All gammaherpesviruses encode a virion glycoprotein positionally homologous to Epstein-Barr virus gp350. These glycoproteins are thought to be involved in cell binding, but little is known of the roles they might play in the whole viral replication cycle. We have analyzed the contribution of murine gammaherpesvirus 68 (MHV-68) gp150 to viral propagation in vitro and host colonization in vivo. MHV-68 lacking gp150 was viable and showed normal binding to fibroblasts and normal single-cycle lytic replication. Its capacity to infect glycosaminoglycan (GAG)-deficient CHO-K1 cells and NS0 and RAW264.7 cells, which express only low levels of GAGs, was paradoxically increased. However, gp150-deficient MHV-68 spread poorly through fibroblast monolayers, with reduced cell-free infectivity, consistent with a deficit in virus release. Electron microscopy showed gp150-deficient virions clustered on infected-cell plasma membranes. MHV-68-infected cells showed reduced surface GAG expression, suggesting that gp150 prevented virions from rebinding to infected cells after release by making MHV-68 infection GAG dependent. Surprisingly, gp150-deficient viruses showed only a transient lag in lytic replication in vivo and established normal levels of latency. Cell-to-cell virus spread and the proliferation of latently infected cells, for which gp150 was dispensable, therefore appeared to be the major route of virus propagation in an infected host.Herpesviruses are large, complex pathogens that use a range of different glycoproteins to spread between cells and between hosts (34). A common first step in infection is virion adsorption to cell surface glycosaminoglycans (GAGs), such as heparan sulfate, which are widespread on epithelial surfaces (5). Herpesvirus-GAG interactions are thought to concentrate virions on cell surfaces and so promote specific protein receptor binding, which is then followed by membrane fusion (32). Cell binding by purified virions and recombinant viral glycoproteins has been studied extensively in vitro, but relatively little is known about how individual herpesvirus glycoproteins contribute to infection of the natural host. Here simple fluid-filled spaces are rare, extracellular matrix is abundant, and viral spread is probably limited to defined anatomical pathways. A significant role for direct cell-to-cell spread in herpesvirus infections is implied by pseudorabies virus that lacks glycoprotein D and infects cells poorly as free virus propagating efficiently in mice (28) and by herpes simplex virus that lacks glycoprotein E (required only for cell-to-cell spread of virus) disseminates inefficiently from the rat cornea (14).Our understanding of in vivo glycoprotein function is especially limited with gammaherpesviruses such as Epstein-Barr virus (EBV) and the Kaposi's sarcoma-associated herpesvirus (KSHV), whose narrow host ranges have largely precluded studies of pathogenesis. In vitro lytic replication systems have also proved difficult to establish. Nonetheless, it is important for practical purposes, suc...

Herpesviruses characteristically persist in immune hosts as latent genomes, but to transmit infection they must reactivate and replicate lytically. The interaction between newly formed virions and pre-existing antibody is therefore likely to be a crucial determinant of viral fitness. Murine gammaherpesvirus-68 (MHV-68) behaves as a natural pathogen of conventional, inbred mice and consequently allows such interactions to be analysed experimentally in a relatively realistic setting. Here, monoclonal antibodies (mAbs) were derived from MHV-68-infected mice and all those recognizing infected-cell surfaces were tested for their capacity to neutralize MHV-68 virions. All of the neutralizing mAbs identified were specific for the viral glycoprotein H (gH)-gL heterodimer and required both gH and gL to reproduce their cognate epitopes. Based on antibody interference, there appeared to be two major neutralization epitopes on gH-gL. Analysis of a representative mAb indicated that it blocked infection at a post-binding step -either virion endocytosis or membrane fusion.

The murine gamma-herpesvirus-68 MK3 protein inhibits CD8(+) T cell recognition by ubiquitinating the cytoplasmic tails of classical MHC class I heavy chains. Here we show that MK3 also provides the first example of a protein that degrades tapasin and TAP. The degradation was MK3 RING finger dependent and primarily affected TAP. MK3 associated with TAP1 in the absence of tapasin or TAP2, suggesting that TAP1 was a primary binding partner in the peptide loading complex. TAP2 also played a major role in MK3 stability and function. By degrading TAP, therefore, MK3 limited its own expression. However, TAP degradation also broadened the MK3 inhibitory repertoire and achieved a remarkable resistance to MHC class I upregulation by interferon-gamma, suggesting that it represents a specific adaptation to immune evasion in lymphoid tissue.

BackgroundHerpesviruses can be neutralized in vitro but remain infectious in immune hosts. One difference between these settings is the availability of immunoglobulin Fc receptors. The question therefore arises whether a herpesvirus exposed to apparently neutralizing antibody can still infect Fc receptor+ cells.Principal FindingsImmune sera blocked murine gamma-herpesvirus-68 (MHV-68) infection of fibroblasts, but failed to block and even enhanced its infection of macrophages and dendritic cells. Viral glycoprotein-specific monoclonal antibodies also enhanced infection. MHV-68 appeared to be predominantly latent in macrophages regardless of whether Fc receptors were engaged, but the infection was not abortive and new virus production soon overwhelmed infected cultures. Lytically infected macrophages down-regulated MHC class I-restricted antigen presentation, endocytosis and their response to LPS.ConclusionsIgG Fc receptors limit the neutralization of gamma-herpesviruses such as MHV-68.

The lytic cycle functions of gammaherpesviruses have received relatively little attention to date, at least in part due to the lack of a convenient experimental model. The murine gammaherpesvirus 68 (MHV-68) now provides such a model and allows the roles of individual lytic cycle gammaherpesvirus proteins to be evaluated in vivo. We have used MHV-68 to determine the contribution of a gammaherpesvirus thymidine kinase (TK) to viral lytic replication and latency establishment. MHV-68 mutants with a disrupted TK gene grew normally in vitro but showed a severe attenuation of replication in the lungs after intranasal inoculation, with lytic titers at least 1,000-fold lower than those of wild-type and revertant viruses. Nevertheless, the establishment of latency by the TK-deficient mutants, while delayed, was not prevented by their lytic replication deficit. The viral TK clearly plays a crucial role in the capacity of MHV-68 to replicate efficiently in its natural host but does not seem to be essential to establish a persistent infection. The potential of TK-deficient mutants as gammaherpesvirus vaccines is discussed.