The Vmw175 binding site in the IE-1 promoter has no apparent role in the expression of Vmw110 during herpes simplex virus type 1 infection

The immediate-early (IE) genes of herpes simplex virus type 1 (HSV-1) are the first to be expressed during infection in tissue culture. Since they are transcribed at abnormally high levels in the absence of IE protein synthesis they appear to be subject to repression during normal infection. One of the major HSV-1 regulatory proteins, Vmw175 (the product of IE gene 3), is required for normal IE gene regulation since mutations which inactivate it lead to abnormally high levels of IE gene expression. The mechanism of repression of the IE-3 promoter requires both the ability of Vmw175 to bind to DNA and the presence of a Vmw175 recognition DNA binding sequence at the cap site of the IE-3 promoter. A similar Vmw175 DNA binding sequence has been defined within the IE-1 promoter. This paper describes the construction of a variant of HSV-1 with a mutation within the IE-1 Vmw175 DNA binding site. Although the mutation destroyed the ability of Vmw175 to bind to the site, and greatly reduced the ability of Vmw175 to repress the IE-1 promoter in transfection assays, the mutation had no effect on the levels of Vmw110 expression during normal HSV-1 infection.     

Analysis of sequences involved in IE2 transactivation of a baculovirus immediate-early gene promoter and identification of a new regulatory motif

Opep-2 is a unique baculovirus early gene that has only been identified in the Orgyia pseudotsugata multiple capsid nucleopolyhedrovirus (OpMNPV). Previous analyses have shown this gene is expressed at very early times post-infection (p.i.) but is shut down by 36-48 h p.i. The promoter of opep-2 therefore, represents a class of early genes that is temporally regulated. In this study, a detailed analysis of the opep-2 promoter is performed to analyze the role individual motifs play in early gene expression. A new 13 base pair regulatory element was identified and shown to be essential in controlling high-level expression of this gene. In addition, mutational analysis revealed that GATA and CACGTG motifs, which have been shown to bind cellular factors in Sf9 and Ld652Y cells, played minor roles in influencing opep-2 expression in the absence of other viral factors. The OpMNPV transactivator IE2 causes a significant activation of the opep-2 promoter. Cotransfection of an extensive number of promoter deletions and mutations did not show any sequence specificity for IE2 transactivation. This is the first detailed analysis of the sequence requirements for IE2 transactivation, and these results suggest that IE2 does not bind directly to specific elements in the opep-2 promoter.     

The Tup1-Ssn6 general repressor is involved in repression of IME1 encoding a transcriptional activator of meiosis in Saccharomyces cerevisiae

Ime1 plays a pivotal role in the initiation of meiosis in a/α diploid cells of Saccharomyces cerevisiae. In the absence of glucose and nitrogen, IME1 expression is greater in a/α cells than in either a or α cells and therefore only a/α, but not a/a or α/α, cells are committed to sporulation. It is known that IME1 expression is positively regulated by Mck1, Rim1, Ime4 and the Swi-Snf complex but other factors may also be involved. In addition, Rme1 is assumed to repress IME1 expression. To provide more details of the repression of expression of IME1, we have isolated mutants in which the IME1p-PHO5 fusion gene integrated at the ura3 locus is expressed in α cells under nutritionally rich conditions. We found that mutations occurred in TUP1, SSN6, SIN4 and RGR1, among which TUP1 and SSN6 were identified for the first time as negative regulators of IME1 expression. Deletion of the Rme1-binding site from the IME1 promoter did not result in activation of the expression of IME1 under nutritionally rich conditions, suggesting that Rme1 does not function as a DNA-binding protein with the Tup1-Ssn6 repression complex. We also demonstrated that the 294-bp fragment from nucleotide position –914 to –621 and the 301-bp fragment from nucleotide position –1215 to –915 of the IME1 promoter region contain elements acting as URS and UAS in TUP1 ⁺ and tup1 mutant cells, respectively. These findings indicate that IME1 is negatively regulated by the Tup1-Ssn6 repressor complex through two distinct upstream regions in conjunction with unidentified DNA-binding proteins. Received: 14 November 1997 / 12 January 1998     

Genetic dissection of the Streptococcus pyogenes M1 protein: regions involved in fibronectin binding and intracellular invasion.

Entry of serotype M1 Streptococcus pyogenes into host cells depends on binding of the host glycoprotein fibronectin (Fn) by the bacterial M1 protein. The present study was undertaken to localize the Fn binding region in M1 and assess other potential functions of M1. A set of recombinant M1 protein fragments were assayed for their capacities to bind Fn and inhibit ingestion of streptococci by epithelial cells. M1 protein, M6 protein and internally-deleted derivatives of M1 were expressed on the surface of Lactococcus lactis. Lactococci that expressed M1 or M6 protein bound Fn and were efficiently taken up by epithelial cells. Deletion of both the N-terminal A and B repeats regions of M1 abrogated Fn binding and intracellular invasion. Deletion of either the A domain (M1DeltaA) or B repeats (M1DeltaB) significantly reduced, but did not completely eliminate, Fn binding indicating that M1 protein may possess two independent Fn binding sites. Fn binding by the M1DeltaA or M1DeltaB proteins was insufficient for efficient invasion, however, suggesting that M protein binding alters the structure of Fn that, in turn, affects the interaction between Fn and epithelial cells. Although expression of M1, M6 or M1DeltaB proteins led to aggregation of lactococcal cells, aggregation did not significantly contribute to invasion efficiency.     

Seroprevalence of HSV-1 and HSV-2 in Barbados

Type-specific enzyme-linked immunosorbent assays, based upon recombinant glycoprotein G (gG), were used to detect antibodies to HSV-1 and HSV-2, in a small Caribbean island population. A blinded serosurvey was performed on samples from 184 blood donors, 122 pregnant women, and 120 HIV-positive patients. The seroprevalence of HSV-1 and HSV-2 was 81% and 34%, respectively, in blood donors, 84% and 40% in the antenatal population and 89% and 77% in the HIV-positive group. As expected the majority of adults were seropositive against HSV-1. However, the HSV-2 seroprevalence was significantly higher in HIV-infected adults than in the other groups. These findings support the need for prospective epidemiological studies in this population.     

Expression of Epstein-Barr virus BZLF1 immediate-early protein induces p53 degradation independent of MDM2, leading to repression of p53-mediated transcription

The Epstein-Barr virus (EBV) lytic program elicits ATM-dependent DNA damage response, resulting in phosphorylation of p53 at N-terminus, which prevents interaction with MDM2. Nevertheless, p53-downstream signaling is blocked. We found here that during the lytic infection p53 was actively degraded in a proteasome-dependent manner even with a reduced level of MDM2. BZLF1 protein enhanced the ubiquitination of p53 in SaOS-2 cells. The degradation of p53 was observed even in the presence of Nutlin-3, an inhibitor of p53-MDM2 interaction, and also in mouse embryo fibroblasts lacking mdm2 gene, indicating that the BZLF1 protein-induced degradation of p53 was independent of MDM2. Furthermore, Nutlin-3 increased the level of p53 in the latent phase of EBV infection but not in the lytic phase. Although p53 level is regulated by MDM2 in the latent phase, it might be mediated by the BZLF1 protein-associated E3 ubiquitin ligase in the lytic phase for efficient viral propagation.