Nonviral heterogeneous sequences are present at the 5'' ends of one species of snowshoe hare bunyavirus S complementary RNA.

Analyses of the 5' ends of snowshoe hare bunyavirus plus sense S RNA species (including mRNA) recovered from infected cells have revealed two types of termini. These include ends that are essentially exact copies of the 3' end of the viral S RNA and others that are similar, but additionally have 13-14 nucleotide extensions that are heterogeneous in sequence. The former probably represent replicative plus sense RNA species, the latter mRNA species that have host cell derived primer sequences.     

Sequence-specific binding of the influenza virus RNA polymerase to sequences located at the 5' ends of the viral RNAs.

The enzymatic activity of recombinant influenza virus RNA polymerase is strictly dependent on the addition of a template RNA containing 5' and 3' viral sequences. Here we report the analysis of the binding specificity and physical characterization of the complex by using gel shift, modification interference, and density gradient techniques. The 13S complex binds specifically to short synthetic RNAs that mimic the partially double stranded panhandle structures found at the termini of both viral RNA and cRNA. The polymerase will also bind independently to the single-stranded 5' or 3' ends of viral RNA. It binds most strongly to specific sequences within the 5' end but is unable to bind these sequences in the context of a completely double stranded structure. Modification interference analysis identified the short sequence motifs at the 5' ends of the viral RNA and cRNA templates that are critical for binding.     

Capped poly(A) leaders of variable lengths at the 5'' ends of vaccinia virus late mRNAs.

Evidence for capped poly(A) leaders of variable lengths located immediately upstream of the translation initiation codon was obtained by direct analyses of a major late mRNA species. A decapping-recapping method was used to specifically substitute a radioactively labeled phosphate for an unlabeled one within the cap structure. RNase H-susceptible sites were made by hybridizing synthetic oligodeoxyribonucleotides to the mRNA encoding a late major structural protein of 11 kilodaltons. Sequences of the type m7G(5')pppAmp (Ap)nUpG. . ., where n varies from a few to more than 40 nucleotides, were deduced by analysis of the length and sequence of RNase H, RNase T1, and RNase U2 digestion products.     

Functions of the sequences at the ends of the inverted repeats of pseudorabies virus.

Two mutants were constructed to explore the functions of the sequences at the end of the S terminus of pseudorabies virus (PrV). In mutant vYa, 17 bp from the internal inverted repeat, as well as adjacent sequences from the L component, were deleted. In mutant v135/9, 143 bp from the internal inverted repeat (including sequences with homology to the pac-1 site of herpes simplex virus), as well as adjacent sequences from the L component, were deleted. Our aim in constructing these mutants was to ascertain whether equalization of the terminal regions of the S component would occur, whether genome termini that lack either the terminal 17 or 143 bp would be generated as a result of equalization of the repeats (thereby identifying the terminal nucleotides that may include cleavage signals), and whether inversion of the S component would occur (thereby ascertaining the importance of the deleted sequences in this process). The results obtained show the following (i) The removal of the terminal 17 or 143 bp of the internal S component, including the sequences with homology to the pac-1 site, does not affect the inversion of the Us. (ii) The equalization of both the vYa and the v135/9 inverted repeats occurs at high frequency, the terminal repeats being converted and becoming similar to the mutated internal inverted repeat. (iii) Mutants in which the 17 terminal base pairs (vYa) have been replaced by unrelated sequences are viable. However, the 143 terminal base pairs appear to be essential to virus survival; concatemeric v135/9 DNA with equalized, mutant-type, inverted repeats accumulates, but mature virions with such equalized repeats are not generated at high frequency. Since concatemeric DNA missing the 143 bp at both ends of the S component is not cleaved, the terminal 143 bp that include the sequences with homology to the pac-1 site are necessary for efficient cleavage. (iv) v135/9 intracellular DNA is composed mainly of arrays in which one S component (with two equalized inverted repeats both having the deletion) is bracketed by two L components in opposite orientations and in which two L components are in head-to-head alignment.(ABSTRACT TRUNCATED AT 250 WORDS)     

Structure of the 5'' untranslated regulatory region of ferritin mRNA studied in solution.

Ferritin mRNAs are the first eukaryotic mRNAs for which a conserved, translational regulatory sequence has been identified. The sequence of twenty-eight nucleotides, called the IRE (iron regulatory element), is found in the 5'-noncoding region and is required for enhanced translation of ferritin mRNA by excess cellular iron; regulation occurs at initiation. The prediction of secondary structure in the IRE is a hairpin loop. We now report an analysis of the IRE structure in solution studied in natural ferritin mRNAs [H and H'(M) subunits] by primer extension, after modification or cleavage by dimethyl sulfate, RNAases T1 and V1, and the chemical nuclease 1, 10-phenanthroline-copper (OPCu) which cleaves single-stranded and bulged regions of RNA. Overall, the structure in solution of the ferritin mRNA regulatory region is a hairpin loop, with magnesium-sensitive features, in which half the stem is provided by the IRE and half by flanking regions; only secondary structure is conserved in the flanking regions. Predicted bulges or internal loops along the stem were clearly detected by OPCu but were missed by the more bulky probe RNAase T1, indicating the efficacy of OPCu in probing subtle features of RNA structure. Magnesium-dependent deviations from the predicted structure were observed in the stem between the hairpin loop and the bulge at C6. The location of the IRE in relation to the initiator AUG or the cap is variable in different ferritin mRNAs. However, the number of nucleotides in the base-paired flanking regions of known ferritin mRNAs is proportional to the distance of the IRE from the cap and places the secondary/tertiary structure 8-10 nucleotides from the cap where interference with initiation is likely.     

Studies of defective interfering RNAs of Sindbis virus with and without tRNAAsp sequences at their 5'' termini.

Three of six independently derived defective interfering (DI) particles of Sindbis virus generated by high-multiplicity passaging in cultured cells have tRNAAsp sequences at the 5' terminus of their RNAs (Monroe and Schlesinger, J. Virol. 49:865-872, 1984). In the present work, we found that the 5'-terminal sequences of the three tRNAAsp-negative DI RNAs were all derived from viral genomic RNA. One DI RNA sample had the same 5'-terminal sequence as the standard genome. The DI RNAs from another DI particle preparation were heterogeneous at the 5' terminus, with the sequence being either that of the standard 5' end or rearrangements of regions near the 5' end. The sequence of the 5' terminus of the third DI RNA sample consisted of the 5' terminus of the subgenomic 26S mRNA with a deletion from nucleotides 24 to 67 of the 26S RNA sequence. These data showed that the 5'-terminal nucleotides can undergo extensive variations and that the RNA is still replicated by virus-specific enzymes. DI RNAs of Sindbis virus evolve from larger to smaller species. In the two cases in which we followed the evolution of DI RNAs, the appearance of tRNAAsp-positive molecules occurred at the same time as did the emergence of the smaller species of DI RNAs. In pairwise competition experiments, one of the tRNAAsp-positive DI RNAs proved to be the most effective DI RNA, but under identical conditions, a second tRNAAsp-positive DI RNA was unable to compete with the tRNAAsp-negative DIs. Therefore, the tRNAAsp sequence at the 5' terminus of a Sindbis DI RNA is not the primary factor in determining which DI RNA becomes the predominant species in a population of DI RNA molecules.     

The 5'' ends of yeast killer factor RNAs are pppGp.

The 5' nucleotides of the double-stranded RNAs of yeast killer factor have been isolated by digestion with pancreatic, T1 and T2 RNase followed by two-dimensional electrophoresis. They were identified by bacterial alkaline phosphatase and snake venom phosphodiesterase digestions. Both the larger double-stranded RNA (L, of 2.5 x 10(6) daltons) and the smaller double-stranded RNA (M, of 1.4 x 10(6) daltons) have the 5' end groups pppGp. These 5' ends are dissimilar to those of the double-stranded RNAs of animal viruses but may be characteristic of the 5' ends of the double-stranded RNAs of fungal viruses.     

Cell-free translation of influenza virus mRNA.

Cytoplasmic poly (A)-rich RNA extracted from fowl plague virus-infected cells was found to program efficiently the translation of two major peptides in the wheat germ cell-free system. These peptides have the same electrophoretic mobility, on polyacrylamide gels, as the two major virion proteins M and NP. [35S] methionine tryptic peptide analysis by one-dimensionalthin-layer ionophoresis and finger printing by two-dimensional thin-layer ionophoresis and chromatography show a high degree of similarity between the two in vitro products and the authentic viral proteins M and NP. Although virion RNA is devoid of any poly (A) sequence, it is confirmed here that the viral complementary cytoplasmic RNA contains poly (A) stretches of varying lengths. Intact purified virion was found to promote the synthesis of very low amounts of the same NP and M proteins in this cell-free system. Quantitative aspects of data would indicate that this is due to minute amounts of complementary viral RNA associated with the virion or with the virion RNA itself. In conclusion, it is shown diectly by cell-free translation of authentic viral products that the influenza virion is "negative stranded" (Baltimore, 1971), at least for its two major structural proteins.