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Here we propose a new general method for directly determining
RNA sequence based on the use of the RNA-dependent RNA polymerase
from bacteriophage φ6 and the chain terminators (RdRP
sequencing). The following properties of the polymerase render
it appropriate for this application: (1) the φ6 polymerase
can replicate a number of single-stranded RNA templates in vitro.
(2) In contrast to the primer-dependent DNA polymerases utilized
in the sequencing procedure by Sanger et al. (Proc Natl Acad Sci
USA, 1977, 74:5463–5467), it initiates nascent
strand synthesis without a primer, starting the polymerization on
the very 3′-terminus of the template. (3) The polymerase can
incorporate chain-terminating nucleotide analogs into the nascent
RNA chain to produce a set of base-specific termination products. Consequently, 3′ proximal or even complete sequence of many
target RNA molecules can be rapidly deduced without prior sequence
information. The new technique proved useful for sequencing
several synthetic ssRNA templates. Furthermore, using genomic
segments of the bluetongue virus we show that RdRP sequencing
can also be applied to naturally occurring dsRNA templates.
This suggests possible uses of the method in the RNA virus
research and diagnostics.
PRD1 is a ds-DNA bacteriophage from the Tectiviridae family with an unusual structural feature: the viral genome is enclosed by a protein-rich membrane, which is in turn enclosed by an external icosahedral protein shell (capsid). Three-dimensional reconstructions from cryo-electron microscopy (cryo-EM) images have revealed the structure of the PRD1 capsid at moderate resolution (28 Å), while X-ray crystallographic studies have recently provided a high resolution (1.85 Å) picture of the major coat protein, P3. We have now combined these results from different imaging methods to obtain a more detailed understanding of the virion organization. The combination has been made in a cyclic process: a preliminary fitting of the atomic structure of P3 to each one of its independent positions in the cryo-EM maps of the capsids provided initial models that could be used to improve the reconstructions; the refined maps then served as a base frame for an optimized fit. This process allows us to study the viral particle structure at “quasi-atomic” resolution.
Bacteriophage φ6 genome consists of three segments
of double-stranded RNA. During maturation, single-stranded
copies of these segments are packaged into preformed polymerase
complex particles. Only φ6 RNA is packaged, and each
particle contains only one copy of each segment. An in
vitro packaging and replication assay has been developed
for φ6, and the packaging signals (pac sites) have
been mapped to the 5′ ends of the RNA segments. In
this study, we propose secondary structure models for the
pac sites of φ6 single-stranded RNA segments. Our
models accommodate data from structure-specific chemical
modifications, free energy minimizations, and phylogenetic
comparisons. Previously reported pac site deletion studies
are also discussed. Each pac site possesses a unique architecture,
that, however, contains common structural elements.
Bacteriophage φ6 has a double-stranded RNA
genome composed of three linear segments, L, M, and S.
The innermost particle in the virion of φ6, like in
the other dsRNA viruses, is an RNA-dependent RNA polymerase
complex, which carries out all the functions needed for
the replication of the viral genome. Empty polymerase complexes
can package the single-stranded copies of the viral genome
segments, replicate the packaged segments into double-stranded
form (minus strand synthesis), and then produce new plus
strands (transcripts) from the double-stranded RNA templates.
The three viral genomic segments contain unique packaging
signals at their 5′ ends, and minus strand synthesis
initiation is dependent on the sequence at the 3′
end. Here we have constructed chimeric segments that have
the packaging signal from one segment and the minus strand
synthesis initiation signal from another segment. Using
purified recombinant polymerase complexes and single-stranded
chimeric and original RNA segments, we have analyzed the
packaging and replication regulation operating in in vitro
conditions. We show that the 5′ end of the L genome
segment in single-stranded form is needed to switch from
the packaging to the minus strand synthesis and the same
sequence is required in double-stranded form to switch
on plus strand synthesis. In addition we have constructed
deletions to the M segment to analyze the possible regulatory
role of the internal noncoding area of this segment.
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