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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".

The DA strain of Theiler's virus persists in the central nervous system of mice and causes chronic inflammation and demyelination. On the other hand, the GDVII strain causes an acute encephalitis and does not persist in surviving animals. Series of recombinants between infectious cDNA clones of the genomes of DA and GDVII viruses have been constructed. The analysis of the phenotypes of the recombinant viruses has shown that determinants of persistence and demyelination are present in the capsid proteins of DA virus. Chimeric viruses constructed by the different research groups gave consistent results, with one exception. Chimeras GD1B-2A/DAFL3 and GDlB-2C/DAFL3, which contain part of capsid protein VP2, capsid proteins VP3 and VP1, and different portions of P2 of GDVII in a DA background, were able to persist and cause demyelination. Chimera R4, whose genetic map is identical to that of GD1B-2A/DAFL3, was not. After exchanging the viral chimeras between laboratories and verifying each other's observations, new chimeras were generated in order to explain this difference. Here we report that the discrepancy can be attributed to a single amino acid difference in the sequence of the capsid protein VP2 of the two parental DA strains. DAFL3 (University of Chicago) and the chimeras derived from it, GD1B-2A/DAFL3 and GDIB-2C/DAFL3, contain a Lys at position 141, while TMDA (Institut Pasteur) and R4, the chimera derived from it, contain an Asn in that position. This amino acid is located at the tip of the EF loop, on the rim of the depression spanning the twofold axis of the capsid. These results show that a single amino acid change can confer the ability to persist and demyelinate to a chimeric Theiler's virus, and they pinpoint a region of the viral capsid that is important for this phenotype.

The rate at which a membrane protein is internalized from the plasma membrane can be regulated by revealing a latent internalization signal in response to an appropriate stimulus. Internalization of the synaptic vesicle membrane protein, synaptotagmin 1, is controlled by two distinct regions of its intracytoplasmic C2B domain, an internalization signal present in the 29 carboxyterminal (CT) amino acids and a separate regulatory region. We have now characterized the internalization motif by mutagenesis and found that it involves an essential tryptophan in the last beta strand of the C2B domain, a region that is distinct from the AP2-binding site previously described. Internalization through the tryptophan-based motif is sensitive to eps15 and dynamin mutants and is therefore likely to be clathrin mediated. A tryptophan-tophenylalanine mutation had no effect on internalization of the CT domain alone, but completely inhibited endocytosis of the folded C2B domain. This result suggests that recognition of sorting motifs can be influenced by their structural context. We conclude that endocytosis of synaptotagmin 1 requires a novel type of internalization signal that is subject to regulation by the rest of the C2B domain.

Human cytomegalovirus (HCMV) infection is usually benign in healthy individuals but can cause life-threatening disease in those with compromised immune systems. Approved drugs available to treat HCMV disease, including ganciclovir, cidofovir, and foscarnet, have significant toxicities that limit their use in certain patient populations. LJP538 and LJP539 are human monoclonal antibodies that are being evaluated as immunoglobulin therapeutics. The antibodies target glycoproteins gB and the gH/ gL/UL128/UL130/UL131a pentameric complex, respectively. Here we present an in vitro characterization of these antibodies. We show that LJP538 and LJP539 are more potent than a marketed immunoglobulin at inhibiting HCMV infection of various cell lines relevant to pathogenesis. We find that LJP538 and LJP539 are active against a panel of clinical isolates in vitro and demonstrate minor-to-moderate synergy in combination. Passage of HCMV in the presence of LJP538 or LJP539 alone resulted in resistance-associated mutations that mapped to the target genes. However, no loss of susceptibility to the combination of antibodies was observed for >400 days in culture. Finally, the binding regions of LJP538 and LJP539 are conserved among clinical isolates. Taken together, these data support the use of LJP538 and LJP539 in combination for clinical trials in HCMV patients.

Cell surface heparan sulfate (HS) is an important co-receptor for many cytokines, chemokines, and growth factors. Here we report that splenic murine B cells express very little HS, and that upon infection with either gammaherpesvirus (MHV68) or betaherpesvirus (MCMV), HS is rapidly upregulated at the surface of B cells. HS upregulation was not observed in mice deficient for the type-I IFN (IFN-I) receptor. Additionally, treatment of wild-type mice with the IFN-I inducer polyinosine polycytidylic acid (poly I:C) triggered HS expression at the B cell surface. Similarly, incubation of purified splenic B cells with IFN-I, TLR ligands, or B cell receptor stimulators ex vivo resulted in a drastic increase in HS surface expression. We found that IFN-I induced an increase in the surface expression of HS-modified syndecan 4 as well as that of an unidentified HSPG. Finally, IFN-I treatment increased B cell responsiveness to APRIL, a cytokine involved in B cell survival and T cell-independent B cell responses. Enzymatic removal of HS from IFN-I treated B cells inhibited APRIL signaling. Altogether, our results indicate that upon herpesvirus infection in mice, HS is rapidly upregulated at the surface of B cells due to the action of IFN-I, potentially increasing B cell responsiveness to cytokines. Induction of HS expression at the B cell surface by stimulators of the innate immune response is likely playing a key role in the development of a robust immune response.