Lack of Heparan Sulfate Expression in B-Cell Lines: Implications for Kaposi's Sarcoma-Associated Herpesvirus and Murine Gammaherpesvirus 68 Infections

Abstract

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...

Tiled Microarray Identification of Novel Viral Transcript Structures and Distinct Transcriptional Profiles during Two Modes of Productive Murine Gammaherpesvirus 68 Infection

“…In addition, transcriptional profiles of newly infected B cells in cell culture and, ideally, in the context of the host would also provide significant insight regarding cell type-dependent impacts on viral gene expression. These studies would likely require deep sequencing technology since B cells have low permissivity for de novo infection (39).…”

Early Events in Kaposi's Sarcoma-Associated Herpesvirus Infection of Target Cells

“…KSHV infection is inhibited by soluble heparin but not by chondroitin sulfates A and C (3). Due to the low level of expression of Ext1, a key glycosyltransferase enzyme in HS biosynthesis, HS is not expressed in appreciable levels in many B-cell lines and in primary B cells (25). The refraction of primary B cells for KSHV infection may be due to the lack of HS as the expression of HS in BJAB cells (an EBV-negative B-cell line) increased the binding of KSHV to the cell surface (25).…”

“…Due to the low level of expression of Ext1, a key glycosyltransferase enzyme in HS biosynthesis, HS is not expressed in appreciable levels in many B-cell lines and in primary B cells (25). The refraction of primary B cells for KSHV infection may be due to the lack of HS as the expression of HS in BJAB cells (an EBV-negative B-cell line) increased the binding of KSHV to the cell surface (25). Interestingly, BJAB cells from one laboratory did not express any HS, while cells from another laboratory expressed abundant HS, which could explain some discrepancies reported for the infection of BJAB cells (25).…”

“…The refraction of primary B cells for KSHV infection may be due to the lack of HS as the expression of HS in BJAB cells (an EBV-negative B-cell line) increased the binding of KSHV to the cell surface (25). Interestingly, BJAB cells from one laboratory did not express any HS, while cells from another laboratory expressed abundant HS, which could explain some discrepancies reported for the infection of BJAB cells (25). KSHV gB, gpK8.1A, ORF4, and gH bind to cell surface HS molecules (2,8,21,33,54,55).…”

Infection of Lymphoblastoid Cell Lines by Kaposi's Sarcoma-Associated Herpesvirus: Critical Role of Cell-Associated Virus

“…The inability of BJAB cells to support KSHV entry may be due, at least in part, to their lack of cell surface heparin sulfate, which is known to promote herpesvirus adsorption (1,6). When a plasmid encoding functions for heparin sulfate was transfected into BJAB cells (23), the cell-free virus could bind to BJAB cells without apparent infection. Furthermore, our work and that of others indicate that BJAB cells have additional postentry blocks to KSHV lytic growth.…”