Kaposi's sarcoma-associated herpesvirus (KSHV) and its murine homolog, murine gammaherpesvirus 68 (MHV68), are lymphotropic viruses that establish latent infection in their host. Surprisingly, while B cells are the main viral reservoir in vivo, B-cell lines are poorly permissive to infection by either MHV68 or KSHV. Here, we report that most B-cell lines express very little to no cell surface heparan sulfate (HS), a glycosaminoglycan that is essential for infection by these viruses. We found that Ext1, a key enzyme in the biosynthesis of HS, was expressed at a low level in these cells. Transfection of B-cell lines with Ext1 restored high HS expression at the cell surface. Overexpression of Ext1 in murine A20 and M12 B-cell lines increased MHV68 surface binding and enhanced the efficiency of infection. Finally, although it was not sufficient to allow efficient infection, the expression of HS on BJAB cells promoted KSHV binding at the cell surface. Thus, our results indicate that MHV68 and KSHV cycles are blocked in B-cell lines at the binding step due to a lack of surface HS.One of the characteristics of gammaherpesviruses is their tropism for B lymphocytes, where they establish latency (i.e., limited viral gene expression) and persist during the whole life of their host. Kaposi's sarcoma-associated herpesvirus (KSHV, also known as human herpesvirus 8) is a gammaherpesvirus associated with both lymphoid and nonlymphoid cell tumors in humans, mostly in immunodeficient patients. KSHV is the etiologic agent of Kaposi's sarcoma, an AIDS-associated skin cancer, as well as B-cell lymphoproliferative disorders such as primary effusion lymphoma and Castleman disease (9, 10, 39). Studies of KSHV are limited by the lack of cell lines able to support productive infection as well as the strict restriction in host range. Murine gammaherpesvirus 68 (MHV68) is phylogenetically related to KSHV (13,48). MHV68 infects mice, where it establishes latency mostly in B cells (15,16,42), and has been associated with lymphoproliferative diseases in longterm-infected mice (41) or immunodeficent mice (44). Moreover, unlike KSHV, MHV68 replicates efficiently in vitro in different fibroblast and epithelial cell lines. Thus, MHV68 provides a small-animal model for the analysis of gammaherpesvirus pathogenesis both in vitro and in vivo (37,40,47).Researchers in the field have been puzzled by the fact that while B cells are the main viral reservoir in vivo, B-cell lines are mostly resistant to infection by KSHV and MHV68. Even though KSHV does not replicate efficiently in cell lines, it can establish latent infection in a variety of adherent cell lines (4). However, B-cell lines appear to be among the most resistant cell lines (4,8,24,35). Even more striking, whereas numerous cell lines are highly permissive for the MHV68 productive cycle, B-cell lines are poorly infected. MHV68 viral transcript (orf73) could be detected by reverse transcription (RT)-PCR (17) or real-time RT-PCR (unpublished observations) after infection of the A20 murine B-cel...
One characteristic linking members of the synaptotagmin family to endocytosis is their ability to bind the heterotetrameric AP2 complex via their C2B domain. By using CD4/synaptotagmin 1 chimeras, we found that the internalization signal of synaptotagmin 1 lies at the extreme COOH-terminus of the protein and can function in the absence of the C2B domain that contains the AP2 binding site. However, although not essential for internalization, the C2B domain of synaptotagmin 1 appeared to control the recognition of the internalization motif. By mutagenesis, two sites have been identified that modify regulation by the C2B domain in the neuroendocrine PC12 cell line. Mutation of a dilysine motif in the β sandwich core of the domain eliminates endocytosis. This site is known to be a site of protein–protein interaction. Mutations in the calcium binding region, or in its close proximity, also affect internalization in PC12 cells. In fibroblasts, the C2B domain inhibits the COOH-terminal internalization signal, resulting in an absence of internalization in those cells. Thus, internalization of synaptotagmin 1 is controlled by the presence of a latent internalization signal in the COOH-terminal region and a regulatory region in the C2B domain. We propose that internalization of synaptotagmin 1 is regulated in this way to allow it to couple the processes of endocytosis and calcium-mediated exocytosis in cells of the neuroendocrine lineage.
Most strains of Theiler's virus (TMEV) cause a persistent infection of the central nervous system of the mouse and a chronic demyelinating disease considered a model for multiple sclerosis. Two strains, on the contrary, cause an acute encephalitis and kill mice in a matter of days. We sequenced the leader and capsid coding region of three persistent (TO4, WW, and Yale) isolates and one neurovirulent (FA) isolate of TMEV. We compared these sequences and those already published for other isolates (DA, BeAn, GDVII, and Vilyuisk). The results suggest that virulent and persistent strains did not evolve as two separate groups, but rather that neurovirulent strains arose from a subgroup of persistent strains. The sequences of viruses isolated in different geographic areas and at different times were highly homologous, a surprising finding for an RNA virus. This suggests that severe constraints are imposed on the genome during the viral life cycle. The sequences of the TO4 and WW strains were identical, suggesting that the latter came from a laboratory contamination. The genomes of all the persistent strains sequenced so far contain an alternate open reading frame in the L region, which has been shown, in the case of the DA strain, to code for an 18-kDa protein called "I".
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