15 research outputs found
Structural features of adenovirus 2 virus-associated RNA required for binding to the protein kinase DAI
The double-stranded RNA activated protein kinase DAI contains an RNA binding domain consisting of two copies of a double-stranded RNA binding motif. We have investigated the role of RNA structure in the interaction between DAI and the structured single-stranded RNA, adenovirus VA RNAI, which inhibits DAI activation. Mutations in the apical stem, terminal stem, and central domain of the RNA were tested to assess the contribution of these elements to DAI binding in vitro. The data demonstrate that over half a turn of intact apical stem is required for the interaction and that there is a correlation between the binding of apical stem mutants and their ability to function both in vivo and in vitro. There was also evidence of preference for GC-rich sequence in the proximal region of the apical stem. In the central domain the correlation between binding and function of mutant RNAs was poor, suggesting that at least some of this region plays no direct role in binding to DAI, despite its functional importance. Exceptionally, central domain mutations that encroached on the phylogenetically conserved stem 4 of VA RNA disrupted binding, and complementary mutations in this sequence partially restored binding. Measurement of the binding of wild-type VA RNAI to DAI and p20, a truncated form of the protein containing the RNA binding domains alone, under various ionic conditions imply that the major interactions are electrostatic and occur via the protein's RNA binding domain. However, differences between full-length DAI and p20 in their binding to mutants in the conserved stem suggest that regions outside the RNA binding domain also participate in the binding. The additional interactions are likely to be non-ionic, and may be important for preventing DAI activation during virus infection
Envelope 2 protein phosphorylation sites S75 & 277 of hepatitis C virus genotype 1a and interferon resistance: A sequence alignment approach
<p>Abstract</p> <p>Background</p> <p>Hepatitis C is a major health problem affecting more than 200 million individuals in world including Pakistan. Current treatment regimen consisting of interferon alpha and ribavirin does not always succeed to eliminate virus completely from the patient's body.</p> <p>Results</p> <p>Interferon induced antiviral protein kinase R (PKR) has a role in the hepatitis C virus (HCV) treatment as dsRNA activated PKR has the capacity to phosphorylate the serine and threonine of E2 protein and dimerization viral RNA. E2 gene of hepatitis C virus (HCV) genotype 1 has an active role in IFN resistance. E2 protein inhibits and terminates the kinase activity of PKR by blocking it in protein synthesis and cell growth. This brings forward a possible relation of E2 and PKR through a mechanism via which HCV evades the antiviral effect of IFN.</p> <p>Conclusion</p> <p>A hybrid in-silico and wet laboratory approach of motif prediction, evolutionary and structural anlysis has pointed out serine 75 and 277 of the HCV E2 gene as a promising candidate for the serine phosphorylation. It is proposed that serine phosphorylation of HCV E2 gene has a significant role in interferon resistance.</p
Synthesis and purification of single-stranded RNA for use in experiments with PKR and in cell-free translation systems
The biosynthesis of RNA in vitro using bacteriophage RNA polymerases has opened up many avenues of research. Large amounts of specific RNA species can be readily produced but small amounts of contaminants that are simultaneously generated can interfere with biological assays, PKR, a ribosome-associated and double-stranded (ds) RNA-dependent protein kinase, is an important regulator of the initiation of protein synthesis. It can be activated by very low concentrations of dsRNA and inhibited by small structured RNAs or high concentrations of dsRNA. The best-studied inhibitor of PKR activation is adenovirus VA RNA1. Its gene was cloned into a plasmid under the control of the T7 RNA polymerase promoter, and the optimization of VA RNA transcription is described. A dsRNA by-product of the transcription reaction activates PKR in kinase autophosphorylation assays, and hence a purification protocol that allows the separation and removal of dsRNA contaminants was developed. A scheme to analyze the RNA product with specific nucleases is discussed. In a reticulocyte cell-free translation system the activation of PKR by dsRNA contaminating a synthetic mRNA preparation is likely to lead to shut-off of translation. An assay to directly visualize and measure the level of PKR phosphorylation in the lysate is detailed
Role of the apical stem in maintaining the structure and function of adenovirus virus-associated RNA.
Adenovirus virus-associated (VA) RNAI maintains efficient protein synthesis during the late phase of infection by preventing the activation of the double-stranded-RNA-dependent protein kinase, DAI. A secondary structure model for VA RNAI predicts the existence of two stems joined by a complex stem-loop structure, the central domain. The structural consequences of mutations and compensating mutations introduced into the apical stem lend support to this model. In transient expression assays for VA RNA function, foreign sequences inserted into the apical stem were fully tolerated provided that the stem remained intact. Mutants in which the base of the apical stem was disrupted retained partial activity, but truncation of the apical stem abolished the ability of the RNA to block DAI activation in vitro, suggesting that the length and position of the stem are both important for VA RNA function. These results imply that VA RNAI activity depends on secondary structure at the top of the apical stem as well as in the central domain and are consistent with a two-step mechanism involving DAI interactions with both the apical stem and the central domain
Mutational analysis of the central domain of adenovirus virus-associated RNA mandates a revision of the proposed secondary structure.
Protein synthesis in adenovirus-infected cells is regulated during the late phase of infection. The rate of initiation is maintained by a small viral RNA, virus-associated (VA) RNAI, which prevents the phosphorylation of eukaryotic initiation factor eIF-2 by a double-stranded RNA-activated protein kinase, DAI. On the basis of nuclease sensitivity analysis, a secondary-structure model was proposed for VA RNA. The model predicts a complex stem-loop structure in the central part of the molecule, the central domain, joining two duplexed stems. The central domain is required for the inhibition of DAI activation and participates in the binding of VA RNA to DAI. To assess the significance of the postulated stem-loop structure in the central domain, we generated compensating, deletion, and substitution mutations. A substitution mutation which disrupts the structure in the central domain abolishes VA RNA function in vitro and in vivo. Base-compensating mutations failed to restore the function or structure of the mutant, implying that the stem-loop structure may not exist. To confirm this observation, we tested mutants with alterations in the hypothetical loop and short stem that constitute the main features of the wild-type model structure. The upper part of the hypothetical loop could be deleted without abolishing the ability of the RNA to block DAI activation in vitro, whereas other loop mutations were deleterious for function and caused major rearrangements in the molecule. Base-compensating mutations in the stem did not restore the expected base pairing, even though the mutant RNAs were still functional in vitro. Surprisingly, a mutant with a noncompensating substitution mutation in the stem was more effective than wild-type VA RNAI in DAI inhibition assays but was ineffective in vivo. The structural and functional consequences of these mutations do not support the proposed model structure for the central domain, and we therefore suggest an alternative structure in which tertiary interactions may play a significant role in shaping the specificity of VA RNA function in the infected cell. Discrepancies between the functionality of mutant forms of VA RNA in vivo and in vitro are consistent with the existence of additional roles for VA RNA in the cell
Removal of double-stranded contaminants from RNA transcripts: synthesis of adenovirus VA RNAI from a T7 vector.
Bacteriophage RNA polymerases are widely used to synthesize defined RNAs on a large scale in vitro. Unfortunately, the RNA product contains a small proportion of contaminating RNAs, including complementary species, which can lead to errors of interpretation. We cloned the gene encoding Ad2 VA RNAI into a vector containing a T7 RNA polymerase promoter in order to generate large quantities of VA RNA for the study of its interaction with the dsRNA-dependent protein kinase DAI. Exact copies of VA RNAI were synthesized efficiently, but were contaminated with small amounts of dsRNA which activated DAI and confounded interpretation of kinase assays. We therefore developed a method to remove the dsRNA contaminants, allowing VA RNAI and mutants to be tested for their ability to activate or inhibit DAI. This method appears to be generally applicable
Transcription elongation factor P-TEFb is required for HIV-1 Tat transactivation in vitro
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Calicivirus translation initiation requires an interaction between VPg and eIF4E
Unlike other positive-stranded RNA viruses that use either a 5'-cap structure or an internal ribosome entry site to direct translation of their messenger RNA, calicivirus translation is dependent on the presence of a protein covalently linked to the 50 end of the viral genome (VPg). We have shown a direct interaction of the calicivirus VPg with the cap-binding protein eIF4E. This interaction is required for calicivirus mRNA translation, as sequestration of eIF4E by 4E-BP1 inhibits translation. Functional analysis has shown that VPg does not interfere with the interaction between eIF4E and the cap structure or 4E-BP1, suggesting that VPg binds to eIF4E at a different site from both cap and 4E-BP1. This work lends support to the idea that calicivirus VPg acts as a novel 'cap substitute' during initiation of translation on virus mRNA