Kaposi's sarcoma (KS) is the most common HIV-associated cancer worldwide and is associated with high levels of morbidity and mortality in some regions. Antiretroviral (ARV) combination regimens have had mixed results for KS progression and resolution. Anecdotal case reports suggest that protease inhibitors (PIs) may have effects against KS that are independent of their effect on HIV infection. As such, we evaluated whether PIs or other ARVs directly inhibit replication of Kaposi's sarcoma-associated herpesvirus (KSHV), the gammaherpesvirus that causes KS. Among a broad panel of ARVs tested, only the PI nelfinavir consistently displayed potent inhibitory activity against KSHV in vitro as demonstrated by an efficient quantitative assay for infectious KSHV using a recombinant virus, rKSHV.294, which expresses the secreted alkaline phosphatase. This inhibitory activity of nelfinavir against KSHV replication was confirmed using virus derived from a second primary effusion lymphoma cell line. Nelfinavir was similarly found to inhibit in vitro replication of an alphaherpesvirus (herpes simplex virus) and a betaherpesvirus (human cytomegalovirus). No activity was observed with nelfinavir against vaccinia virus or adenovirus. Nelfinavir may provide unique benefits for the prevention or treatment of HIV-associated KS and potentially other human herpesviruses by direct inhibition of replication.

Bacteria commonly exchange genetic information by the horizontal transfer of conjugative plasmids. In gram-negative conjugation, a relaxase enzyme is absolutely required to prepare plasmid DNA for transit into the recipient via a type IV secretion system. Here we report a mutagenesis of the F plasmid relaxase gene traI using in-frame, 31-codon insertions. Phenotypic analysis of our mutant library revealed that several mutant proteins are functional in conjugation, highlighting regions of TraI that can tolerate insertions of a moderate size. We also demonstrate that wild-type TraI, when overexpressed, plays a dominant-negative regulatory role in conjugation, repressing plasmid transfer frequencies ϳ100-fold. Mutant TraI proteins with insertions in a region of approximately 400 residues between the consensus relaxase and helicase sequences did not cause conjugative repression. These unrestrictive TraI variants have normal relaxase activity in vivo, and several have wild-type conjugative functions when expressed at normal levels. We postulate that TraI negatively regulates conjugation by interacting with and sequestering some component of the conjugative apparatus. Our data indicate that the domain responsible for conjugative repression resides in the central region of TraI between the protein's catalytic domains.Much of the lateral gene transfer between bacteria occurs through the action of conjugative plasmids that encode all of the functions necessary for their hosts to transmit them to recipient cells. Plasmid transfer is achieved through direct cell contact and active transport of DNA by the donor. In gram-negative conjugation systems, typified by the F plasmid of Escherichia coli, only one strand of DNA is translocated, so single-strand cleavage and unwinding of the substrate DNA must occur prior to transfer (9). Strand scission is performed by plasmid-encoded "relaxases" that cleave their cognate plasmid at a specific site called nic within the origin-of-transfer region (oriT). In the case of the F and related plasmids, the accessory proteins TraM, TraY, and integration host factor also bind at oriT as part of the relaxosome complex (15,17,19,32). DNA unwinding is usually performed by a separate helicase, though in some systems, such as the F and R388 plasmids of E. coli, relaxase and helicase activities are both present in the relaxase (11,27,42). After making a single-stranded break at nic, the relaxase is thought to deliver the DNA to a type IV secretory apparatus that can translocate it across the recipient cell membrane. The relaxase remains covalently bound to the nic site and religates the scission once DNA transfer is complete. The replication of single-stranded plasmid DNA in the donor and recipient regenerates the double-stranded DNA plasmid in both cells.The F plasmid relaxase TraI is a well-studied model of structure and function for relaxases. The single-stranded DNA-cleaving activity of TraI is present in the first ϳ310 residues of this 1,756-residue protein, while the helicase motifs are locat...

Type IV secretory systems are a group of bacterial transporters responsible for the transport of proteins and nucleic acids directly into recipient cells. Such systems play key roles in the virulence of some pathogenic organisms and in conjugation-mediated horizontal gene transfer. Many type IV systems require conserved "coupling proteins," transmembrane polypeptides that are critical for transporting secreted substrates across the cytoplasmic membrane of the bacterium. In vitro evidence suggests that the functional form of coupling proteins is a homohexameric, ring-shaped complex. Using a library of tagged mutants, we investigated the structural and functional organization of the F plasmid conjugative coupling protein TraD by coimmunoprecipitation, cross-linking, and genetic means. We present direct evidence that coupling proteins form stable oligomeric complexes in the membranes of bacteria and that the formation of some of these complexes requires other F-encoded functions. Our data also show that different regions of TraD play distinct roles in the oligomerization process. We postulate a model for in vivo oligomerization and discuss the probable participation of individual domains of TraD in each step.

We used the maltose transport complex MalFGK 2 of the Escherichia coli cytoplasmic membrane as a model for the study of the assembly of hetero-oligomeric membrane protein complexes. Analysis of other membrane protein complexes has led to a general model in which a unique, ordered pathway is followed from subunit monomers to a final oligomeric structure. In contrast, the studies reported here point to a fundamentally different mode for assembly of this transporter. Using coimmunoprecipitation and quantification of interacting partners, we found that all subunits of the maltose transport complex efficiently form heteromeric complexes in vivo. The pairwise complexes were stable over time, suggesting that they all represent assembly intermediates for the final MalFGK 2 transporter. These results indicate that several paths can lead to assembly of this oligomer. We also characterized MalF and MalG mutants that caused reduced association between some or all of the subunits of the complex with this assay. The mutant analysis highlights some important motifs for subunit contacts and suggests that the promiscuous interactions between these Mal proteins contribute to the efficiency of complex assembly. The behaviors of the wild type and mutant proteins in the co-immunoprecipitations support a model of multiple assembly pathways for this complex.

Biological fabrication routes can provide a way to overcome the limitations presented by current chemistry-based nanoparticle arrangement and assembly methods. Many recent assembly strategies utilize DNA as the templating molecule by patterning gold nanoparticles on DNA through chemical conjugation via, for example, a sulfhydryl bond.[1] Reliance upon this chemistry, however, limits applications because it acts indiscriminately on several different metals and is only useful for some noble-metal nanoparticles. Strategies that covalently link nanoparticles to proteins or DNA risk denaturation or distortion of native protein, distortion of DNA, and/or disruption of the plasmonic or photonic properties unique to nanoparticles.[2] We present a strategy for nanoparticle patterning on DNA that utilizes the biologically based self-assembly properties of DNA-binding proteins to facilitate the targeted immobilization of nanoparticles on DNA. Here we show that a derivative of the DNA-binding protein TraI spontaneously organizes colloidal gold nanoparticles on DNA through an engineered gold-binding peptide motif. This system, based solely on specific, noncovalent, biologically determined interactions, represents significant progress on the route to spontaneously ordered assembly of nanoparticles important for downstream applications in nanoelectronics and photonics.TraI (192 kDa, 1756 residues) is the E. coli F-plasmid-encoded relaxase/helicase that harbors sequence-specific single-stranded-DNA-binding activity (relaxase domain) and nonspecific single and double-stranded-DNA-binding activity (helicase domain).[3] We engineered TraI with a gold-binding motif at an internal permissive site after residue Q369 to direct the assembly of gold nanoparticles (AuNPs) on DNA through noncovalent interactions. Permissive sites, regions of proteins that tolerate a wide variety of amino acid additions without disrupting native protein function, were previously identified through transposon/epitope tag mutagenesis in TraI. [4,5] This study utilizes TraI's nonspecific DNA-binding activity as the first step toward optimization of this biologically based nanoparticletemplating strategy. Because TraI is also capable of sequencespecific DNA binding, this work paves the path to the final step of the biologically based strategy: addressable, targeted immobilization of nanoparticles on DNA.Inorganic binding peptides identified by several groups [6,7]