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Kaposi's sarcoma-associated herpesvirus (KSHV; human herpesvirus 8) belongs to the subfamily of Gammaherpesvirinae and is the etiological agent of Kaposi's sarcoma as well as of two lymphoproliferative diseases: primary effusion lymphoma and multicentric Castleman disease. The KSHV life cycle is divided into a latent and a lytic phase and is highly regulated by viral immunomodulatory proteins which control the host antiviral immune response. Among them is a group of proteins with homology to cellular interferon regulatory factors, the viral interferon regulatory factors 1-4. The KSHV vIRFs are known as inhibitors of cellular interferon signaling and are involved in different oncogenic pathways. Here we characterized the role of the second vIRF protein, vIRF2, during the KSHV life cycle. We found the vIRF2 protein to be expressed in different KSHV positive cells with early lytic kinetics. Importantly, we observed that vIRF2 suppresses the expression of viral early lytic genes in both newly infected and reactivated persistently infected endothelial cells. This vIRF2-dependent regulation of the KSHV life cycle might involve the increased expression of cellular interferon-induced genes such as the IFIT proteins 1, 2 and 3, which antagonize the expression of early KSHV lytic proteins. Our findings suggest a model in which the viral protein vIRF2 allows KSHV to harness an IFN-dependent pathway to regulate KSHV early gene expression.

Several human pathogens and the plant pathogen Agrobacterium tumefaciens use a type IV secretion system for translocation of effector proteins into host cells. How effector proteins are selected for transport is unknown, but a C-terminal transport signal is present in the proteins translocated by the A. tumefaciens VirB/D4 type IV secretion system. We characterized this signal in the virulence protein VirF by alanine scanning and further site-directed mutagenesis. The Cre recombinase was used as a reporter to measure the translocation efficiency of Cre-Vir fusions from A. tumefaciens to Arabidopsis. The data unambiguously showed that positive charge is an essential characteristic of the C-terminal transport signal. We increased the sensitivity of this translocation assay by modifying the Cre-induced readout in host cells from kanamycin resistance to GFP expression. This improvement allowed us to detect translocation of the VirD2 relaxase protein in the absence of transferred DNA, showing that attachment to the transferred DNA is not essential for transport by the VirB/D4 system. We also found another translocated effector protein, namely the VirD5 protein encoded by the tumor-inducing plasmid. According to secondary structure predictions, the C termini of all VirB/D4-translocated proteins identified so far are unstructured; however, they contain a characteristic hydropathic profile. Based on sequence alignments and mutational analysis of VirF, we conclude that the C-terminal transport signal for recruitment and translocation of effector proteins by the A. tumefaciens VirB/D4 system is hydrophilic and has a net positive charge with a consensus motif of R-X(7)-R-X-R-X-R-X-X(n)>.

Abstract The Rns protein of enterotoxigenic Escherichia coli (ETEC) and the VirF protein of Shigella flexneri are members of the AraC family of transcription regulators. Rns is required for positive activation of the CS1 fimbrial genes, while VirF is a positive regulator of an invasion gene regulon. The amino acid sequences of the proteins are 36% identical, and both proteins activate transcription in response to increases in temperature. Here, we show that Rns is capable of complementing fully a null mutation in the S. flexneri virF gene. However, the VirF protein cannot replace Rns as an activator of CS1 gene expression in ETEC. This failure is not due to the absence from ETEC of a co-factor required by VirF since it also occurs when the CS1 system is moved into an S. flexneri genetic background. Nor is it a function of growth medium composition or a failure in virF gene expression. Instead, these findings point to important differences in the mechanisms by which these related transcription factors regulate gene expression in Gram-negative pathogens.

Kaposi sarcoma-associated herpesvirus vIRF is a viral transcription factor that inhibits interferon signaling and transforms NIH 3T3 cells, but does not bind interferon-stimulated response element (ISRE) DNA sequences. Here we show that induction of the MYC protooncogene is required for cell transformation by vIRF, and that vIRF increases MYC transcription up to 15-fold through specific promoter interactions at an ISRE sequence called the plasmacytoma repressor factor (PRF) element. These effects are resistant to cycloheximide but are inhibited by a dominant-negative ISRE-binding protein, indicating that vIRF acts together with a cellular cofactor at the PRF element to directly transactivate MYC. The coadaptor CREB-binding protein (CBP) binds vIRF and synergizes transactivation of MYC, but, unexpectedly, closely related histone acetyltransferases p300 and P/CAF potently suppress vIRF transactivation. On the basis of the prediction that other interferon-inhibiting viral transforming proteins behave similarly, we found that Epstein-Barr virus-induced nuclear antigen 2 (EBNA2) also binds p300/CBP, and that both EBNA2 and adenovirus E1A transactivate MYC through the PRF element. For E1A, P/CAF coactivates MYC, whereas both p300 and CBP suppress E1A transactivation. For EBNA2, both P/CAF and CBP coactivate the MYC promoter, whereas p300 suppresses EBNA2 transactivation. These findings demonstrate that viral transforming proteins can activate as well as inhibit transcription through coadaptor interactions. At some promoters CBP and p300 have previously unrecognized, competitive antagonism to each other. While all three viral proteins target the same promoter element, each has a different coadaptor use profile. These findings are consistent with cellular MYC repression playing a role in innate immunity as well as in control of cell proliferation.

The transfer of T-DNA from Agrobacterium to plant cells is mediated by a system which involves the virB operon of the Ti plasmid. We report that VirE2 and VirD2, two T-DNA-associated proteins, as well as VirF, a protein known to be secreted into plant cells, are present in the periplasm and supernatant fractions of growing cells of Agrobacterium as are VirJ and ChvE, two known periplasmic proteins. Two cytoplasmic proteins, Ros and chloramphenicol acetyl transferase, and a VirE2 :: green fluorescent protein construct were not detected in the above fraction. Export of VirE2 into the culture supernatant did not require any Ti plasmid genes, except for VirE1, a specific chaperone for VirE2. The levels of the VirE2 and VirD2 proteins in the supernatant increased significantly when cells were grown at 19 degrees C as compared with 28 degrees C. When Agrobacterium expressed the oncogenic suppressive activity protein (Osa), VirE2 and VirF proteins could not be detected in the supernatant or the periplasm and the level of VirD2 was greatly reduced. However, oncogenic suppressive activity protein did not block the accumulation of VirJ and ChvE in the periplasm. Our data suggest that VirD2, VirE2, and VirF are transported across the cytoplasmic membrane by a specific pathway, independent of virB. Thus, transfer of the T-complex of Agrobacterium may take place in two steps, the first mediated by an unidentified pathway and the second by the virB system.

Kaposi's sarcoma-associated herpesvirus/human herpesvirus 8 (KSHV/HHV8) has been etiologically associated with several malignancies including Kaposi's sarcoma and primary effusion lymphoma. Oncogenic viral interferon regulatory factor (vIRF) encoded by KSHV ORF-K9 is a homologue of cellular interferon regulatory factor (IRF), and has been demonstrated to inhibit type I/II interferon signal transduction and transform NIH3T3 cells through the interactions with IRF-1, IRF-3, and CBP/p300 proteins. To counteract vIRF's pathogenic role, we have developed five ribozymes targeting ORF-K9 mRNA to suppress vIRF expression. The vIRF RNA substrates were cleaved up to 80% in a substrate-specific manner in transcript cleavage assays in vitro. In a transient transfection assay, two of the ribozymes efficiently suppressed the expression of vIRF protein measured by dual-color immunofluorescence assay that simultaneously detects the expression of both vIRF protein and ribozyme. Flow cytometry analysis showed that these ribozymes reduced vIRF expression up to 76%. A mutant ribozyme had no cleavage activity in vitro, but exhibited antisense effect in vivo. These results suggest that the ribozymes may provide a new approach for functional knockout of vIRF gene, and are potential candidates of antiviral therapy for KSHV-related malignancies.

The Rns protein of enterotoxigenic Escherichia coli (ETEC) and the VirF protein of Shigella flexneri are members of the AraC family of transcription regulators. Rns is required for positive activation of the CS1 fimbrial genes, while VirF is a positive regulator of an invasion gene regulon. The amino acid sequences of the proteins are 36% identical, and both proteins activate transcription in response to increases in temperature. Here, we show that Rns is capable of complementing fully a null mutation in the S. flexneri virF gene. However, the VirF protein cannot replace Rns as an activator of CS1 gene expression in ETEC. This failure is not due to the absence from ETEC of a co-factor required by VirF since it also occurs when the CS1 system is moved into an S. flexneri genetic background. Nor is it a function of growth medium composition or a failure in virF gene expression. Instead, these findings point to important differences in the mechanisms by which these related transcription factors regulate gene expression in Gram-negative pathogens.

Abstract We have previously reported the identification of a gene from Mycobacterium tuberculosis, H37Rv, which on the basis of its nucleotide sequence encoded a protein product of 38 kDa. This 38-Kda mycobacterial protein designated as VirS exhibits homology with the VirF protein of Shigella, the VirFy protein of Yersinia and the Cfad, Rns and FapR proteins from various enterotoxigenic Escherichia coli strains. In this communication, we show the close sequence and structural similarities of the VirS protein with VirF, VirFy, Cfad, Rns and FapR and describe the results of our studies on the characterization of the virS gene promoter and its expression in E. coli and mycobacteria. virS was present exclusively in the species belonging to the M. tuberculosis complex as revealed by Southern blot and PCR analysis. Our findings suggest the involvement of virS in the regulation of pathogenesis of M. tuberculosis.

VirF, an AraC-like activator, is required to trigger a regulatory cascade that initiates the invasive program of Shigella spp., the etiological agents of bacillary dysentery in humans. VirF expression is activated upon entry into the host and depends on many environmental signals. Here, we show that the virF mRNA is translated into two proteins, the major form, VirF 30 (30 kDa), and the shorter VirF 21 (21 kDa), lacking the N-terminal segment. By site-specific mutagenesis and toeprint analysis, we identified the translation start sites of VirF 30 and VirF 21 and showed that the two different forms of VirF arise from differential translation. Interestingly, in vitro and in vivo translation experiments showed that VirF 21 is also translated from a leaderless mRNA (llmRNA) whose 5′ end is at position +309/+310, only 1 or 2 nucleotides upstream of the ATG84 start codon of VirF 21 . The llmRNA is transcribed from a gene-internal promoter, which we identified here. Functional analysis revealed that while VirF 30 is responsible for activation of the virulence system, VirF 21 negatively autoregulates virF expression itself. Since VirF 21 modulates the intracellular VirF levels, this suggests that transcription of the llmRNA might occur when the onset of the virulence program is not required. We speculate that environmental cues, like stress conditions, may promote changes in virF mRNA transcription and preferential translation of llmRNA. IMPORTANCE Shigella spp. are a major cause of dysentery in humans. In bacteria of this genus, the activation of the invasive program involves a multitude of signals that act on all layers of the gene regulatory hierarchy. By controlling the essential genes for host cell invasion, VirF is the key regulator of the switch from the noninvasive to the invasive phenotype. Here, we show that the Shigella virF gene encodes two proteins of different sizes, VirF 30 and VirF 21 , that are functionally distinct. The major form, VirF 30 , activates the genes necessary for virulence, whereas the minor VirF 21 , which shares the C-terminal two-thirds of VirF 30 , negatively autoregulates virF expression itself. VirF 21 is transcribed from a newly identified gene-internal promoter and, moreover, is translated from an unusual leaderless mRNA. The identification of a new player in regulation adds complexity to the regulation of the Shigella invasive process and may help development of new therapies for shigellosis. Shigella spp. are a major cause of dysentery in humans. In bacteria of this genus, the activation of the invasive program involves a multitude of signals that act on all layers of the gene regulatory hierarchy. By controlling the essential genes for host cell invasion, VirF is the key regulator of the switch from the noninvasive to the invasive phenotype. Here, we show that the Shigella virF gene encodes two proteins of different sizes, VirF 30 and VirF 21 , that are functionally distinct. The major form, VirF 30 , activates the genes necessary for virulence, whereas the minor VirF 21 , which shares the C-terminal two-thirds of VirF 30 , negatively autoregulates virF expression itself. VirF 21 is transcribed from a newly identified gene-internal promoter and, moreover, is translated from an unusual leaderless mRNA. The identification of a new player in regulation adds complexity to the regulation of the Shigella invasive process and may help development of new therapies for shigellosis.

The viral interferon regulatory factors (vIRFs) of KSHV are known to dysregulate cell signaling pathways to promote viral oncogenesis and to block antiviral immune responses to facilitate infection. However, it remains unknown to what extent each vIRF plays a role in gene regulation. To address this, we performed a comparative analysis of the protein structures and gene regulation of the four vIRFs. Our structure prediction analysis revealed that despite their low amino acid sequence similarity, vIRFs exhibit high structural homology in both their DNA-binding domain (DBD) and IRF association domain. However, despite this shared structural homology, we demonstrate that each vIRF regulates a distinct set of KSHV gene promoters and human genes in epithelial cells. We also found that the DBD of vIRF1 is essential in regulating the expression of its target genes. We propose that the structurally similar vIRFs evolved to possess specialized transcriptional functions to regulate specific genes.