Chronic encephalomyelitis virus exhibits cellular tropism and evades pDCs by binding to sialylated integrins as the cell surface receptors.

Theiler's murine encephalomyelitis virus (TMEV) causes a chronic demyelinating disease similar to multiple sclerosis in mice. Although sialic acids have been shown to be essential for TMEV attachment to the host, the surface receptor has not been identified. While type I interferons play a pivotal role in the elimination of the chronic infectious Daniel (DA) strain, the role of plasmacytoid dendritic cells (pDCs) is controversial. We herein found that TMEV binds to conventional DCs but not to pDCs. A glycomics analysis showed that the sialylated N-glycan fractions were lower in pDCs than in conventional DCs, indicating that pDCs are not susceptible to TMEV infection due to the low levels of sialic acid. TMEV capsid proteins contain an integrin recognition motif, and dot blot assays showed that the integrin proteins bind to TMEV and that the viral binding was reduced in the desialylated αX ß2 . αX ß2 protein suppressed TMEV replication in vivo, and TMEV co-localized with integrin αM at the cell membrane and TLR 3 in the cytoplasm, suggesting that αM serves as the viral attachment and entry. These results show that the chronic encephalomyelitis virus utilizes sialylated integrins as cell surface receptors, leading to cellular tropism to evade pDC activation.

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.

Failure in activation of the canonical NF-κB pathway by human T-cell leukemia virus type 1 Tax in non-hematopoietic cell lines.

Human T-cell leukemia virus type 1 (HTLV-1) Tax (Tax1) plays crucial roles in leukemogenesis in part through activation of NF-κB. In this study, we demonstrated that Tax1 activated an NF-κB binding (gpκB) site of the gp34/OX40 ligand gene in a cell type-dependent manner. Our examination showed that the gpκΒ site and authentic NF-κB (IgκB) site were activated by Tax1 in hematopoietic cell lines. Non-hematopoietic cell lines including hepatoma and fibroblast cell lines were not permissive to Tax1-mediated activation of the gpκB site, while the IgκB site was activated in those cells in association with binding of RelB. However RelA binding was not observed in the gpκB and IgκB sites. Our results suggest that HTLV-1 Tax1 fails to activate the canonical pathway of NF-κB in non-hematopoietic cell lines. Cell type-dependent activation of NF-κB by Tax1 could be associated with pathogenesis by HTLV-1 infection.

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.

Intrinsic temperature sensitivity of influenza C virus hemagglutinin-esterase-fusion protein.

Influenza C virus replicates more efficiently at 33°C than at 37°C. To determine whether hemagglutinin-esterase-fusion protein (HEF), a surface glycoprotein of influenza C virus, is a restricting factor for this temperature sensitivity, we analyzed the biological and biochemical properties of HEF at 33°C and 37°C. We found that HEF exhibits intrinsic temperature sensitivities for surface expression and fusion activity.

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.

Saffold virus, a novel human Cardiovirus with unknown pathogenicity.

Although cardioviruses have been thought to mainly infect rodents, a novel human cardiovirus, designated Saffold virus (SAFV), was identified in 2007. SAFV is grouped with Theiler-like rat virus and Theiler's murine encephalomyelitis virus (TMEV) in the species Theilovirus of the genus Cardiovirus of the family Picornaviridae. Eight genotypes of SAFV have now been identified. SAFV has been isolated from nasal and stool specimens from infants presenting with respiratory and gastrointestinal symptoms as well as from children with nonpolio acute flaccid paralysis; however, the relationship of SAFV to this symptomatology remains unclear. Of note, the virus has also been isolated from the cerebrospinal fluid specimens of patients with aseptic meningitis. This finding is of interest since TMEV is known to cause a multiple sclerosis-like syndrome in mice. The involvement of SAFV in various diseases (e.g., respiratory illness, gastrointestinal illness, neurological diseases, and type I diabetes) is presently under investigation. In order to clarify the pathogenicity of SAFV, additional epidemiological studies are required. Furthermore, identification of the SAFV cellular receptor will help establish an animal model for SAFV infection and help clarify the pathogenesis of SAFV-related diseases. In addition, investigation of the tissue-specific expression of the receptor may facilitate development of a novel picornavirus vector, which could be a useful tool in gene therapy for humans. The study of viral factors involved in viral pathogenicity using a reverse genetics technique will also be important.

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.

In vivo expression of the HBZ gene of HTLV-1 correlates with proviral load, inflammatory markers and disease severity in HTLV-1 associated myelopathy/tropical spastic paraparesis (HAM/TSP).

BACKGROUND: Recently, human T-cell leukemia virus type 1 (HTLV-1) basic leucine zipper factor (HBZ), encoded from a minus strand mRNA was discovered and was suggested to play an important role in adult T cell leukemia (ATL) development. However, there have been no reports on the role of HBZ in patients with HTLV-1 associated inflammatory diseases. RESULTS: We quantified the HBZ and tax mRNA expression levels in peripheral blood from 56 HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) patients, 10 ATL patients, 38 healthy asymptomatic carriers (HCs) and 20 normal uninfected controls, as well as human leukemic T-cell lines and HTLV-1-infected T-cell lines, and the data were correlated with clinical parameters. The spliced HBZ gene was transcribed in all HTLV-1-infected individuals examined, whereas tax mRNA was not transcribed in significant numbers of subjects in the same groups. Although the amount of HBZ mRNA expression was highest in ATL, medium in HAM/TSP, and lowest in HCs, with statistical significance, neither tax nor the HBZ mRNA expression per HTLV-1-infected cell differed significantly between each clinical group. The HTLV-1 HBZ, but not tax mRNA load, positively correlated with disease severity and with neopterin concentration in the cerebrospinal fluid of HAM/TSP patients. Furthermore, HBZ mRNA expression per HTLV-1-infected cell was decreased after successful immunomodulatory treatment for HAM/TSP. CONCLUSION: These findings suggest that in vivo expression of HBZ plays a role in HAM/TSP pathogenesis.

Impaired function of human T-lymphotropic virus type 1 (HTLV-1)-specific CD8+ T cells in HTLV-1-associated neurologic disease.

Despite abundant activated virus-specific cytotoxic T lymphocytes (CTLs), patients with human T-lymphotropic virus type 1 (HTLV-1)-associated myelopathy/tropical spastic paraparesis (HAM/TSP) showed a significantly higher frequency of infected T cells than did healthy virus carriers (HVCs). Here, we demonstrate that at a given proviral load, the frequency of CD8(+) T cells that are negative for specific costimulatory molecules was significantly higher in HAM/TSP than in age-matched HVCs and uninfected healthy controls (HCs), whereas the frequency of intracellular perforin-positive CD8(+) T cells was significantly lower in both HAM/TSP and HVCs than in HCs. An inverse correlation between HTLV-1 proviral load (PVL) and percent perforin-positive CD8(+) T cells were observed only in disease-protective allele HLA-A*02-positive HVCs, but not in HAM/TSP patients, whether HLA-A*02 positive or negative, nor in HLA-A*02-negative HVCs. Significantly lower perforin expression was observed in HTLV-1-specific than in cytomegalovirus-specific CD8(+) T cells. Majority of HTLV-1-specific CD8(+) T cells in HVCs showed a CD28(-)CD27(+) phenotype, whereas HAM/TSP showed a CD28(-)CD27(-) phenotype. HTLV-1-specific CD8(+) T cells from HAM/TSP patients showed significantly lower degranulation than HVCs by CD107a mobilization assay. These findings suggest that an impaired function of HTLV-1-specific CTLs is associated with failing antiviral control and disease HAM/TSP.