A retroviral RNA secondary structure required for efficient initiation of reverse transcription

Genetic evidence is presented which suggests the existence of an important structural element in the 5' noncoding region of avian retrovirus RNA. The proposed structure, which we term the U5-leader stem, is composed of sequences in the middle of U5 and in the leader, flanking the primer-binding site. U5 and leader mutations which would disrupt this structure caused a partial replication defect. However, nucleotide substitutions in the leader, which would structurally compensate for a U5 deletion mutation, restored normal replication. Analysis of replication intermediates of viruses with the above mutations suggests that the U5-leader stem is required for efficient DNA synthesis in vivo and for initiation of DNA synthesis from the tRNA(Trp) primer in melittin-activated virions. However, this structure does not appear to be required for binding of the tRNA(Trp) primer to viral RNA. These results support a role for the U5-leader stem structure, independent of its primary sequence, in the initiation of retroviral replication.     

RNA polymerases during differentiation of avian erythrocytes

DNA-dependent RNA polymerase was analysed during the terminal differentiation stages of avian erythrocytes. It was found that the mature duck erythrocyte, although quiescent in RNA synthesis, contains clearly measurable quantities of RNA polymerase B (or II). Immature polychromatic erythrocytes, derived from anemic ducks and actively synthesizing hemoglobin mRNA, additionally contain significant amounts of RNA polymerase A (or I) and C (or III) previously not detected in these cells. These latter classes of enzymes, although present, are apparently not engaged in RNA synthesis in polychromatic erythrocytes.     

Structural organization of viral RNA-dependent RNA polymerases

This review describes available data on the structure of viral RNA-dependent RNA polymerases (RdRP) obtained from X-ray analysis and discusses the functional significance of the structural elements of these enzymes. Because most of the studies done to date relate to RdRP structures of picorna-, flavi-, and caliciviruses, here we consider mostly the structures of RdRP of these groups of viruses, and also include information about polymerases of other virus families.     

Tentative identification of RNA-dependent RNA polymerases of dsRNA viruses and their relationship to positive strand RNA viral polymerases

Amino acid sequence stretches similar to the four most conserved segments of positive strand RNA viral RNA-dependent RNA polymerases have been identified in proteins of four dsRNA viruses belonging to three families, i.e. P2 protein of bacteriophage phi 6 (Cystoviridae), RNA 2 product of infectious bursa disease virus (Birnaviridae), lambda 3 protein of reovirus, and VP1 of bluetongue virus (Reoviridae). High statistical significance of the observed similarity was demonstrated, allowing identification of these proteins as likely candidates for RNA-dependent RNA polymerases. Based on these observations, and on the previously reported sequence similarity between the RNA polymerases of a yeast dsRNA virus and those of positive strand RNA viruses, a possible evolutionary relationship between the two virus classes is discussed.     

Delivery of short hairpin RNA sequences by using a replication-competent avian retroviral vector

While recent studies have demonstrated that retroviral vectors can be used to stably express short hairpin RNA (shRNA) to inhibit gene expression, these studies have utilized replication-defective retroviruses. We describe the creation of a replication-competent, Gateway-compatible retroviral vector capable of expressing shRNA that inhibits the expression of specific genes.