Cloning and sequence analysis of dsRNA segments 5, 6 and 7 of a novel non-group A, B, C adult rotavirus that caused an outbreak of gastroenteritis in China.

A diarrhoeal outbreak among adults in China was caused by a new rotavirus, termed ADRV-N, that does not react with antisera directed against group A, B or C rotaviruses [Zhonghua Liu Xing Bing Xue Za Zhi (Chin. Epidemiol.) 19 (1998) 336]. ADRV-N can be propagated in cell cultures [Zhonghua Yi Xue Za Zhi (Natl. Med. J. China) 82 (2002) 14]. We present the complete sequences for ADRV-N genome segments 5 and 6, and a full ORF sequence of genome segment 7. The deduced amino acid sequences suggest that these segments encode NSP1, VP6 and NSP3, respectively. These three ADRV-N genome segments have a unique -ACCCC-3' terminal sequence. The 5'-GG- terminus of segments 5 and 6 is the same as that of other rotaviruses. The amino acid similarity between VP6 and NSP3 of ADRV-N and the cognate sequences of their closest counterpart, group B IDIR, was 37 and 35%, respectively. The ADRV-N NSP1 has a double-stranded RNA binding motif (DSRM) and a putative autoproteolytic cleavage motif upstream from the DSRM. The putative ADRV-N NSP3 has a truncated C-terminus compared to the cognate protein of group B rotaviruses. All the available data demonstrate that ADRV-N differs significantly from the known rotaviruses and strongly suggest that ADRV-N is the first recognized member of a new group of rotaviruses infecting humans.

Comparison of polymerase subunits from double-stranded RNA bacteriophages.

The family Cystoviridae comprises several bacteriophages with double-stranded RNA (dsRNA) genomes. We have previously purified the catalytic polymerase subunit (Pol) of one of the Cystoviridae members, bacteriophage phi6, and shown that the protein can catalyze RNA synthesis in vitro. In this reaction, both bacteriophage-specific and heterologous RNAs can serve as templates, but those containing 3' termini from the phi6 minus strands are favored. This provides a molecular basis for the observation that only plus strands, not minus strands, are transcribed from phi6 dsRNA segments in vivo. To test whether such a regulatory mechanism is also found in other dsRNA viruses, we purified recombinant Pol subunits from the phi6-related bacteriophages phi8 and phi13 and assayed their polymerase activities in vitro. The enzymes catalyze template-dependent RNA synthesis using both single-stranded-RNA (ssRNA) and dsRNA templates. However, they differ from each other as well as from phi6 Pol in certain biochemical properties. Notably, each polymerase demonstrates a distinct preference for ssRNAs bearing short 3'-terminal sequences from the virus-specific minus strands. This suggests that, in addition to other factors, RNA transcription in Cystoviridae is controlled by the template specificity of the polymerase subunit.

Primer-independent RNA sequencing with bacteriophage phi6 RNA polymerase and chain terminators.

Here we propose a new general method for directly determining RNA sequence based on the use of the RNA-dependent RNA polymerase from bacteriophage phi6 and the chain terminators (RdRP sequencing). The following properties of the polymerase render it appropriate for this application: (1) the phi6 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.

A mechanism for initiating RNA-dependent RNA polymerization.

In most RNA viruses, genome replication and transcription are catalysed by a viral RNA-dependent RNA polymerase. Double-stranded RNA viruses perform these operations in a capsid (the polymerase complex), using an enzyme that can read both single- and double-stranded RNA. Structures have been solved for such viral capsids, but they do not resolve the polymerase subunits in any detail. Here we show that the 2 A resolution X-ray structure of the active polymerase subunit from the double-stranded RNA bacteriophage straight phi6 is highly similar to that of the polymerase of hepatitis C virus, providing an evolutionary link between double-stranded RNA viruses and flaviviruses. By crystal soaking and co-crystallization, we determined a number of other structures, including complexes with oligonucleotide and/or nucleoside triphosphates (NTPs), that suggest a mechanism by which the incoming double-stranded RNA is opened up to feed the template through to the active site, while the substrates enter by another route. The template strand initially overshoots, locking into a specificity pocket, and then, in the presence of cognate NTPs, reverses to form the initiation complex; this process engages two NTPs, one of which acts with the carboxy-terminal domain of the protein to prime the reaction. Our results provide a working model for the initiation of replication and transcription.

The polymerase subunit of a dsRNA virus plays a central role in the regulation of viral RNA metabolism.

Bacteriophage φ6 has a three-segmented double-stranded (ds) RNA genome, which resides inside a polymerase complex particle throughout the entire life cycle of the virus. The polymerase subunit P2, a minor constituent of the polymerase complex, has previously been reported to replicate both φ6-specific and heterologous single-stranded (ss) RNAs, giving rise to dsRNA products. In this study, we show that the enzyme is also able to use dsRNA templates to perform semi-conservative RNA transcription in vitro without the assistance of other proteins. The polymerase synthesizes predominantly plus-sense copies of φ6 dsRNA, medium and small segments being more efficient templates than the large one. This distribution of the test-tube reaction products faithfully mimics viral transcription in vivo. Experiments with chimeric ssRNAs and dsRNAs show that short terminal nucleotide sequences can account for the difference in efficiency of RNA synthesis. Taken together, these results suggest a model explaining important aspects of viral RNA metabolism regulation in terms of enzymatic properties of the polymerase subunit.

Crystallization and preliminary X-ray crystallographic studies on the bacteriophage phi6 RNA-dependent RNA polymerase.

The RNA-dependent RNA polymerase (P2) from bacteriophage Phi6 has been cloned and the protein overexpressed in Escherichia coli to produce an active enzyme. A fully substituted selenomethionyl version of the protein has also been produced. Crystals of both proteins have been grown; most belong to the monoclinic space group P2(1), with unit-cell parameters a = 105.9, b = 94.0, c = 140.9 A, beta = 101.4 degrees, but some are trigonal (space group P3(1) or P3(2)), with unit-cell parameters a = b = 110.1, c = 159.4 A, gamma = 120 degrees. Both crystal forms occur in the same crystallization drop and are morphologically indistinguishable. Native data sets have been collected from both types of crystals to better than 3 A resolution.

Co-translational folding of an eukaryotic multidomain protein in a prokaryotic translation system.

Continuous monitoring of the enzymatic activity of newly synthesized firefly luciferase in Escherichia coli cell-free translation system was performed to record folding kinetics of this multidomain eukaryotic protein in the prokaryotic cytosol. Whereas in vitro refolding of denatured luciferase in prokaryotic cytosol occurred with a low yield of active enzyme and took about an hour, the enzyme acquired its native structure immediately upon release from the ribosome, as seen from the immediate halt of active luciferase accumulation upon blocking of translation with inhibitors. The nascent luciferase was also capable of acquiring the active conformation prior to release from the ribosome, when its C terminus was extended with a polypeptide segment. Specific enzymatic activity of the firefly luciferase was found to be equally high irrespective of whether this protein was synthesized in eukaryotic or prokaryotic translation systems. The data presented demonstrate the fundamental ability of prokaryotic cytosol to support effective co-translational protein folding in general and co-translational folding of multidomain proteins in particular.

Replicase activity of purified recombinant protein P2 of double-stranded RNA bacteriophage phi6.

In nature, synthesis of both minus- and plus-sense RNA strands of all the known double-stranded RNA viruses occurs in the interior of a large protein assembly referred to as the polymerase complex. In addition to other proteins, the complex contains a putative polymerase possessing characteristic sequence motifs. However, none of the previous studies has shown template-dependent RNA synthesis directly with an isolated putative polymerase protein. In this report, recombinant protein P2 of double-stranded RNA bacteriophage phi6 was purified and demonstrated in an in vitro enzymatic assay to act as the replicase. The enzyme efficiently utilizes phage-specific, positive-sense RNA substrates to produce double-stranded RNA molecules, which are formed by newly synthesized, full-length minus-strands base paired with the plus-strand templates. P2-catalyzed replication is also shown to be very effective with a broad range of heterologous single-stranded RNA templates. The importance and implications of these results are discussed.

Cell-free immunology: construction and in vitro expression of a PCR-based library encoding a single-chain antibody repertoire.

A novel cloning-independent strategy has been developed to generate a combinatorial library of PCR fragments encoding a murine single-chain antibody repertoire and express it directly in a cell-free system. The new approach provides an effective alternative to the techniques involving in vivo procedures of preparation and handling large libraries of antibodies. The possible use of the described strategy in the ribosome display is discussed.

Enzymatic activity of the ribosome-bound nascent polypeptide.

Firefly luciferase was shown to be completely folded and thus enzymatically active immediately upon release from the ribosome [Kolb et al. (1994) EMBO J. 13, 3631-3637]. However, no luciferase activity was observed while full-length luciferase was attached to the ribosome as a peptidyl-tRNA, probably because the C-terminal portion of the enzyme is masked by the ribosome and/or ribosome-associated proteins. Here we have demonstrated that the ribosome-bound enzyme acquires the enzymatic activity when its C-terminus is extended by at least 26 additional amino acid residues. The results demonstrate that the acquisition of the final native conformation by a nascent protein does not need the release of the protein from the ribosome.

Cotranslational folding of proteins.

Many unfolded polypeptides are capable of refolding into their native structure upon the removal of the denaturant. However, the folding of the mature protein during renaturation does not accurately reflect the folding process of nascent proteins in the interior of the cell. This view resulted from the discovery of molecular chaperones known to modulate protein folding. Recent publications discussing the possible role and mechanisms of chaperone action suggest that folding in vivo may be a posttranslational process. Here we discuss data that indicate the final native structure and biological activity can be attainted can be nascent protein on the ribosome, thus supporting the cotranslational folding hypothesis.

Folding of firefly luciferase during translation in a cell-free system.

In vitro synthesis of firefly luciferase and its folding into an enzymatically active conformation were studied in a wheat germ cell-free translation system. A novel method is described by which the enzymatic activity of newly synthesized luciferase can be monitored continuously in the cell-free system while this protein is being translated from its mRNA. It is shown that ribosome-bound polypeptide chains have no detectable enzymatic activity, but that this activity appears within a few seconds after luciferase has been released from the ribosome. In contrast, the renaturation of denatured luciferase under identical conditions occurs with a half-time of 14 min. These results support the cotranslational folding hypothesis which states that the nascent peptides start to attain their native tertiary structure during protein synthesis on the ribosome.