Melioidosis is a severe disease that can be difficult to diagnose because of its diverse clinical manifestations and a lack of adequate diagnostic capabilities for suspected cases. There is broad ...interest in improving detection and diagnosis of this disease not only in melioidosis-endemic regions but also outside these regions because melioidosis may be underreported and poses a potential bioterrorism challenge for public health authorities. Therefore, a workshop of academic, government, and private sector personnel from around the world was convened to discuss the current state of melioidosis diagnostics, diagnostic needs, and future directions.
The bacteriophage Mu immunity repressor is a conformationally sensitive sensor that can be interconverted between forms resistant to and sensitive to degradation by ClpXP protease. Protease-sensitive ...repressor molecules with an altered C-terminal sequence promote rapid degradation of the wild-type repressor by inducing its C-terminal end to become exposed. Here we determined that the last 5 C-terminal residues (CTD5) of the wild-type repressor contain the motif required for recognition by the ClpX molecular chaperone, a motif that is strongly dependent upon the context in which it is presented. Although attachment of the 11-residue ssrA degradation tag to the C terminus of green fluorescent protein (GFP) promoted its rapid degradation by ClpXP, attachment of 5-27 C-terminal residues of the repressor failed to promote degradation. Disordered peptides derived from 41 and 35 C-terminal residues of CcdA (CcdA41) and thioredoxin (TrxA35), respectively, activated CTD5 when placed as linkers between GFP and repressor C-terminal sequences. However, when the entire thioredoxin sequence was included as a linker to promote an ordered configuration of the TrxA35 peptide, the resulting substrate was not degraded. In addition, a hybrid tag, in which CTD5 replaced the 3-residue recognition motif of the ssrA tag, was inactive when attached directly to GFP but active when attached through the CcdA41 peptide. Thus, CTD5 is sufficient to act as a recognition motif but has requirements for its presentation not shared by the ssrA tag. We suggest that activation of CTD5 may require presentation on a disordered or flexible domain that confers ligand flexibility.
Melioidosis is a severe disease that can be difficult to diagnose because of its diverse clinical manifestations and a lack of adequate diagnostic capabilities for suspected cases. There is broad ...interest in improving detection and diagnosis of this disease not only in melioidosis-endemic regions but also outside these regions because melioidosis may be underreported and poses a potential bioterrorism challenge for public health authorities. Therefore, a workshop of academic, government, and private sector personnel from around the world was convened to discuss the current state of melioidosis diagnostics, diagnostic needs, and future directions.
Hepatitis delta virus (HDV) is a unique human pathogen whose RNA structures are critical to viral function. The genome is a circular single-stranded RNA with high levels of intramolecular ...complementarity (~ 74%) leading to formation of the unbranched rod structure characteristic of HDV RNA. The complementary antigenome, which has similar structural features, encodes the open reading frame from which mRNAs of the two forms of HDV protein are transcribed: the short form of the delta antigen (HDAg-S) is absolutely required for initiation and maintenance of viral replication, and the long form (HDAg-L) is necessary and sufficient for virus particle production in cells expressing hepatitis B surface antigens (HBsAg). HDV is considered a subviral satellite of hepatitis B virus (HBV) due to its dependence on HBsAg. In fact, HDV also relies on host functions to complete its replication cycle. Exploitation of cellular functions may be directly related to the structure of the ribonucleoprotein complex (RNP) formed through interaction of HDV RNA and viral proteins. This dissertation analyzes the effects of HDV RNA structure on RNA-protein interactions and viral replication. The first half examines the direct biochemical interaction between native bacterially expressed HDAg and in vitro transcribed HDV RNA. These data identify features of the RNA structure that are critical to binding by HDAg. Through electrophoretic mobility shift assay, it was discovered that HDV RNAs containing at least 311 total nucleotides (nt) were stably bound while those with fewer than 298 nt were not bound. This feature was shown to be biologically relevant in transfected cells. Furthermore, micrococcal nuclease digestion indicates the size of the RNP protected is the same as that formed by the smallest RNA bound by HDAg. The second half of this dissertation focuses on effects of RNA structure on viral function specifically, replication. Mutational analysis, specifically designed to preserve the predicted RNA structure of the interior of the unbranched rod structure, indicates sensitivity to nucleotide deletion. Deletions of various sizes (totaling 6 – 53 nt) in 6 different unrelated segments showed reduced viral replication. These results, and others, suggest most of the HDV RNA structure is pivotal to RNP formation and viral function.
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