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

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

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

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

Abstract 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

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

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