133 research outputs found
Analysis of RNA-Protein interactions involved in calicivirus translation and replication
The interaction of host-cell nucleic acid-binding proteins with the genomes of
positive-stranded RNA viruses is known to play a role in the translation and
replication of many viruses. To date, however, the characterisation of similar
interactions with the genomes of members of the Caliciviridae family has been
limited to in vitro binding analysis. In this study, feline calicivirus (FCV) and murine
norovirus (MNV) have been used as model systems to identify and characterise the
role of host-cell factors that interact with the viral RNA and RNA structures that
regulate virus replication. It was demonstrated that RNA-binding proteins such as
polypyrimidine tract-binding protein (PTB), poly(C)-binding proteins (PCBPs) and
La protein interact with the extremities of MNV and FCV genomic and subgenomic
RNAs. PTB acted as a negative-regulator in FCV translation and is possibly involved
in the switch between translation and replication during the late stages of the
infection, as PTB is exported from the nucleus to the cytoplasm, where calicivirus
replication takes place. Furthermore, using the MNV reverse-genetics system,
disruption of 5' end stem-loops reduced infectivity ~15-20 fold, while disruption of an
RNA structure that is suspected to be part of the subgenomic RNA synthesis promoter
and an RNA structure at the 3' end completely inhibited virus replication. Restoration
of infectivity by repair mutations in the subgenomic promoter region and the recovery
of viruses that contained repressor mutations within the disrupted structures, in both
the subgenomic promoter region and the 3’ end, confirmed a functional role for these
RNA secondary structures. Overall this study has yielded new insights into the role of
RNA structures and RNA-protein interactions in the calicivirus life cycle
Assessing end-user awareness of social engineering and phishing
Social engineering is a significant problem involving technical and nontechnical ploys in order to acquire information from unsuspecting users. This paper presents an assessment of user awareness of such methods in the form of email phishing attacks. Our experiment used a webbased survey, which presented a mix of 20 legitimate and illegitimate emails, and asked participants to classify them and explain the rationale for their decisions. This assessment shows that the 179 participants were 36% successful in identifying legitimate emails, versus 45% successful in spotting illegitimate ones. Additionally, in many cases, the participants who identified illegitimate emails correctly could not provide convincing reasons for their selections
Where are we in understanding salamander locomotion: biological and robotic perspectives on kinematics
Salamanders have captured the interest of biologists and roboticists for decades because of their ability to locomote in different environments and their resemblance to early representatives of tetrapods. In this article, we review biological and robotic studies on the kinematics (i.e., angular profiles of joints) of salamander locomotion aiming at three main goals: (i) to give a clear view of the kinematics, currently available, for each body part of the salamander while moving in different environments (i.e., terrestrial stepping, aquatic stepping, and swimming), (ii) to examine what is the status of our current knowledge and what remains unclear, and (iii) to discuss how much robotics and modeling have already contributed and will potentially contribute in the future to such studies
Where are we in understanding salamander locomotion: biological and robotic perspectives on kinematics
Salamanders have captured the interest of biologists and roboticists for decades because of their ability to locomote in different environments and their resemblance to early representatives of tetrapods. In this article, we review biological and robotic studies on the kinematics (i.e., angular profiles of joints) of salamander locomotion aiming at three main goals: (i) to give a clear view of the kinematics, currently available, for each body part of the salamander while moving in different environments (i.e., terrestrial stepping, aquatic stepping, and swimming), (ii) to examine what is the status of our current knowledge and what remains unclear, and (iii) to discuss how much robotics and modeling have already contributed and will potentially contribute in the future to such studie
The murine norovirus core subgenomic RNA promoter consists of a stable stem-loop that can direct accurate initiation of RNA synthesis.
UNLABELLED: All members of the Caliciviridae family of viruses produce a subgenomic RNA during infection. The subgenomic RNA typically encodes only the major and minor capsid proteins, but in murine norovirus (MNV), the subgenomic RNA also encodes the VF1 protein, which functions to suppress host innate immune responses. To date, the mechanism of norovirus subgenomic RNA synthesis has not been characterized. We have previously described the presence of an evolutionarily conserved RNA stem-loop structure on the negative-sense RNA, the complementary sequence of which codes for the viral RNA-dependent RNA polymerase (NS7). The conserved stem-loop is positioned 6 nucleotides 3' of the start site of the subgenomic RNA in all caliciviruses. We demonstrate that the conserved stem-loop is essential for MNV viability. Mutant MNV RNAs with substitutions in the stem-loop replicated poorly until they accumulated mutations that revert to restore the stem-loop sequence and/or structure. The stem-loop sequence functions in a noncoding context, as it was possible to restore the replication of an MNV mutant by introducing an additional copy of the stem-loop between the NS7- and VP1-coding regions. Finally, in vitro biochemical data suggest that the stem-loop sequence is sufficient for the initiation of viral RNA synthesis by the recombinant MNV RNA-dependent RNA polymerase, confirming that the stem-loop forms the core of the norovirus subgenomic promoter. IMPORTANCE: Noroviruses are a significant cause of viral gastroenteritis, and it is important to understand the mechanism of norovirus RNA synthesis. Here we describe the identification of an RNA stem-loop structure that functions as the core of the norovirus subgenomic RNA promoter in cells and in vitro. This work provides new insights into the molecular mechanisms of norovirus RNA synthesis and the sequences that determine the recognition of viral RNA by the RNA-dependent RNA polymerase.This is the author's accepted manuscript. The final version is available from the American Society for Microbiology at http://jvi.asm.org/content/89/2/1218.lon
Polypyrimidine tract binding protein functions as a negative regulator of feline calicivirus translation.
Positive strand RNA viruses rely heavily on host cell RNA binding proteins for various aspects of their life cycle. Such proteins interact with sequences usually present at the 5' or 3' extremities of the viral RNA genome, to regulate viral translation and/or replication. We have previously reported that the well characterized host RNA binding protein polypyrimidine tract binding protein (PTB) interacts with the 5'end of the feline calicivirus (FCV) genomic and subgenomic RNAs, playing a role in the FCV life cycle.We have demonstrated that PTB interacts with at least two binding sites within the 5'end of the FCV genome. In vitro translation indicated that PTB may function as a negative regulator of FCV translation and this was subsequently confirmed as the translation of the viral subgenomic RNA in PTB siRNA treated cells was stimulated under conditions in which RNA replication could not occur. We also observed that PTB redistributes from the nucleus to the cytoplasm during FCV infection, partially localizing to viral replication complexes, suggesting that PTB binding may be involved in the switch from translation to replication. Reverse genetics studies demonstrated that synonymous mutations in the PTB binding sites result in a cell-type specific defect in FCV replication.Our data indicates that PTB may function to negatively regulate FCV translation initiation. To reconcile this with efficient virus replication in cells, we propose a putative model for the function of PTB in the FCV life cycle. It is possible that during the early stages of infection, viral RNA is translated in the absence of PTB, however, as the levels of viral proteins increase, the nuclear-cytoplasmic shuttling of PTB is altered, increasing the cytoplasmic levels of PTB, inhibiting viral translation. Whether PTB acts directly to repress translation initiation or via the recruitment of other factors remains to be determined but this may contribute to the stimulation of viral RNA replication via clearance of ribosomes from viral RNA
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