Role of herpes simplex virus type 1 UL46 and UL47 in alpha TIF-mediated transcriptional induction: characterization of three viral deletion mutants

Abstract

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 Effect of Herpes Simplex Virus Type 1 L-Particles on Virus Entry, Replication, and the Infectivity of Naked Herpesvirus DNA

“…The wild-type (WT) viruses used were HSV-1 (strain 17), PRV, and EHV-1 (strain Ab4). Several mutants of HSV-1 strain 17 were also used: in1814 an insertion mutant in the UL48 gene reading frame that eliminates ␣-TIF transinducing activity but retains the protein's structural role (Ace et al, 1988;1989); DL1403 a mutant lacking Vmw IE 110K, having the RL2 gene deleted (Stow and Stow, 1986); FxE, a mutant without the zinc finger motif of the IE 110K protein (Everett, 1989); 17(41 Ϫ ), which lacks the vhs gene UL41 (Fenwick and Everett, 1990); tsK a mutant in gene RS1 which makes a temperature-sensitive IE175K transactivating protein (Marsden et al, 1976;Preston, 1981); and three HSV-1 (strain F) mutants ⌬46, ⌬47, and ⌬46/47 deleted in UL46, UL47, and both UL46 and 47 genes, respectively (Zhang et al, 1991;Zhang and McKnight, 1993).…”

An 85-kilodalton herpes simplex virus type 1 alpha trans-induction factor (VP16)-VP13/14 fusion protein retains the transactivation and structural properties of the wild-type molecule during virus infection

“…Virus and cells. All viruses, cell lines, and conditions for viral infection, [35S]Met labeling, and thymidine kinase assays have been described elsewhere (16,30,75,76). The purity of each UL47 deletion mutant stock was confirmed before use by Southern blot analysis as described previously (75,76).…”

“…SDS-PAGE and Western immunoblot analysis. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), autoradiograms, and Western blot analyses were done as described previously (76). Rabbit polyclonal antibodies against HSV-1 VP13/14 (R220/5), ICP4 (N15), and ICP8 (367) were the gifts of D. Meredith, N. DeLuca, and P. Kinchington, respectively.…”

“…In this report, we characterize the functional activities of an otTIF fusion protein, designated otTIF-f, which contains 61 amino acids of another HSV-1 protein, VP13/14, fused in frame to its C terminus. oTIF-f is expressed by a virus mutant (R[F]UL47A2) in lieu of the wild-type oLTIF (76). We demonstrate that potential alterations in conformation and amino acid interactions resulting from this perturbation do not significantly alter otTIF-mediated Cl complex formation, transactivation, or virion assembly.…”

The herpes simplex virus type 1 particle: structure and molecular functions

“…It has recently been proposed that 65KTIF is associated with V P l l / 12 and VP13/14 in the virion (268). Both sets of proteins may modulate the transinducing activity in transient expression systems (147), but in the context of virus only the UL47 proteins (VPl3 / 14) seem to have an effect (267).…”