Gamma-herpesviruses persist in lymphocytes and cause disease by driving their proliferation. Lymphocyte infection is therefore a key pathogenetic event. Murid Herpesvirus-4 (MuHV-4) is a rhadinovirus that like the related Kaposi's Sarcoma-associated Herpesvirus persists in B cells in vivo yet infects them poorly in vitro. Here we used MuHV-4 to understand how virion tropism sets the path to lymphocyte colonization. Virions that were highly infectious in vivo showed a severe post-binding block to B cell infection. Host entry was accordingly an epithelial infection and B cell infection a secondary event. Macrophage infection by cell-free virions was also poor, but improved markedly when virion binding improved or when macrophages were co-cultured with infected fibroblasts. Under the same conditions B cell infection remained poor; it improved only when virions came from macrophages. This reflected better cell penetration and correlated with antigenic changes in the virion fusion complex. Macrophages were seen to contact acutely infected epithelial cells, and cre/lox-based virus tagging showed that almost all the virus recovered from lymphoid tissue had passed through lysM + and CD11c + myeloid cells. Thus MuHV-4 reached B cells in 3 distinct stages: incoming virions infected epithelial cells; infection then passed to myeloid cells; glycoprotein changes then allowed B cell infection. These data identify new complexity in rhadinovirus infection and potentially also new vulnerability to intervention.
Rhadinoviruses cause lymphocytic cancers. Their infection of lymphocytes is therefore an important therapeutic target. How this occurs is unclear. One prevalent hypothesis has been that virions directly infect lymphocytes when they enter new hosts. Here we show that host entry by Murid Herpesvirus-4, a close relative of the Kaposi's Sarcoma-associated Herpesvirus, is an epithelial rather than a lymphocyte infection: the mucosal lymphoid colonization typical of acute infectious mononucleosis only occurred later. Macrophages were closely associated with the acutely infected epithelium, and most if not all of the virus reaching B cells showed evidence of previous myeloid cell infection. Macrophage-derived virions showed a greatly enhanced capacity for lymphocyte infection that was associated with antigenic changes in the viral fusion proteins. Thus host colonization required epithelial and myeloid infections before there was lymphocyte infection. The implication is that each of these infection events could be independently targeted to limit viral persistence.