Biomedical subjects

Lisa O Roberts

Publications and source records attributed to Lisa O Roberts.

10 recordsLinked to original sources

PubMed2006

Caliciviruses differ in their functional requirements for eIF4F components.

Two classes of viruses, namely members of the Potyviridae and Caliciviridae, use a novel mechanism for the initiation of protein synthesis that involves the interaction of translation initiation factors with a viral protein covalently linked to the viral RNA, known as VPg. The calicivirus VPg proteins can interact directly with the initiation factors eIF4E and eIF3. Translation initiation on feline calicivirus (FCV) RNA requires eIF4E because it is inhibited by recombinant 4E-BP1. However, to date, there have been no functional studies carried out with respect to norovirus translation initiation, because of a lack of a suitable source of VPg-linked viral RNA. We have now used the recently identified murine norovirus (MNV) as a model system for norovirus translation and have extended our previous studies with FCV RNA to examine the role of the other eIF4F components in translation initiation. We now demonstrate that, as with FCV, MNV VPg interacts directly with eIF4E, although, unlike FCV RNA, translation of MNV RNA is not sensitive to 4E-BP1, eIF4E depletion, or foot-and-mouth disease virus Lb protease-mediated cleavage of eIF4G. We also demonstrate that both FCV and MNV RNA translation require the RNA helicase component of the eIF4F complex, namely eIF4A, because translation was sensitive (albeit to different degrees) to a dominant negative form and to a small molecule inhibitor of eIF4A (hippuristanol). These results suggest that calicivirus RNAs differ with respect to their requirements for the components of the eIF4F translation initiation complex.

Animals↗

PubMed2006

The mitochondrial pathway of apoptosis is triggered during feline calicivirus infection.

Feline calicivirus (FCV) belongs to the family Caliciviridae and is an important pathogen of the upper respiratory tract of cats. Recent studies have shown that cells infected with FCV undergo apoptosis, as evidenced by caspase activation, chromatin condensation and cleavage of poly(ADP-ribose) polymerase. Here, the upstream events were investigated in order to define the molecular mechanism of apoptosis in FCV-infected cells. It was shown that FCV induced translocation of phosphatidylserine to the cell outer membrane and release of cytochrome c from mitochondria at about 6-8 h post-infection. These events were preceded by the loss of mitochondrial membrane potential and Bax translocation from the cytosol to mitochondria between 4 and 6 h after infection. Release of cytochrome c from mitochondria triggered the activation of caspase-9 and the subsequent activation of the executioner caspase, caspase-3. These results suggest that the mitochondrial pathway of apoptosis is triggered during FCV infection.

Animals↗

PubMed2006

Feline calicivirus replication: requirement for polypyrimidine tract-binding protein is temperature-dependent.

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 characterization of similar interactions with the genomes of members of the family Caliciviridae has been limited to in vitro binding analysis. In this study, Feline calicivirus (FCV) has been used as a model system to identify and characterize the role of host-cell factors that interact with the viral RNA. It was demonstrated that polypyrimidine tract-binding protein (PTB) interacts specifically with the 5' sequences of the FCV genomic and subgenomic RNAs. Using RNA interference it was shown that PTB is required for efficient FCV replication in a temperature-dependent manner. siRNA-mediated knockdown of PTB resulted in a 15- to 100-fold reduction in virus titre, as well as a concomitant reduction in viral RNA and protein synthesis at 32 degrees C. In addition, virus-induced cytopathic effect was significantly delayed as a result of an siRNA-mediated reduction in PTB levels. A role for PTB in the calicivirus life cycle was more apparent at temperatures above and below 37 degrees C, fitting with the hypothesis that PTB functions as an RNA chaperone, potentially aiding the folding of RNA into functional structures. This is the first functional demonstration of a host-cell protein interacting with a calicivirus RNA.

5' Untranslated Regions↗

PubMed2005

Stabilized baculovirus vector expressing a heterologous gene and GP64 from a single bicistronic transcript.

The efficient scale-up of recombinant protein production in insect-cell bioreactors using baculovirus expression vectors is hampered by reductions in yield with increasing viral passage, the so-called passage effect. This phenomenon is characterized by the generation and subsequent accumulation of defective interfering baculoviruses (DIs), which interfere with the replication of genomically intact virus. A novel baculovirus expression vector is presented equipped with a bicistronic expression cassette that allows the simultaneous expression of the recombinant gene (GFP, first cistron) and an essential baculovirus gene (GP64, second cistron) from a single messenger RNA (mRNA). The translation of GP64 is mediated by an internal ribosome entry site (IRES) element from Rhopalosiphum padi virus (RhPV) while the native GP64 gene is deleted. In this way, a dominant selection pressure is placed on the entire bicistronic mRNA and hence on the maintenance of the foreign gene. The bicistronic expression vector was superior to the control baculovirus vector in that GFP expression remained at much higher levels upon continued virus passage. The versatility of this stabilized vector was demonstrated by its ability to propagate in a number of cell lines including Sf21, Sf9 and High Five cells. This novel baculovirus vector is especially valuable for large-scale recombinant protein production in insect-cell bioreactors where the number of viral passages is high.

Baculoviridae↗

PubMed2005

A cross-kingdom internal ribosome entry site reveals a simplified mode of internal ribosome entry.

Rhopalosiphum padi virus (RhPV) is an insect virus of the Dicistroviridae family. Recently, the 579-nucleotide-long 5' untranslated region (UTR) of RhPV has been shown to contain an internal ribosome entry site (IRES) that functions efficiently in mammalian, plant, and insect in vitro translation systems. Here, the mechanism of action of the RhPV IRES has been characterized by reconstitution of mammalian 48S initiation complexes on the IRES from purified components combined with the toeprint assay. There is an absolute requirement for the initiation factors eIF2 and eIF3 and the scanning factor eIF1 to form 48S complexes on the IRES. In addition, eIF1A, eIF4F (or the C-terminal fragment of eIF4G), and eIF4A strongly stimulated the assembly of this complex, whereas eIF4B had no effect. Although the eIF4-dependent pathway is dominant in the RhPV IRES-directed cell-free translation, omission of either eIF4G or eIF4A or both still allowed the assembly of 48S complexes from purified components with approximately 23% of maximum efficiency. Deletions of up to 100 nucleotides throughout the 5'-UTR sequence produced at most a marginal effect on the IRES activity, suggesting the absence of specific binding sites for initiation factors. Only deletion of the U-rich unstructured 380-nucleotide region proximal to the initiation codon resulted in a complete loss of the IRES activity. We suggest that the single-stranded nature of the RhPV IRES accounts for its strong but less selective potential to bind key mRNA recruiting components of the translation initiation apparatus from diverse origins.

5' Untranslated Regions↗

PubMed2004

Cleavage of eukaryotic initiation factor eIF4G and inhibition of host-cell protein synthesis during feline calicivirus infection.

Caliciviruses are small, non-enveloped, positive-stranded RNA viruses that are pathogenic for both animals and man. Although their capsid structure and genomic organization are distinct from picornaviruses, they have similarities to these viruses in their non-structural proteins. Picornaviruses induce a rapid inhibition of host-cell cap-dependent protein synthesis and this is mainly achieved through cleavage of eIF4G and/or dephosphorylation of 4E-BP1. In this study, the effect of calicivirus infection was examined on host-cell protein synthesis in order to determine whether they also induce host shut-off. We report that infection of cells with feline calicivirus (FCV) leads to the inhibition of cellular protein synthesis. This is accompanied by the cleavage of the eukaryotic translation initiation factors eIF4GI and eIF4GII in a manner reminiscent of that induced by picornaviruses. However, the cleavages occur at different sites. The potential mechanisms of these cleavage events and the implications for the translation of calicivirus mRNA are discussed.

Animals↗

PubMed2004

The Rhopalosiphum padi virus 5' internal ribosome entry site is functional in Spodoptera frugiperda 21 cells and in their cell-free lysates: implications for the baculovirus expression system.

Cap-independent internal initiation of translation occurs on a number of viral and cellular mRNAs and is directed by internal ribosome entry site (IRES) elements. Rhopalosiphum padi virus (RhPV) is a member of the Dicistroviridae. These viruses have single-stranded, positive-sense RNA genomes that contain two open reading frames, both preceded by IRES elements. Previously, the activity of the RhPV 5' UTR IRES has been demonstrated in mammalian, Drosophila and wheat germ in vitro translation systems. It is now shown that this IRES also functions within Spodoptera frugiperda (Sf21) cells which are widely used in the baculovirus expression system, and in a novel Sf21 cell-based lysate system. Inclusion of the RhPV IRES in a dicistronic reporter mRNA transcript increased translation of the second cistron 23-fold within Sf21 cells. In contrast, the encephalomyocarditis virus IRES was inactive in both systems. The RhPV IRES therefore has the potential to be utilized in insect cell expression systems.

5' Untranslated Regions↗

PubMed2004

A bunyamwera virus minireplicon system in mosquito cells.

Artificial minigenomes are powerful tools for studying the replication and transcription of negative-strand RNA viruses. Bunyamwera virus (BUN; genus Orthobunyavirus, family Bunyaviridae) is an arbovirus that shows fundamental biological differences when replicating in mammalian versus mosquito cells. To study BUN RNA synthesis in mosquito cells, we developed a bacteriophage T7 RNA polymerase-based minireplicon system similar to that described previously for mammalian cells. An Aedes albopictus C6/36-derived mosquito cell line stably expressing T7 RNA polymerase was established. Viral proteins and artificial minigenomes (containing Renilla luciferase as a reporter) were transcribed and expressed in these cells from transfected T7 promoter-containing plasmids. Transcription of the minigenome required two viral proteins, the nucleocapsid protein N and the RNA-dependent RNA polymerase L, a situation similar to that in mammalian cells. However, unlike the situation in mammalian cells, the viral polymerase was not inhibited by the viral nonstructural protein NSs. We also report that promoter strength is different for vertebrate versus invertebrate cells. The development of this system opens the way for a detailed comparison of bunyavirus replication in cells of disparate phylogeny.

Aedes↗

PubMed2003

Caspase-mediated cleavage of the feline calicivirus capsid protein.

Feline calicivirus (FCV) is responsible for an acute upper respiratory tract disease in cats. The FCV capsid protein is synthesized as a precursor (76 kDa) that is post-translationally processed into the mature 62 kDa capsid protein by removal of the N-terminal 124 amino acids. Our previous studies have also detected a 40 kDa protein, related to the FCV capsid protein, produced during infection. Here we demonstrate that cleavage of the FCV capsid protein, during infection of cells in culture, was prevented by caspase inhibitors. In addition, caspase-2, -3 and -7 were activated during FCV infection, as shown by pro-form processing, an increase in N-acetyl-Asp-Glu-Val-Asp-7-amido-4-trifluoromethylcoumarin cleavage activity and in situ poly(ADP-ribose) polymerase cleavage. Caspase activation coincided with the induction of apoptosis and capsid cleavage to the 40 kDa fragment. An in vitro cleavage assay, using recombinant human caspases and in vitro-derived FCV capsid protein, revealed that caspase-2, and to a lesser extent caspase-6, cleaved the capsid protein to generate a 40 kDa fragment. Taken together, these results suggest that FCV triggers apoptosis within infected cells and that caspase-induced capsid cleavage occurs concomitantly with apoptosis. The possible role of capsid cleavage in the pathogenesis of FCV infection is discussed.

Animals↗

PubMed2003

Apoptosis in cultured cells infected with feline calicivirus.

Caliciviruses are important pathogens of man and animals; feline calicivirus (FCV) is responsible for an acute upper respiratory tract disease in cats. To date, little is known about the mechanism of cell damage induced by these viruses. We set out to determine if apoptosis played any role in cell death in FCV infection of cultured cells. We demonstrate that caspase-2, -3, and -7 were activated during FCV infection, as evidenced by pro-form processing and an increase in acetyl-Asp-Glu-Val-Asp-7-amido-4-trifluoromethylcoumarin cleavage activity, as well as cleavage of poly(ADP-ribose)polymerase. Caspase activation coincided with the condensation of chromatin. At about 8 h post infection we also detected cleavage of the FCV capsid protein; this was prevented by caspase inhibitors. Taken together these results suggest that FCV triggers apoptosis within infected cells and that caspases are involved in the cleavage of the capsid protein.

Animals↗