Interaction between gH/gL and the fusion protein gB is likely a conserved feature of the entry mechanism for all herpesviruses. Human cytomegalovirus (HCMV) gH/gL can be bound by gO or by the set of proteins UL128, UL130, and UL131, forming gH/ gL/gO and gH/gL/UL128-131. The mechanisms by which these complexes facilitate entry are poorly understood. Mutants lacking UL128-131 replicate well on fibroblasts but fail to enter epithelial/endothelial cells, and this has led to the general assumption that gH/gL/UL128-131 promotes gB-mediated fusion on epithelial/endothelial cells whereas gH/gL/gO provides this function on fibroblasts. This was challenged by observations that gO-null mutants were defective on all of these cell types, suggesting that entry into epithelial/endothelial cells requires both of the gH/gL complexes, but the severe replication defect of the gO mutants precluded detailed analysis. We previously reported that the ratio of gH/gL/gO and gH/gL/UL128-131 in the virion envelope varied dramatically among HCMV strains. Here, we show that strains not only differ in the ratio, but also vary in the total amount of gH/gL in the virion. Cell-type-specific particle-to-PFU ratios of HCMV strains that contained different amounts of gH/gL/gO and gH/gL/UL128-131 were determined. Infection of both fibroblasts and epithelial cells was generally correlated with the abundance of gH/gL/gO, but not with that of gH/gL/UL128-131. The low infectivity of virions rich in gH/gL/UL128-131 but low in gH/gL/gO could be overcome by treatment with the chemical fusogen polyethylene glycol (PEG), strongly arguing that gH/gL/gO provides the conserved herpesvirus gH/gL entry function of promoting gB-mediated fusion for entry into all cell types, whereas gH/gL/UL128-131 acts through a distinct mechanism to allow infection of select cell types. IMPORTANCEThe functions of HCMV gH/gL complexes in entry are unclear. Unlike the well-studied Epstein-Barr virus (EBV), where gH/gL and gH/gL/gp42 complexes both seem capable of promoting gB fusion during entry into different cell types, our studies here suggest that for HCMV, gH/gL/gO promotes gB fusion on all cell types, whereas gH/gL/UL128-131 broadens virus tropism through a distinct, as yet unknown mechanism. To our knowledge, this is the first suggestion of a herpesvirus gH/gL that does not act by promoting gB fusion, which might make HCMV a useful model to study the fundamental mechanisms by which herpesvirus gH/gL regulates gB fusion. Moreover, gH/gL/UL128-131 is a candidate vaccine target. Our findings help to explain the cell-type-dependent virus neutralization exhibited by anti-gH/gL/UL128-131 antibodies and underscore the importance of gH/ gL/gO as another important part of vaccine or therapeutic strategies. P rimary infection of healthy adults by human cytomegalovirus (HCMV) is usually subclinical or mildly symptomatic but leads to lifelong persistent or latent infection. Primary infection or reactivation of HCMV in immunocompromised hosts, such those infected with HIV and t...

Herpesvirus glycoprotein complex gH/gL provides a core entry function through interactions with the fusion protein gB and can also influence tropism through receptor interactions. The Epstein-Barr virus gH/gL and gH/gL/gp42 serve both functions for entry into epithelial and B cells, respectively. Human cytomegalovirus (HCMV) gH/gL can be bound by the UL128-131 proteins or gO. The phenotypes of gO and UL128-131 mutants suggest that gO-gH/gL interactions are necessary for the core entry function on all cell types, whereas the binding of UL128-131 to gH/gL likely relates to a distinct receptor-binding function for entry into some specific cell types (e.g., epithelial) but not others (e.g., fibroblasts and neurons). There are at least eight isoforms of gO that differ by 10 to 30% of amino acids, and previous analysis of two HCMV strains suggested that some isoforms of gO function like chaperones, disassociating during assembly to leave unbound gH/gL in the virion envelope, while others remain bound to gH/gL. For the current report, we analyzed the gH/gL complexes present in the virion envelope of several HCMV strains, each of which encodes a distinct gO isoform. Results indicate that all strains of HCMV contain stable gH/gL/gO trimers and gH/gL/UL128-131 pentamers and little, if any, unbound gH/gL. TR, TB40/e, AD169, and PH virions contained vastly more gH/gL/gO than gH/gL/UL128-131, whereas Merlin virions contained mostly gH/gL/UL128-131, despite abundant unbound gO remaining in the infected cells. Suppression of UL128-131 expression during Merlin replication dramatically shifted the ratio toward gH/gL/gO. These data suggest that Merlin gO is less efficient than other gO isoforms at competing with UL128-131 for binding to gH/gL. Thus, gO diversity may influence the pathogenesis of HCMV through effects on the assembly of the core versus tropism gH/gL complexes.

53acids between TR and ME, we analyzed recombinant viruses in which the UL74(gO) ORF was 54 swapped. TR virions were >40-fold more infectious than ME. Transcriptional repression of 55 UL128-131 enhanced infectivity of ME to the level of TR, despite still far lower levels of 56 gH/gL/gO. Swapping the UL74(gO) ORF had no effect on either TR or ME. A quantitative 57 immunoprecipitation approach revealed that gH/gL expression was within 4-fold between TR 58 and ME, but gO expression was 20-fold less by ME, and suggested differences in mRNA

Varicella–zoster virus (VZV) is the causative agent of chicken pox (varicella) and shingles (zoster). Although considered benign diseases, both varicella and zoster can cause complications. Zoster is painful and can lead to post herpetic neuralgia. VZV has also been linked to stroke, related to giant cell arteritis in some cases. Vaccines are available but the attenuated vaccine is not recommended in immunocompromised individuals and the efficacy of the glycoprotein E (gE) based subunit vaccine has not been evaluated for the prevention of varicella. A hallmark of VZV pathology is the formation of multinucleated cells termed polykaryocytes in skin lesions. This cell–cell fusion (abbreviated as cell fusion) is mediated by the VZV glycoproteins gB, gH and gL, which constitute the fusion complex of VZV, also needed for virion entry. Expression of gB, gH and gL during VZV infection and trafficking to the cell surface enables cell fusion. Recent evidence supports the concept that cellular processes are required for regulating cell fusion induced by gB/gH–gL. Mutations within the carboxyl domains of either gB or gH have profound effects on fusion regulation and dramatically restrict the ability of VZV to replicate in human skin. This loss of regulation modifies the transcriptome of VZV infected cells. Furthermore, cellular proteins have significant effects on the regulation of gB/gH–gL-mediated cell fusion and the replication of VZV, exemplified by the cellular phosphatase, calcineurin. This review provides the current state-of-the-art knowledge about the molecular controls of cell fusion-dependent pathogenesis caused by VZV.

6cytomegalovirus, influencing the assembly of gH/gL complexes and virion 7 infectivity. ABSTRACT 45Tropism of human cytomegalovirus (HCMV) is influenced by the envelope glycoprotein 46 complexes gH/gL/gO and gH/gL/UL128-131. During virion assembly, gO and the UL128-131 47 proteins compete for binding to gH/gL in the ER. This assembly process clearly differs among 48 strains since Merlin (ME) virions contain abundant gH/gL/UL128-131 and little gH/gL/gO, 49 whereas TR contains much higher levels of total gH/gL, mostly in the form of gH/gL/gO, but 50 much less gH/gL/UL128-131 than ME. Remaining questions include 1) what are the 51 mechanisms behind these assembly differences, and 2) do differences reflect in vitro culture 52 adaptations or natural genetic variations? Since the UL74(gO) ORF differs by 25% of amino 53 acids between TR and ME, we analyzed recombinant viruses in which the UL74(gO) ORF was 54 swapped. TR virions were >40-fold more infectious than ME. Transcriptional repression of 55 UL128-131 enhanced infectivity of ME to the level of TR, despite still far lower levels of 56 gH/gL/gO. Swapping the UL74(gO) ORF had no effect on either TR or ME. A quantitative 57 immunoprecipitation approach revealed that gH/gL expression was within 4-fold between TR 58 and ME, but gO expression was 20-fold less by ME, and suggested differences in mRNA 59 transcription, translation or rapid ER-associated degradation of gO. Trans-complementation of 60 gO expression during ME replication gave 6-fold enhancement of infectivity beyond the 40-fold 61 effect of UL128-131 repression alone. Overall, strain variations in assembly of gH/gL 62 complexes result from differences in expression of gO and UL128-131, and selective 63 advantages for reduced UL128-131 expression during fibroblast propagation are much stronger 64 than for higher gO expression. 65 IMPORTANCE 66Specific genetic differences between independently isolated HCMV strains may result from 67 purifying selection on de novo mutations arising during propagation in culture, or random 68 sampling among the diversity of genotypes present in clinical specimens. Results presented 69 indicate that while reduced UL128-131 expression may confer a powerful selective advantage 70 during cell-free propagation of HCMV in fibroblast cultures, selective pressures for increased gO 71 expression are much weaker. Thus, variation in gO expression among independent strains may 72 represent natural genotype variability present in vivo. This may have important implications for 73 virus-host interactions such as immune recognition, and underscores the value of studying 74 molecular mechanisms of replication using multiple HCMV strains.75 INTRODUCTION 76 Human cytomegalovirus (HCMV) is widely spread throughout the world, found in 77 approximately 60% of adults in developed countries and 100% in developing countries 78 (reviewed in (1-3) (4)). Immunocompromised individuals such as those infected with HIV 79 patients, or transplant recipients under antirejection treatments can suffer HCMV re...