trans-Repression of protein expression dependent on the Epstein-Barr virus promoter Wp during latency.

An ordered silencing of Epstein-Barr virus (EBV) latency gene transcription is critical for establishment of persistent infection within B lymphocytes, yet the mechanisms responsible and the role that the virus itself may play are unclear. Here we describe two B-cell superinfection models with which to address these problems. In the first, Burkitt lymphoma (BL) cells that maintain latency I, when superinfected, initially supported transcription from the common EBNA promoters Wp and Cp (latency III) but ultimately transitioned to latency I (Cp/Wp silent), an essential requirement for establishment of EBV latency in vivo. We used this model to test whether the early lytic-cycle gene BHLF1, implicated in silencing of the Cp/Wp locus, is required to establish latency I. Upon superinfection with EBV deleted for the BHLF1 locus, however, we have demonstrated that BHLF1 is not essential for this aspect of EBV latency. In the second model, BL cells that maintain Wp-restricted latency, a variant program in which Cp is silent but Wp remains active, sustained the latency III program of transcription from the superinfecting-virus genomes, failing to transition to latency I. Importantly, there was substantial reduction in Wp-mediated protein expression from endogenous EBV genomes, in the absence of Cp reactivation, that could occur independent of a parallel decrease in mRNA. Thus, our data provide evidence of a novel, potentially posttranscriptional mechanism for trans-repression of Wp-dependent gene expression. We suggest that this may ensure against overexpression of the EBV nuclear antigens (EBNAs) prior to the transcriptional repression of Wp in cis that occurs upon activation of Cp.

N-terminal mutants of herpes simplex virus type 2 gH are transported without gL but require gL for function.

Glycoprotein H (gH) is conserved among all herpesviruses and is essential for virus entry and cell fusion along with gL, gB, and, in most alphaherpesviruses, gD. Within the gH/gL heterodimer, it is thought that gH accounts for the fusion function and gL acts as a chaperone for the folding and transport of gH. Here, we found that the N terminus of gH2 contains important elements involved in both its folding and its transport. Our conclusions are based on the phenotypes of a series of gH deletion mutants in which the signal sequence (residues 1 to 18) was retained and N-terminal residues were removed up to the number indicated. The first mutant, gH2Delta29 (deletion of residues 19 to 28), like wild-type (WT) gH, required gL for both transport and function. To our surprise, two other mutants (gH2Delta64 and gH2Delta72) were transported to the cell surface independent of gL but were nonfunctional, even when complexed with gL. Importantly, a fourth mutant (gH2Delta48) was transported independent of gL but was functional only when complexed with gL. Using a panel of monoclonal antibodies against gH2, we found that when gH2Delta48 was expressed alone, its antigenic structure differed from that of gH2Delta48/gL or gH2-WT/gL. Mutation of gH2 residue R39, Y41, W42, or D44 allowed gL-independent transport of gH. Our results also show that gL is not merely required for gH transport but is also necessary for the folding and function of the complex. Since gH2Delta64/gL and gH2Delta72/gL were nonfunctional, we hypothesized that residues critical for gH/gL function lie within this deleted region. Additional mutagenesis identified L66 and L72 as important for function. Together, our results highlight several key gH residues: R39, Y41, W42, and D44 for gH transport and L66 and L72 for gH/gL structure and function.

Epitope mapping of herpes simplex virus type 2 gH/gL defines distinct antigenic sites, including some associated with biological function.

The gH/gL complex plays an essential role in virus entry and cell-cell spread of herpes simplex virus (HSV). Very few immunologic reagents were previously available to either identify important functional regions or gain information about structural features of this complex. Therefore, we generated and characterized a panel of 31 monoclonal antibodies (MAbs) against HSV type 2 (HSV-2) gH/gL. Fourteen MAbs bound to a conformation-dependent epitope of the gH2/gL2 complex, and all blocked virus spread. The other 17 MAbs recognized linear epitopes of gH (12) or gL (5). Interestingly, two of the gL MAbs and six of the gH MAbs were type common. Overlapping synthetic peptides were used to map MAbs against linear epitopes. These data, along with results of competition analyses and functional assays, assigned the MAbs to groups representing eight distinct antigenic sites on gH (I to VIII) and three sites on gL (A, B, and C). Of most importance, the MAbs with biological activity mapped either to site I of gH2 (amino acids 19 to 38) or to sites B and C of gL2 (residues 191 to 210). Thus, these MAbs constitute a novel set of reagents, including the first such reagents against gH2 and gL2 as well as some that recognize both serotypes of each protein. Several recognize important functional domains of gH2, gL2, or the complex. We suggest a common grouping scheme for all of the known MAbs against gH/gL of both HSV-1 and HSV-2.