still remains unclear, although it was identified as a novel human cardiovirus in 2007. In order to encourage the molecular pathogenetic studies of SAFV, we generated an infectious cDNA clone of SAFV type 3 (SAFV-3). The present study demonstrated that the synthesis of the full-length infectious RNA by T7 RNA polymerase was terminated by a homologous sequence motif with the human preproparathyroid hormone (PTH) signal in the SAFV-3 genome. To obtain the infectious RNA using T7 promoter, a variant of T7 RNA polymerase, which fails to recognize the PTH signal, was useful. This study will provide a valuable technical insight into the reverse genetics of SAFV.

Palmitoylation of CM2 is dispensable to influenza C virus replication.

CM2 is the second membrane protein of influenza C virus. The significance of the posttranslational modifications of CM2 remains to be clarified in the context of viral replication, although the positions of the modified amino acids on CM2 have been determined. In the present study, using reverse genetics we generated rCM2-C65A, a recombinant influenza C virus lacking CM2 palmitoylation site, in which cysteine at residue 65 of CM2 was mutated to alanine, and examined viral growth and viral protein synthesis in the recombinant-infected cells. The rCM2-C65A virus grew as efficiently as did the parental virus in cultured HMV-II cells as well as in embryonated chicken eggs. The synthesis and biochemical features of HEF, NP, M1 and mutant CM2 in the rCM2-C65A-infected HMV-II cells were similar to those in the parental virus-infected cells. Furthermore, membrane flotation analysis of the infected cells revealed that equal amount of viral proteins was recovered in the plasma membrane fractions of the rCM2-C65A-infected cells to that in the parental virus-infected cells. These findings indicate that defect in palmitoylation of CM2 does not affect transport and maturation of HEF, NP and M1 as well as CM2 in virus-infected cells, and palmitoylation of CM2 is dispensable to influenza C virus replication.

The anti-apoptotic protein L(*) of Theiler's murine encephalomyelitis virus (TMEV) contains a mitochondrial targeting signal.

L(*) protein of TMEV is out-of-frame with the viral polyprotein from an alternative initiation codon AUG, 13 nucleotides downstream from the authentic polyprotein AUG. Anti-apoptotic activity of L(*) was demonstrated by both 'loss of function' and 'gain of function' experiments. However, the precise mechanism(s) of anti-apoptotic activity of L(*) remains to be clarified. In this study, L(*) was demonstrated to be localized to mitochondria. It was also shown by the GFP fusion protein that N-terminal sequence of L(*) may contain a mitochondrial targeting signal (MTS). Surprisingly, L(*)((5-70))-GFP and L(*)((41-70))-GFP were localized to mitochondria although L(*)((1-70))-GFP was distributed in the cytosol, suggesting L(*) has an MTS between amino acid (AA) positions 41 and 70, and that L(*)((1-4)) inhibits its mitochondrial targeting. Furthermore, L(*)((1-70))-GFP was localized to the mitochondria by co-expression of L(*)((65-156)), indicating that L(*)((65-156)) suppresses the inhibition of mitochondrial targeting by L(*)((1-4)). These results suggest that the intra- or inter-molecular interaction of L(*) regulates its mitochondrial localization. It is also suggested that L(*) may inhibit the intrinsic apoptosis through the localization to mitochondria.

Opposite effects of two nonstructural proteins of Theiler's murine encephalomyelitis virus regulates apoptotic cell death in BHK-21 cells.

Theiler's murine encephalomyelitis virus is divided into two subgroups, TO and GDVII, inducing subgroup-specific diseases. In order to investigate the role(s) of nonstructural proteins of TMEV, L and L(∗), leaders of two subgroups, were separately expressed with or without L(∗) in BHK-21 cells. Expression of L increased the number of apoptotic cells. L(∗)/BHK-21 cells constitutively expressing L(∗) showed the decrease in cell death induced by L. These results suggest that L and L(∗) regulate apoptosis during viral infection and contribute to TMEV subgroup-specific biological activities.

Cytokine/chemokine profile in J774 macrophage cells persistently infected with DA strain of Theiler's murine encephalomyelitis virus (TMEV).

Theiler's murine encephalomyelitis virus (TMEV) is a picornavirus and persists in the spinal cords of mice, followed by inflammatory demyelinating disease. Viral persistence is a key determinant for the TMEV-induced demyelination. Macrophages are thought to serve as the site of TMEV persistence during the chronic demyelinating phase. We previously demonstrated that two nonstructural proteins of TMEV, L and L(*), were important for virus growth in J774.1 macrophage cells. However, the key factors of macrophage cells related to virus persistence and demyelination remain poorly understood. The inflammatory response is heavily dependent on cytokine and chemokine production by cell of both the immune system and the central nervous system (CNS). In this study, we established the macrophage cells persistently infected with DA strain, and then analyzed the cytokine expression pattern in those cells. The present results are the first to demonstrate the up-regulation of B-lymphocyte chemoattractant (BLC) and granulocyte colony-stimulating factor (G-CSF) in the macrophage cells persistently infected with DA strain. Furthermore, up-regulation of interleukin (IL)-10 and down-regulation of interferon (IFN)-alpha 4, IFN-beta, and IFN-gamma were shown in those cells. The data suggest that these cytokines/chemokines may contribute to the virus persistence and the acceleration of TMEV-induced demyelination.

Activation of the PI3K-Akt pathway by human T cell leukemia virus type 1 (HTLV-1) oncoprotein Tax increases Bcl3 expression, which is associated with enhanced growth of HTLV-1-infected T cells.

Bcl3 is a member of the IkappaB family that regulates genes involved in cell proliferation and apoptosis. Recent reports indicated that Bcl3 is overexpressed in HTLV-1-infected T cells via Tax-mediated transactivation, and acts as a negative regulator of viral transcription. However, the role of Bcl3 in cellular signal transduction and the growth of HTLV-1-infected T cells have not been reported. In this study, we showed that the knockdown of Bcl3 by short hairpin RNA inhibited the growth of HTLV-1-infected T cells. Although phosphatidylinositol-3 kinase (PI3K) inhibitor reduced Bcl3 expression, inactivation of glycogen synthase kinase 3 (GSK3), an effector kinase of the PI3K/Akt signaling pathway, restored Bcl3 expression in Tax-negative but not in Tax-positive T cells. Our results indicate that the overexpression of Bcl3 in HTLV-1-infected T cells is regulated not only by transcriptional but also by post-transcriptional mechanisms, and is involved in overgrowth of HTLV-1-infected T cells.

Leader (L) of Theiler's murine encephalomyelitis virus (TMEV) is required for virus growth in a murine macrophage-like cell line.

Theiler's murine encephalomyelitis virus is divided into two subgroups on the basis of their different biological activities. GDVII subgroup strains cause acute and fatal encephalomyelitis in mice, while TO or DA subgroup strains cause non-fatal polioencephalomyelitis in weanling mice followed by virus persistence and demyelination in the spinal cords. Nonstructural leader (L) protein is encoded at the most N-terminus of the polyprotein. The L coding region of TO or DA subgroup strains has another out-of-frame open reading frame, which produces another nonstructural protein, L*. L* protein is reported to be essential for virus growth in macrophage cells. In the present report, we studied the role of L protein in virus growth in macrophage-like cell line, J774-1, by using a series of deletion mutant viruses. In J774-1 cells (the absence of L* protein), the mutant virus [deleting the entire L coding region (Delta L), N-terminal zinc-finger domain (Delta Z), acidic domain (Delta A), or C-terminal serine/threonine (S/T)-rich domain (DeltaS/T)] did not grow. The mutant virus disrupting zinc-finger motif (L(cys)) did not grow, either. However, in L*-expressing J774-1 cells (the presence of L* protein), L(cys), Delta Z and DeltaS/T had a rescue of the growth activity, while Delta L or Delta A had no rescue. The data suggest that L protein is required for virus growth in J774-1 cells and also suggest that the site responsible for virus growth in those cells, is the acidic domain of L protein.

Different subcellular localization of Theiler's murine encephalomyelitis virus leader proteins of GDVII and DA strains in BHK-21 cells.

The highly virulent GDVII strain of Theiler's murine encephalomyelitis virus causes acute and fatal encephalomyelitis, whereas the DA strain causes mild encephalomyelitis followed by a chronic inflammatory demyelinating disease with virus persistence. The differences in the amino acid sequences of the leader protein (L) of the DA and GDVII strains are greater than those for any other viral protein. We examined the subcellular distribution of DA L and GDVII L tagged with the FLAG epitope in BHK-21 cells. Wild-type GDVII L was localized predominantly in the cytoplasm, whereas wild-type DA L showed a nucleocytoplasmic distribution. A series of the L mutant experiments demonstrated that the zinc finger domain, acidic domain, and C-terminal region of L were necessary for the nuclear accumulation of DA L. A GDVII L mutant with a deletion of the serine/threonine (S/T)-rich domain showed a nucleocytoplasmic distribution, in contrast to the predominant cytoplasmic distribution of wild-type GDVII L. A chimeric DA/GDVII L, D/G, which encodes the N region of DA L including the zinc finger domain and acidic domain, followed by the GDVII L sequence including the S/T-rich domain, was distributed exclusively throughout the cytoplasm but not in the nucleus, as observed with wild-type GDVII L. Another chimeric L, G/D (which is the converse of the D/G construct), accumulated in the nucleus as well as the cytoplasm, as was observed for wild-type DA L. The findings suggest that the differential distribution of DA L and GDVII L is determined primarily by the S/T-rich domain. The S/T-rich domain may be important for the viral activity through the regulation of the subcellular distribution of L.

Theiler's murine encephalomyelitis virus leader protein amino acid residue 57 regulates subgroup-specific virus growth on BHK-21 cells.

Strains of Theiler's murine encephalomyelitis virus (TMEV) are divided into two subgroups, TO and GDVII. TMEV strains show subgroup-specific virus growth and cell tropism and induce subgroup-specific diseases. Using site-directed mutagenesis, we demonstrated that the amino acid at position 57 of the leader protein (L(57)), which is located at the most N-terminal part of the polyprotein, regulates subgroup-specific virus growth on BHK-21 cells. Further study suggested that L(57) may regulate viral RNA encapsidation, although it does not affect the synthesis of viral proteins or the assembly of viral intermediates.

Leader (L) and L* proteins of Theiler's murine encephalomyelitis virus (TMEV) and their regulation of the virus' biological activities.

Theiler's murine encephalomyelitis virus (TMEV) is divided into two subgroups on the basis of their different biological activities. GDVII subgroup strains produce fatal poliomyelitis in mice without virus persistence or demyelination. In contrast, TO subgroup strains induce demyelinating disease with virus persistence in the spinal cords of weanling mice. Two proteins, whose open reading frames are located in the N-terminus of the polyprotein, recently have been reported to be important for TMEV biological activities. One is leader (L) protein and is processed from the most N-terminus of the polyprotein; its function is still unknown. Although the homology of capsid proteins between DA (a representative strain of TO subgroup) and GDVII strains is over 94% at the amino acid level, that of L shows only 85%. Therefore, L is thought to be a key protein for the subgroup-specific biological activities of TMEV. Various studies have demonstrated that L plays important roles in the escape of virus from host immune defenses in the early stage of infection. The second protein is a 17-18 kDa protein, L*, which is synthesized out-of-frame with the polyprotein. Only TO subgroup strains produce L* since GDVII subgroup strains have an ACG rather than AUG at the initiation site and therefore do not synthesize L*. 'Loss and gain of function' experiments demonstrate that L* is essential for virus growth in macrophages, a target cell for TMEV persistence. L* also has been demonstrated to be necessary for TMEV persistence and demyelination. Further analysis of L and L* will help elucidate the pathomechanism(s) of TMEV-induced demyelinating disease.

Identification of the homolog of cell-counting factor in the cellular slime mold Dictyostelium discoideum.

Genes for the cell-counting factors in Dictyostelium discoideum, countin and countin2, are considered to control the size of the multicellular structure of this organism. A novel gene, countin3, that is homologous to countin and countin2 genes (49 and 39% identity in amino acid sequence, respectively) was identified in the D. discoideum genome. The expression of countin3 was observed in the vegetatively growing cells, decreased in the aggregating stage, increased in the mid-developmental stage and decreased again in subsequent stages. This expression pattern is different from that of countin and countin2. The distinct expression kinetics of three genes suggests that they would have unique roles in size control of D. discoideum.