Different Subcellular Localization of Theiler's Murine Encephalomyelitis Virus Leader Proteins of GDVII and DA Strains in BHK-21 Cells

Abstract

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 Theiler's murine encephalomyelitis virus (TMEV) belongs to the genus Cardiovirus of the family Picornaviridae, and its strains are divided into two subgroups on the basis of their different biological activities. The neurovirulent strains, such as GDVII and FA, produce acute and fatal encephalomyelitis in mice. The persistent strains, such as TO, DA, BeAn, etc., induce mild and nonfatal encephalomyelitis, followed by a chronic demyelinating disease with virus persistence in the spinal cords of mice. This late demyelinating disease is thought to be an excellent experimental model for the human demyelinating disease multiple sclerosis (MS) (5,17,20).The TMEV genome is a single-stranded RNA molecule and translated as a long precursor polyprotein to yield 12 viral proteins by autoproteolytic cleavage (23). Two subgroup strains of TMEV have a sequence identity of approximately 95% at the amino acid level. The amino acid sequences of the proteins encoded by the P1, P2, and P3 regions of both strains are highly conserved and show 94, 96, and 98% identity, respectively. The genome has another coding region, designated the leader (L), at the most amino-terminal location of the precursor polyprotein. The L coding region encodes 76 amino acids (aa) and shows a low sequence identity of only 85% to the above-described three regions (16,19,22). Therefore, L has the greatest difference in amino acid sequence among any of the viral proteins and may play an important role in subgroupspecific biological activities of TMEV. In this study, we have investigated the subcellular localization of the L proteins of GDVII and DA strains and characterized the functional domains involved in the differential distribution between DA L and GDVII L in BHK-21 cells by a series of deletion mutant and chimeric construct experiments. MATERIALS AND METHODSPlasmid constructs. L is organized into the three domains: (i) the N-terminal atypical zinc finger domain, (ii) the acidic domain, and (iii) the serine/threonine (S/T)-rich domain. The amino acid sequence alignment of both strains is shown in Fig. 1A. Amino acid sequences of the zinc finger and acidic domains of both strains are identical. In contrast, the S/T-rich domain was less conserved. Five out of 13 residues are different. The C-terminal region consisting of 13 aa also contains three different amino acid residues between the strains. Wild-type L (Lwt) and mutated constructs of the DA or GDVII strain are depicted in Fig. 1B. Those cDNAs were generated by PCR amplification using pDAFL3 containing the full-length DA cDNA (24) or pGDVIIFL2 containing the full-length GDVII cDNA (10). L⌬Z, L⌬A, L⌬S/T, and L⌬C lack the zinc finger domain (aa 3 to 14), the acidic domain (aa 30 to 46), th...

Entire genome sequence analysis of genotype IX Newcastle disease viruses reveals their early-genotype phylogenetic position and recent-genotype genome size

“…On the other hand, the insertion of a 6-nt motif is an rare event in NDV evolution in view of the extremely low probability of nucleotide addition or deletion in the genome RNA of Paramyxoviruses [37,38]. Thus, it was most possible that genotype IX NDV was the common origin of genotype V-VIII.…”

AMP-activated protein kinase phosphorylates EMCV, TMEV and SafV leader proteins at different sites

“…The L T -dependent apoptotic phenotypes (2), and L T intracellular localization differences (17) previously assigned to the S 57 (DA) and P 57 (GDVII) sequences, are now logically explained by the phosphorylation status of this residue. We do not yet know if GDVII or other viruses of this subtype use an alternative primary site instead.…”

“…Such viruses are also unable to inhibit stress granule formation (8). Equivalent mutations to the L T Ser/Thr domain (S 51 A, S 53 A, S 54 A) do not have similar effects (14), although a single amino acid change in the L T of GDVII virus (P 57 ) and DA virus (S 57 ) correlates with differential apoptotic phenotypes (2), and L T intracellular localization differences (17). …”

“…L T and L S also become phosphorylated, but at different sites than L E , and in reactions that do not require CK2 (15). Sequence comparisons have suggested S 57 (17) and/or T 63 (14) as possible sites in L T (DA). We now have experimental validation that L T (BeAn) and L S (SafV-2) and are differentially phosphorylated at S 57 (Ser/Thr domain) and T 58 (Theilo domain), respectively.…”

Intracellular localization of Saffold virus Leader (L) protein differs in Vero and HEp-2 cells

“…12 Moreover, the L protein of TMEV was reported to localize either 1 in both the nucleus and cytoplasm (DA strain) or 2 the cytoplasm only (GDVII strain). 13 The L protein of DA strain was known to interfere with the nucleocytoplasmic trafficking of cellular proteins; it promotes the redistribution of a nuclear protein to the cytoplasm and a cytoplasmic protein to the nucleus. 14 It was also demonstrated to inhibit the production of immediate-early alpha/beta interferon at the transcription level, 15 which led to the dysregulation of the whole cellular defense system by probably fostering the persistence of TMEV DA strain.…”