In herpes simplex virus 1-infected cells, the transcription of a genes, the first set of genes to be expressed, is induced by a virion component, the a-trans-induction factor, and requires a cis site. Homologs of the cis site are present in the promoter-regulatory domains of all a genes and bind two cellular proteins designated as aHl and aH2H3. We report that a-trans-induction factor, synthesized in vitro or present in nuclear extracts of infected cells, forms complexes with viral DNA fragments containing its cis-acting site only in the presence of cellular proteins and only under conditions that also enable the binding of the aHi protein to the DNA. The induction of a genes by a-trans-induction factor appears, therefore, to be mediated by the interaction of the viral protein with cellular proteins at its cis-acting site.

The five a genes of herpes simplex virus 1 are the first set of genes to be expressed after infection. Previous studies have shown that a genes resident in eukaryotic cells are induced by infection with herpes simplex virus 1 or 2 but not by other herpesviruses and indicate that the a trans-inducing factor was a structural component of the virion. This factor induces genes linked to a bona fide promoter and containing at the 5' end a small sequence derived from the promoterregulatory domains of a genes. We report the sequence of a small DNA fragment shown preViously to be capable of expressing the a trans-inducing factor in transient expression systems. The only gene encoded in its entirety in this fragment is predicted to specify a 479 amino acid protein with a Mr of 53,053. The precise termini of the 1.74-kilobase mRNA specifyfing this protein were determined in our 5' and 3' S1 nuclease protection studies.

Epstein-Barr virus (EBV)-associated posttransplant lymphoproliferative disease (PTLD) is an uncommon but potentially fatal complication of immunosuppression in solid-organ transplant recipients. A semiquantitative DNA polymerase chain reaction assay was developed to amplify a unique 269-bp region of the EBNA-1 gene in peripheral blood lymphocytes (PBL) using the primers described by Telenti et al (J Clin Microbiol 28:2187, 1990). Serial samples were studied from 23 transplant recipients, 12 of whom were diagnosed with PTLD. The majority of transplant recipients who were EBV seropositive at the time of transplant surgery and who did not develop PTLD (5 of 7, 71%) exhibited less than a 10-fold increase in the levels of EBV-infected PBL over the 0.1 to 5 EBV genomes/10(6) PBL observed in immunocompetent EBV seropositive controls. Transplant recipients who were seronegative at the time of transplantation and who underwent a primary EBV infection but did not develop PTLD exhibited a reduced capacity to control viremia because the levels of EBV-infected PBL were up to 400 times greater than the 1.0 to 50 EBV genomes/10(6) PBL observed in individuals undergoing acute infectious mononucleosis (Rocci et al: N Engl J Med 296:132, 1977). However, all transplant recipients who developed PTLD exhibited a marked elevation of EBV-infected PBL independent of their serologic state at the time of transplantation. Six of the 10 transplant recipients with PTLD exhibited > or = 300,000 EBV genomes/10(5) PBL, two exhibited 10,000 to 50,000 EBV-infected genomes/10(5) PBL, and one each exhibited 2,500 and 500 EBV genomes/10(5) PBL. However, the latter two samples were obtained 4 to 5 weeks after the diagnosis of PTLD and may reflect a decrease in viral load resulting from immunomodulation. Marked decreases in the levels of EBV nuclear antigen-1 (EBNA-1), EBNA-2, and EBNA-LP antibodies correlated with the increase in EBV-infected PBL. Hence, a quantitative difference in circulating EBV viral load and EBNA antibody levels is evident between transplant recipients with and without PTLD and may be useful as a noninvasive prognostic marker with which to monitor and/or predict the development of PTLD.

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The transcriptional induction of the a or immediate-early gene class of herpes simplex virus type 1 effected by the a trans-induction factor (aTIF, ICP25, VP16, Vmw65) requires an a-specific cis-acting site. Increased transcription does not result from the direct, independent binding of aTIF, but rather from an aTIFdependent formation of a protein-DNA complex containing, in addition to aTIF, at least one host cell factor. One of the host factors is a POU domain protein which recognizes an octamer element in the a-specific consensus. There is evidence that aTIF may drive the formation of multiple protein-DNA complexes containing a POU protein and additional host factors. Previously, the gene products of UL46 and UL47 have been implicated in modulating the aTIF-dependent transcriptional induction of a genes. Our current studies have extended these analyses from a transient-expression system to a series of viral deletion mutants. In these studies we demonstrate that neither UL46nor UL47-encoded gene product, either separately or in combination, is required for viral growth in cell culture. The absence of UL47 reduces by up to 80% the ability of the virus to induce an a-regulated thymidine kinase reporter gene resident in 143TK-cells. Autoradiograms of [35S]methionine pulse-labeled infected cell proteins, separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, show that deleting UL46 and/or UL47 has no discernable effect on the synthesis of aTIF or aTIF-containing proteins. Subsequent Western immunoblot analysis, with rabbit anti-aTIF antibodies made to an otTIF-Staphylococcus aureus protein A fusion, demonstrated that the accumulation and steady-state levels of aTIF or aTIF-containing proteins was indistinguishable from that of the thymidine kinase-negative isogenic parental virus, RA305. * Corresponding author. able to complex with aTIF (3,12,21,23,24,32,37,44,49). Complex formation appears to be mediated through the POU domain present in these proteins, which recognizes an octamer element contained within the a element (gyATGN TAATgarattcyttgnggg) (3,12,18,21,24,36). A recent report suggests that aTIF may drive the formation of multiple protein-DNA complexes containing a POU protein and additional host factors, one of which may be cell type specific (21).Previous work has identified a role for at least two additional viral factors, encoded by HSV-1 UL46 and UL47, in aTIF-dependent transcriptional induction (33,34). In transient-expression assays the gene product of UL46 was able to enhance aTIF-mediated induction of an aTK reporter construct 2to 3-fold, while the gene product of UL47 acted to reduce the levels of aTIF-mediated induction 2.5to 3-fold in the presence or absence of UL46. These results were the first report of additional viral factors involved in a-gene induction, and they predicted the presence of a regulatory gene cluster composed of UL46, UL47, and aTIF (UL48) (33,34).The goals of our current study were to investigate the roles of UL46 and UL47 in the context of the virus to determi...

The gene products of herpes simplex virus type 1 UL46 and UL47 enhance the efficiency of aTIF (VP16)-mediated a gene expression through an unknown mechanism of action. To further characterize the function of the UL46-and UL47-encoded proteins during virus infection, a series of isogenic herpes simplex virus type 1 strain F-derived UL46 and UL47 single-deletion mutants and a UL46/47 double-deletion mutant were constructed and compared with the wild type. Analysis of purified virions obtained from the UL46 deletion mutant showed for the first time that UL46 encoded the virion tegument phosphoproteins VP11 and VP12 (VP11/12). Similar analyses of the UL47 deletion mutants confirmed an earlier report by McLean et al. that UL47 also encoded two virion tegument phosphoproteins, VP13 and VP14 (VP13/14) (G. McLean, F.

IL-6, a multifunctional cytokine produced by monocytes, fibro-

Herpes simplex viruses encode a structural protein which induces, in trans, expression of a genes, the first set of genes to be expressed after infection of permissive cells. This protein, designated as the a-trans-inducing factor (a-TIF), maps within the BamHI F fragment, and its gene has been sequenced. In the course of mapping the domain of the oa-TIF gene, it was noted that the intact BamHI fragment was consistently more effective than the complete domain of the a-TIF gene in inducing expression of a genes. Cotransfections of DNA fragments containing an a indicator gene and the a-TIF gene with various regions of the BamHI F DNA fragment revealed that the sequences located 3' to the ot-TIF gene raised the activity of the a-TIF gene to nearly the same level as that of the intact BamHI F fragment. The nucleotide sequence and S1 nuclease mapping analyses revealed the presence of two transcribed open reading frames capable of encoding polypeptides with translated molecular weights of 77,357 and 70,527. To determine whether the effect of these sequences in trans on a-TIF-mediated induction of a genes was due to expression of these genes or competition for transcriptional factors, we constructed plasmids that contained both genes. Into each or both of these genes we inserted, near the translation initiation sites, 14-base-pair linkers carrying translational stop codons (TAG) in all three reading frames. Analyses of these plasmids indicated that the gene encoding the 70,527-molecular-weight polypeptide reduced a-TIF-dependent induction of a genes, whereas the gene encoding the 77,357-molecularweight polypeptide increased this activity. Insertion of the stop codons abolished these activities.