In one of the earliest events in human cytomegalovirus (HCMV)-infected cells, the major immediate-early (IE) protein IE1 initially targets to and then disrupts the nuclear structures known as PML oncogenic domains (PODs) or nuclear domain 10. Recent studies have suggested that modification of PML by SUMO is essential to form PODs and that IE1 both binds to PML and may disrupt PODs by preventing or removing SUMO adducts on PML. In this study, we showed that in contrast to herpes simplex virus type 1 (HSV-1) IE110 (ICP0), the loss of sumoylated forms of PML by cotransfected IE1 was resistant to the proteasome inhibitor MG132 and that IE1 did not reduce the level of unmodified PML. Reduced sumoylation of PML was also observed in U373 cells after infection with wild-type HCMV and proved to require IE1 protein expression. Mutational analysis revealed that the central hydrophobic domain of IE1, including Leu174, is required for both PML binding and loss of PML sumoylation and confirmed that all IE1 mutants tested that were deficient in these functions also failed both to target to PODs and to disrupt PODs. These same mutants were also inactive in several reporter gene transactivation assays and in inhibition of PML-mediated repression. Importantly, a viral DNA genome containing an IE1 gene with a deletion [IE1(⌬290-320)] that was defective in these activities was not infectious when transfected into permissive fibroblast cells, but the mutant IE1(K450R), which is defective in IE1 sumoylation, remained infectious. Our mutational analysis strengthens the idea that interference by IE1 with both the sumoylation of PML and its repressor activity requires a physical interaction with PML that also leads to disruption of PODs. These activities of IE1 also correlate with several unusual transcriptional transactivation functions of IE1 and may be requirements for efficient initiation of the lytic cycle in vivo.Human cytomegalovirus (HCMV), a member of the betaherpesvirus subfamily, typically causes nearly ubiquitous asymptomatic latent or persistent infections. However, primary infections of newborns and reactivation from latent infection in immunocompromised individuals, including recipients of organ transplantation and patients with AIDS, can lead to lifethreatening problems with overt systematic and chronic disease (56, 65). During lytic cycle infection, HCMV gene expression occurs in a three-step sequential fashion with immediate-early (IE or ␣), delayed-early (DE or ␤), and late (L or ␥) kinetics. Among the IE proteins, two nuclear regulatory phosphoproteins, IE1 (or IE72) and IE2 (or IE86), are the first and most abundantly expressed proteins and are synthesized by differential splicing from the same complex overlapping transcription unit within the major IE (MIE) locus (56).The 72-kDa IE1 protein (491 amino acids) is encoded by exons 1, 2, 3, and 4 (UL123), whereas the 86-kDa IE2 protein (579 amino acids) is encoded by exons 1, 2, 3, and 5 (UL122), and the two proteins share 85 amino acids at the N terminus. IE2 is a spe...

Vaccinia virus encodes a multisubunit DNAdependent RNA polymerase (EC 2.7.7.6) that is packaged in the infectious virus particle. This polymerase was found to contain a submolar polypeptide of approximately 85 kDa in addition to the core subunits, which consist of two larger and several smaller polypeptides. The polymerase containing the 85-kDa polypeptide was separated from the core polymerase by column chromatography. Although the core polymerase actively transcribed heterologous single-stranded DNA, only the form with the associated 85-kDa polypeptide could act in conjunction with an early stage-speciflic factor to transcribe double-stranded DNA containing a vaccinia virus early promoter. Peptide sequencing established that the RNA polymerase-associated 85-kDa protein was derived from the vaccinia virus H4L open reading frame, which encodes a 94-kDa polypeptide that we named RAP94. RAP94 is not closely related to prokaryotic a70 or eukaryotic RAP30 RNA polymerase-binding proteins, although there are short regions of sequence similarity. The specific association of RAP94 with viral RNA polymerase was corroborated with antibody raised to a recombinant fusion protein. Unlike the previously defined subunits of vaccinia virus RNA polymerase, RAP94 is synthesized exclusively late in infection, and synthesis could be prevented by a DNA replication inhibitor. The role of RAP94 in mediating specific transcription was demonstrated by using an extract from cells in which the H4L open reading frame had been transiently expressed.The synthesis of mRNA by DNA-dependent RNA polymerases (EC 2.7.7.6) often requires one or more accessory proteins. These factors assist the polymerase in binding to the promoter selectively and initiating transcription. In prokaryotes, this process is regulated by the o-factor protein family (1). In eukaryotes, the role of the o subunit appears to be spread among several proteins, which can be grouped into two broad categories of general transcription factors (2). One class of factors, to which the TATA-motif-binding protein (TFIID) belongs, binds to promoter sequences and forms the initial protein-DNA complex. Factors belonging to the second category, such as RAP30/74 (also known as TFIIF), associate with RNA polymerase II or other protein factors and have no sequence-specific DNA-binding properties (3).A distant relationship between bacterial aofactors and RAP30 has been suggested on the basis of sequence similarities (4).The poxviruses, of which vaccinia virus is the prototype, provide especially favorable systems for combining biochemical and genetic approaches to the study of transcription. These DNA viruses replicate in the cytoplasm and encode most if not all of the proteins needed for viral transcription, including a eukaryotic-like multisubunit DNA-dependent RNA polymerase and stage-specific transcription factors (reviewed in refs. 5 and 6). The early class of vaccinia virus genes is expressed immediately after infection, whereas viral DNA replication precedes the expression of ...

Plasmids capable of undergoing genetic exchange in mitotically dividing Saccharomyces cerevisiae cells were used to measure the length of gene conversion events, to determine patterns of coconversion when multiple markers were present, and to correlate the incidence of reciprocal recombination with the length of conversion tracts. To construct such plasmids, restriction site linkers were inserted both within the HIS3 gene and in the flanking sequences, and two different his3-alleles were placed in a vector. Characterization of the genetic exchanges in these plasmids showed that most occur with the conversion of one his3-allele. Many of these events included coconversions in which more than one marker along the allelic sequence was replaced. The frequency of coconversion decreased with the distance between two markers such that markers further than 1 kilobase apart were infrequently coconverted. From these results the average length of conversion was determined to be approximately 0.5 kllobase. Examination of coconversions involving three or more markers revealed an almost obligatory, simultaneous coconversion pattern of all markers. Thus, when two markers which flank an intervening marker are converted, the intervening marker is 20 times more likely to be converted than to remain unchanged. The results of these studies also showed that the incidence of reciprocal recombination, which accompanies more than 20% of the conversion events, is more frequent when the conversion tract is longer than average.

Glutaredoxins (GRXs), also known as thioltransferases, use glutathione as a cofactor for reduction of disulfides in prokaryotes and eukaryotes. We demonstrate that the vaccinia virus 02L open reading frame encodes a fumctional