Complementation analysis of the flavivirus Kunjin NS3 and NS5 proteins defines the minimal regions essential for formation of a replication complex and shows a requirement of NS3 in cis for virus assembly

We have previously reported successful trans-complementation of defective Kunjin virus genomic RNAs with a range of large lethal deletions in the nonstructural genes NS1, NS3, and NS5 (A. A. Khromykh et al., J. Virol. 74:3253-3263, 2000). In this study we have mapped further the minimal region in the NS5 gene essential for efficient trans-complementation of genome-length RNAs in repBHK cells to the first 316 of the 905 codons. To allow amplification and easy detection of complemented defective RNAs with deletions apparently affecting virus assembly, we have developed a dual replicon complementation system. In this system defective replicon RNAs with a deletion(s) in the nonstructural genes also encoded the puromycin resistance gene (PAC gene) and the reporter gene for beta-galactosidase (beta-Gal). Complementation of these defective replicon RNAs in repBHK cells resulted in expression of PAC and beta-Gal which allowed establishment of cell lines stably producing replicating defective RNAs by selection with puromycin and comparison of replication efficiencies of complemented defective RNAs by beta-Gal assay. Using this system we demonstrated that deletions in the C-terminal 434 codons of NS3 (codons 178 to 611) were complemented for RNA replication, while any deletions in the first 178 codons were not. None of the genome-length RNAs containing deletions in NS3 shown to be complementable for RNA replication produced secreted defective viruses during complementation in repBHK cells. In contrast, structural proteins produced from these complemented defective RNAs were able to package helper replicon RNA. The results define minimal regions in the NS3 and NS5 genes essential for the formation of complementable replication complex and show a requirement of NS3 in cis for virus assembly.     

Structure of the intracellular defective viral RNAs of defective interfering particles of mouse hepatitis virus.

The intracellular defective RNAs generated during high-multiplicity serial passages of mouse hepatitis virus JHM strain on DBT cells were examined. Seven novel species of single-stranded polyadenylic acid-containing defective RNAs were identified from passages 3 through 22. The largest of these RNAs, DIssA (molecular weight [mw], 5.2 X 10(6)), is identical to the genomic RNA packaged in the defective interfering particles produced from these cells. Other RNA species, DIssB1 (mw, 1.9 X 10(6) to 1.6 X 10(6)), DIssB2 (mw, 1.6 X 10(6)), DIssC (mw, 2.8 X 10(6)) DIssD (mw, 0.82 X 10(6)), DIssE (mw, 0.78 X 10(6)), and DIssF (mw, 1.3 X 10(6)) were detected at different passage levels. RNase T1-resistant oligonucleotide fingerprinting demonstrated that all these RNAs were related and had multiple deletions of the genomic sequences. They contained different subsets of the genomic sequences from those of the standard intracellular mRNAs of nondefective mouse hepatitis virus JHM strain. Thus these novel intracellular viral RNAs were identified as defective interfering RNAs of mouse hepatitis virus JHM strain. The synthesis of six of the seven normal mRNA species specific to mouse hepatitis virus JHM strain was completely inhibited when cells were infected with viruses of late-passage levels. However, the synthesis of RNA7 and its product, viral nucleoprotein, was not significantly altered in late passages. The possible mechanism for the generation of defective interfering RNAs was discussed.     

A yeast mutant which accumulates precursor tRNAs.

It has been proposed that the conditional yeast mutant ts136 is defective in the transport of mRNA from the nucleus to the cytoplasm (Hutchinson, Hartwell and McLaughlin, 1969). We have examined ts136 to determine whether it is defective in tRNA biosynthesis. At the restrictive temperature, the mutant accumulates twelve new species of RNA. These species co-migrate on polyacrylamide gels with some of the pulse-labeled precursor tRNAs. Three of the new RNAs (species 1a, 1b and 1c are large enough to contain two tandom tRNAs. Although RNAs 1a, 1b, and 1c do not contain detectable levels of modified and methylated bases, at least one of them hybridizes to DNA from an E. coli plasmid containing a yeast tRNA gene. All the remaining RNAs (2--8) contain modified and methylated bases typical of tRNA. Three of these species were tested and were found to hybridize to tRNA genes. Ribosomal RNA synthesis is also defective in ts136. It is suggested that ts136 may be defective in a nucleolytic activity, which is a prerequisite to RNA transport.     

Wide occurrence of measles virus subgenomic RNAs in attenuated live-virus vaccines

Nine measles vaccine preparations, including four different viral strains, provided by eight different manufacturers were analysed by Northern blot for the nature of their nucleocapsid RNAs. Out of nine preparations, six were shown to contain subgenomic RNAs, along with the full length genomic RNA. Presence or absence of the subgenomic RNAs correlated strictly with the viral strains used. The role of the defective interfering particles in measles virus vaccine attenuation, and in its seroconversion efficacy upon vaccination, as well as the potential hazard of the presence of defective interfering particles in live-virus vaccine preparations, is discussed.     

Evidence for a Novel Mechanism of Influenza Virus-Induced Type I Interferon Expression by a Defective RNA-Encoded Protein

Influenza A virus (IAV) defective RNAs are generated as byproducts of error-prone viral RNA replication. They are commonly derived from the larger segments of the viral genome and harbor deletions of various sizes resulting in the generation of replication incompatible viral particles. Furthermore, small subgenomic RNAs are known to be strong inducers of pattern recognition receptor RIG-I-dependent type I interferon (IFN) responses. The present study identifies a novel IAV-induced defective RNA derived from the PB2 segment of A/Thailand/1(KAN-1)/2004 (H5N1). It encodes a 10 kDa protein (PB2_∆) sharing the N-terminal amino acid sequence of the parental PB2 protein followed by frame shift after internal deletion. PB2_∆ induces the expression of IFNβ and IFN-stimulated genes by direct interaction with the cellular adapter protein MAVS, thereby reducing viral replication of IFN-sensitive viruses such as IAV or vesicular stomatitis virus. This induction of IFN is completely independent of the defective RNA itself that usually serves as pathogen-associated pattern and thus does not require the cytoplasmic sensor RIG-I. These data suggest that not only defective RNAs, but also some defective RNA-encoded proteins can act immunostimulatory. In this particular case, the KAN-1-induced defective RNA-encoded protein PB2_∆ enhances the overwhelming immune response characteristic for highly pathogenic H5N1 viruses, leading to a more severe phenotype in vivo.     

Subviral molecules of RNA associated with plant ss(+)RNA viruses

Plant ss(+)RNA viruses besides their genome RNAs often are associated with additional subviral RNA molecules which occur naturally or are generated de novo during infection. There are such molecules like: satellite, defective, defective interfering and chimeric RNAs. Subviral RNAs can not replicate and encapsidate by oneself. Helper viruses supply the protein complexes that are necessary to these processes. The subviral molecules are characterized by small size. Recombination, deletion and accumulation of mutation are the main ways of arising subviral elements, although the origin of satRNAs is unknown. The unique feature of subviral RNAs is their ability to modify of infection progress caused by helper virus. They can attenuate or enhance the intensity of disease symptoms. The overall influence on disease development depends on three-component complex consisting of: plant host-virus' strain--subviral RNA. This article is a synthetic review of information concerning subviral RNA molecules of plant viruses, their structure, functions and origin.     

Translation of the flavivirus kunjin NS3 gene in cis but not its RNA sequence or secondary structure is essential for efficient RNA packaging

Our previous studies using trans-complementation analysis of Kunjin virus (KUN) full-length cDNA clones harboring in-frame deletions in the NS3 gene demonstrated the inability of these defective complemented RNAs to be packaged into virus particles (W. J. Liu, P. L. Sedlak, N. Kondratieva, and A. A. Khromykh, J. Virol. 76:10766-10775). In this study we aimed to establish whether this requirement for NS3 in RNA packaging is determined by the secondary RNA structure of the NS3 gene or by the essential role of the translated NS3 gene product. Multiple silent mutations of three computer-predicted stable RNA structures in the NS3 coding region of KUN replicon RNA aimed at disrupting RNA secondary structure without affecting amino acid sequence did not affect RNA replication and packaging into virus-like particles in the packaging cell line, thus demonstrating that the predicted conserved RNA structures in the NS3 gene do not play a role in RNA replication and/or packaging. In contrast, double frameshift mutations in the NS3 coding region of full-length KUN RNA, producing scrambled NS3 protein but retaining secondary RNA structure, resulted in the loss of ability of these defective RNAs to be packaged into virus particles in complementation experiments in KUN replicon-expressing cells. Furthermore, the more robust complementation-packaging system based on established stable cell lines producing large amounts of complemented replicating NS3-deficient replicon RNAs and infection with KUN virus to provide structural proteins also failed to detect any secreted virus-like particles containing packaged NS3-deficient replicon RNAs. These results have now firmly established the requirement of KUN NS3 protein translated in cis for genome packaging into virus particles.     

Defective interfering virus associated with A/Chicken/Pennsylvania/83 influenza virus.

The A/Chicken/Pennsylvania/1/83 influenza virus, isolated from a respiratory infection of chickens, is an avirulent H5N2 virus containing subgenomic RNAs (W.J. Bean, Y. Kawaoka, J.M. Wood, J.E. Pearson, and R.G. Webster, J. Virol. 54:151-160, 1985). We show here that defective interfering particles are present in this virus population. The virus had a low ratio of plaque-forming to hemagglutinating units and produced interference with standard virus multiplication in infectious center reduction assays. Subgenomic RNAs were identified as internally deleted polymerase RNAs. We have confirmed that this virus protects chickens from lethal H5N2 influenza virus infection. This protective effect appeared to be due to the inhibition of virulent virus multiplication. Additionally, subgenomic RNAs derived from polymerase RNAs were detected in 5 of 18 RNA preparations from animal influenza virus isolates. Therefore, defective interfering particles are sometimes produced in natural influenza virus infections, not just under laboratory conditions. These particles may be capable of suppressing the pathogenic effect of virulent virus infections in nature.     

The virion host shutoff protein (UL41) of herpes simplex virus 1 is an endoribonuclease with a substrate specificity similar to that of RNase A

Earlier, our laboratory reported that purified glutathione S-transferase-virion host shutoff (GST-vhs) protein exhibited endoribonucleolytic activity in in vitro assays using as substrates in vitro-transcribed regions of IEX-1 mRNA. Here, we report that studies of the cleavage patterns of synthetic RNA oligonucleotides defined the activity of GST-vhs as being similar to that of RNase A. Thus, GST-vhs cleaved the RNA at the 3' end of single-stranded cytidine and uridine residues. Since the GST-mvhs nuclease-defective mutant protein failed to cleave the synthetic RNAs, the results unambiguously attribute the activity to vhs.     

Synthesis of virus-specific polypeptides and genomic RNA during the replicative cycle of Pichinde virus

A stock of plaque-purified Pichinde virus, prepared under conditions designed to limit the amounts of defective interfering virus, was used to infect BHK cells. At daily intervals after infection, cells were examined for infectious and radiolabeled virus particle production and for the synthesis of virus-specific polypeptides. Quantitative comparisons were also made of the concentrations of genomic Pichinde virus L and S RNAs in the cytoplasm of infected cells on different days after infection. Our results showed that virus particle production, rates of protein synthesis, and the intracellular levels of viral genomic RNAs all increased and decreased with similar kinetics, and that this regulation was independent of the cell growth cycle. We were unable to relate these changes in viral macromolecule and virus production to the appearance of readily identifiable defective interfering particles. Our findings suggest that regulation of virus replication early during the replicative cycle of Pichinde virus may not be dependent upon the generation of defective interfering virus.     

Emerging themes in non-coding RNA quality control

Quality control pathways for non-coding RNAs such as tRNAs and rRNAs are widespread. In both prokaryotes and eukaryotes, poly(A) polymerases target aberrant non-coding RNAs for degradation. In yeast, a nuclear complex that includes the poly(A) polymerase Trf4p works together with the exosome in degrading a broad array of non-coding RNAs, several of which are aberrant. Yeast also have additional pathways for the degradation of defective RNAs and other pathways may exist in higher eukaryotes. One possibility is that cells recognize specific, still undiscovered, features common to misfolded RNAs; however, an alternative is that RNA quality control proteins interact with relatively general RNA structures, whereas correctly folded RNAs are sequestered by specific RNA-binding proteins and thus protected from degradation. Recently available structures of protein and ribonucleoprotein complexes involved in non-coding RNA quality control are providing a more detailed understanding of this process.     

Epstein-Barr virus gene expression in P3HR1-superinfected Raji cells.

The pattern of Epstein-Barr virus (EBV) RNAs expressed in Raji cells superinfected with P3HR1 EBV was examined. RNAs whose expression was of an immediate-early type (resistant to treatment of the cells with anisomycin) were identified. These RNAs, encoding the EBV reading frames BZLF1 and BRLF1, were probably expressed from defective virus within the P3HR1 preparation, and some of them were responsible for the induction of the EBV productive cycle in the Raji cells. The structures of the B95-8 RNAs equivalent to the anisomycin-resistant RNAs were determined. The RNA encoding the BZLF1 reading frame contained two splices which extended and modified the reading frame from that previously described.     

Different Classes of Ribonucleic Acid Isolated from Lymphocytic Choriomeningitis Virus

Purified preparations of lymphocytic choriomeningitis virus (LCM virus) contain three classes of RNA. The previously described 18s, 23s, 28s, and 31s RNAs, where the 23s and 31s RNAs are viral-specific, and the 18s and 28s RNAs probably are host RNAs incorporated in the virion. Now, 4s, 5s, and 5.5s RNAs can be isolated as well. Thus five RNAs which migrate by acrylamide gel electrophoresis as ribosome-derived RNA can be isolated from purified LCM virus. This observation further supports the reports that arenaviruses may contain ribosomes. The ribosome-derived RNA can be synthesized both before and after the virus infection. The viral 23s could be a hydrogen-bonded complex forming the 31s RNA, or it could be contained in defective interfering LCM virus particles; these possibilities are examined.     

Defective interfering passages of Sindbis virus: nature of the defective virion RNA.

Defective interfering particles of Sindbis virus contain 20S RNA identical to that found in BHK cells co-infected with standard and defective virions. We have characterized these RNAs by their oligonucleotide fingerprints. Most of the oligonucleotides were identical to those found in the mRNA (26S RNA) that codes for the virion structural proteins. Three oligonucleotides found in 20S RNA were absent from the 26S RNA pattern and may represent sequences from the 5' end of the virion RNA. Previous difficulties in describing the nature of the defective virion RNA were due to the aggregated state of the RNA. Nucleocapsids obtained from standard and defective virions were essentially the same size and had about the same density, suggesting that defective particles contain more than a single molecule of 20S RNA.     

Evolution of defective-interfering double-stranded RNAs of the yeast killer virus.

We have characterized by T1 fingerprint analysis several defective interfering (DI) double-stranded RNAs of the simple yeast virus ScV. A common sequence of about 0.5 to 0.6 kilobase pairs, including both 3' termini of the parental RNA, was present in each DI RNA. Several DI RNAs had novel T1 oligonucleotides not present in their parental RNA.     

Cleavage of mitochondria-like transfer RNAs expressed in Escherichia coli

Mitochondrial (mt) transfer RNAs (tRNAs) often harbor unusual structural features causing their secondary structure to differ from the conventional cloverleaf. tRNAs designed with such irregularities, termed mt-like tRNAs, are active in Escherichia coli as suppressors of reporter genes, although they display low steady-state levels. Characterization of fragments produced during mt-like tRNA processing in vitro and in vivo suggests that these RNAs are not fully processed at their 5' ends and are cleaved internally. These abnormal processing events may account for the low levels of mature mt-like RNAs in vivo and are most likely related to defective processing by RNase P.