Kaposi's sarcoma-associated herpesvirus (KSHV) is a recently discovered human gamma herpesvirus (HHV-8) that plays an important role in Kaposi's sarcoma development. Here, we further characterize the regulation of the early HHV-8 gene, open reading frame 57 (ORF57). ORF57 is a spliced gene consisting of two exons with a 108-bp intron near the 5' end. The ORF57 mRNA can potentially be initiated at two different start sites, and its expression can be significantly stimulated by ORF50, an HHV-8 immediate early gene. The target site for ORF50 transactivation was mapped to a 40-bp fragment compassing nt 81904 to 81943 in the ORF57 promoter. Our study on the regulation of ORF57 expression by ORF50 provides the basis for further studies on the regulation of HHV-8 lytic gene expression.

The ART (Activator of Replication and Transcription) protein of Kaposi's sarcoma-associated herpesvirus (KSHV), or human herpesvirus-8 (HHV-8), is encoded by the ORF50 gene. It is expressed as an immediate-early gene and plays a crucial role in the transition between latency and productive infection. HHV-8 ART is a transcriptional transactivator which can up-regulate viral gene expression. Transient expression assays showed that ART strongly activated ORF57 and K8 promoter-directed gene expression in both CV-1 and BJAB cells. The ART target site was mapped to a 40-bp region compassing nt 81904 to 81943 on the ORF57 promoter. When linked upstream to a heterologous SV40 promoter, this region by itself was able to confer ART responsiveness. This 40-bp segment contains a 16-bp consensus sequence which is also found in the K8 promoter region located between nt 74769 to 74784. Deletion of the fragment including this 16-bp consensus abrogated the ART responsiveness of the K8 promoter. The role of this 16-bp consensus in ART transactivation was further supported by site-directed mutagenesis. Mutations of the conserved nucleotides within the 16-bp consensus in the ORF57 promoter dramatically impaired its responsiveness to ART. Fusion protein analysis with chimeric proteins containing the DNA binding domain of yeast transactivator Gal4 (residues 1 to 147) and different ART segments defined an acidic C-terminal region (amino acids [aa] 527 to 634) as a potent activator. Deletions of this activation domain in the ART protein resulted in a decrease or loss of its ability to activate ORF57 and K8 promoters containing the ART responsive element in transfected cells. How the ART activation domain activates ORF57 and K8 gene expression through the 16-bp consensus sequence remains to be determined.

The spatial distribution of the survival and growth of Listeria monocytogenes during manufacturing and ripening of Camembert cheese was studied. All cheeses were artificially inoculated with L. monocytogenes and ripened in three stages: room temperature (∼20C) and relative humidity (RH) of 60% for 36 h, 13C and RH of 93% for 2 weeks, and 7C and RH of 85% for 3 weeks. During ripening, different locations were analyzed for population of L. monocytogenes. The data were collected on days 1, 5, 10, 15, 20, 25, 30 and 35 of ripening. Results showed that the population of L. monocytogenes on day 1 was 6.06 log10 cfu/g, which was 1.96 log10 cfu/g increasing over the initial inoculation of 4.08 log10 cfu/g. For the subsequent 20 days, the L. monocytogenes population declined to 5.33 log10 cfu/g. Thereafter, the L. monocytogenes population increased to 6.07 log10 cfu/g on day 35 of ripening. Generally, the growth of L. monocytogenes is faster in locations near the surface than in the center. There were no significant differences between batches and sections of cheese, whereas the ripening time and locations had a significant effect on the survival and growth of L. monocytogenes. The survival and growth data for L. monocytogenes during ripening of Camembert cheese can be used to develop dynamic predictive models for possible use in risk assessment and Hazard Analysis and Critical Control Point implementation.

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