Single Internal Deletion Research Articles

First Report of Coinfection of Satellite and Defective RNA of Bamboo mosaic virus in the Bamboo Host in California, U.S.A.

Bamboo mosaic potexvirus (BaMV) was first reported to infect bamboo in Brazil in 1977, and the virus was later identified in Taiwan, the United States, mainland China, India, Australia, and Indonesia (Abe et al. 2019; Elliott and Zettler 1997; M. T. Lin et al. 1977; N. S. Lin et al. 1993, 1995; W. W. Lin et al. 2015). Three leaf samples of 20 plants of Beechey bamboo (Bambusa beecheyana) showing mosaic and yellowish streak symptoms were collected in Palomar College Arboretum (San Marcos, CA) in 2019. A virus was isolated by cesium chloride density gradient (Lin and Chen 1991), and sodium dodecyl sulfate polyacrylamide gel electrophoresis of purified virions showed one major 30-kDa protein. Viral RNAs were extracted from virions, and cDNAs were synthesized using oligo(dT)15 and random primers with AMV and M-MLV RNase H− reverse transcriptase (Promega), respectively. The fragments of genomic and satellite RNA of BaMV were amplified by polymerase chain reaction (PCR) using Q5 high-fidelity DNA polymerase (NEB). The PCR primers and virus characterization are further described in supplementary materials, and amplicons were Sanger sequenced. The viral sequences of three BaMV-Yoshi clones (MT111578, MT111579, and MT111580) were aligned and compared using MAFFT and Phylo.io. The full genomic nucleotide sequence of BaMV-Yoshi shares 79.0 to 80.3% identity with other BaMV isolates in GenBank, whereas amino acids of BaMV-Yoshi ORF4 are only 71.2 to 78.9% identical to others. Phylogenetic analysis revealed that BaMV-Yoshi diverged from a common ancestor earlier than all Asian isolates and became a distinct group by itself (W. W. Lin et al. 2017; Wang et al. 2017). Six clones of BaMV satellite (satBaMV-Y; MT111572 to MT111577) RNAs show 93.2 to 95.1% identity to other satBaMV isolates in GenBank, and they contain noncanonical polyadenylation signal AAUAAU or AAGAAU instead of AAUAAA conserved in satBaMV isolates from Taiwan, China, or San Diego (CA). Northern blotting using ³²P-labeled BaMV 5′ untranslated region-specific riboprobe also detected 1.2- and 0.9-kb RNA species in virions, indicating that they are encapsidated defective RNAs (D RNAs). Two BaMV D RNAs (Y1 and Y2; MT113945 and MT113946) consist of 1,162 and 845 nucleotides in length with a single internal deletion of the helper virus genome, but their RNA recombination sites (Y1, 525/5,730; Y2, 185/5,709) are different from D RNAs generated by serial passages of BaMV-S in tobacco (Yeh et al. 1999). To our knowledge, this is the first report of full-length BaMV genome information outside Asia, and new recombination hotspots of BaMV D RNAs occurring in the bamboo host. This is also the first mixed infection of two BaMV subviral RNAs identified in nature. Contribution of mixed infection of satBaMV and D RNAs to replication competence and evolution of the helper virus warrants further investigation.

Corrected and Republished from: The COP9 Signalosome Interacts with and Regulates Interferon Regulatory Factor 5 Protein Stability.

The transcription factor interferon regulatory factor 5 (IRF5) exerts crucial functions in the regulation of host immunity against extracellular pathogens, DNA damage-induced apoptosis, death receptor signaling, and macrophage polarization. Tight regulation of IRF5 is thus warranted for an efficient response to extracellular stressors and for limiting autoimmune and inflammatory responses. Here we report that the COP9 signalosome (CSN), a general modulator of diverse cellular and developmental processes, associates constitutively with IRF5 and promotes its protein stability. The constitutive CSN/IRF5 interaction was identified using proteomics and confirmed by endogenous immunoprecipitations. The CSN/IRF5 interaction occurred on the carboxyl and amino termini of IRF5; a single internal deletion (Δ455-466) was found to significantly reduce IRF5 protein stability. CSN3 was identified as a direct interacting partner of IRF5, and knockdown of this subunit with small interfering RNAs (siRNAs) resulted in enhanced degradation. Degradation was further augmented by knockdown of CSN1 and CSN3 together. The ubiquitin E1 inhibitor UBEI-41 or the proteasome inhibitor MG132 prevented IRF5 degradation, supporting that its stability is regulated by the ubiquitin-proteasome system. Importantly, activation of IRF5 by the death receptor ligand tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) resulted in enhanced degradation via loss of the CSN/IRF5 interaction. This study defines the CSN as a new interacting partner of IRF5 that controls its stability.

The COP9 Signalosome Interacts with and Regulates Interferon Regulatory Factor 5 Protein Stability

The transcription factor interferon regulatory factor 5 (IRF5) exerts crucial functions in the regulation of host immunity against extracellular pathogens, DNA damage-induced apoptosis, death receptor signaling, and macrophage polarization. Tight regulation of IRF5 is thus warranted for an efficient response toward extracellular stressors and for limiting autoimmune and inflammatory responses. Here we report that the COP9 signalosome (CSN), a general modulator of diverse cellular and developmental processes, associates constitutively with IRF5 and promotes its protein stability. The constitutive CSN/IRF5 interaction was identified using proteomics and confirmed by endogenous immunoprecipitations. The CSN/IRF5 interaction occurred on the carboxyl and amino termini of IRF5; a single internal deletion from amino acids 455 to 466 (Δ455-466) was found to significantly reduce IRF5 protein stability. CSN subunit 3 (CSN3) was identified as a direct interacting partner of IRF5, and knockdown of this subunit with small interfering RNAs resulted in enhanced degradation. Degradation was further augmented by knockdown of CSN1 and CSN3 together. The ubiquitin E1 inhibitor UBEI-41 or the proteasome inhibitor MG132 prevented IRF5 degradation, supporting the idea that its stability is regulated by the ubiquitin-proteasome system. Importantly, activation of IRF5 by the death receptor ligand tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) resulted in enhanced degradation via loss of the CSN/IRF5 interaction. This study defines CSN to be a new interacting partner of IRF5 that controls its stability.

Analysis of external factors that affect formation and accumulation of D-RNAs in Bromovirus infections

Defective RNA molecules (D-RNAs) are being studied in several plant RNA virus groups. In the genus Bromovirus, D-RNAs have been described for Broad bean mottle virus (BBMV), they are formed by a single internal deletion in the RNA2 and have been generated de novo by serial passages. In contrast, in Brome mosaic virus (BMV) D-RNAs are generated by a single or double internal deletion in the RNA3 without serial passages in their hosts. In this work the external effects of the host and the growth temperature in the generation and accumulation of D-RNAs in BBMV, BMV and Cowpea chlorotic mottle virus (CCMV) are studied. The BBMV and BMV D-RNAs were generated and accumulated with or without serial passages of the viruses in different hosts and cultivars. Plants grown at 12, 16, 20 and 24 deg C in a growth chamber and in a greenhouse (22 +/- 5 deg C) generated D-RNAs after being inoculated with virusfree D-RNAs, with and without passages. The D-RNAs observed in BBMV and BMV presented some common characteristics: both were formed de novo after serial passages or without passages, their deletion borders had short repeated and palindrome sequences that favour recombination. In addition, growing inoculated plants at lower temperatures greatly facilitated the generation and accumulation of D-RNAs. The CCMV did not generate defective molecules in cowpea (Vigna unguiculata) and Nicotiana benthamiana plants after several serial passages or without passages. This is the first time that D-RNAs have been generated in BBMV without passages and BMV with serial passages.

A defective RNA associated with bamboo mosaic virus and the possible common mechanisms for RNA recombination in potexviruses.

A naturally occurring 1.1 kb RNA was isolated from purified virions of bamboo mosaic potexvirus isolate S (BaMV-S). This RNA is a defective RNA (D RNA) derived from a single internal deletion of the BaMV genome. A cDNA clone representing the complete nucleotide sequence of the BaMV-S D RNA was generated and its nucleotide sequence was determined. The BaMV D cDNA is 1015 nts in length [excluding the poly(A) tail] and consists of two regions corresponding to 867 nts of the 5' terminus and 148 nts of the 3' terminus of the BaMV genomic RNA. BaMV D cDNA contains a single open reading frame (ORF) encoding a putative 29.7 kDa protein comprised of a fusion of the first 258 amino acids of BaMV ORF 1 and the last 2 amino acids of coat protein. The coding capacity of D RNA was verified by in vitro translation of native BaMV-S D RNA and of 1.1 kb RNA transcribed in vitro from the full-length D cDNA. BaMV D RNA can be reproducibly generated by serial passages of BaMV-S in Nicotiana benthamiana and is the first D RNA in the potexvirus group shown to be generated de novo. Alignments of sequences surrounding the 5' and 3' junction borders of reported potexvirus D RNAs reveal a 65.2-84.6% sequence identity, suggesting that common mechanisms for viral RNA recombination are involved in the generation of potexvirus D RNAs.

Molecular Characterization of Tomato Spotted Wilt Virus Defective Interfering RNAs and Detection of Truncated L Proteins

Junction sites of 25 different defective interfering (DI) RNAs of tomato spotted wilt virus (TSWV) were characterized. The DI RNAs varied in size from 2.0 to 5.2 kilobases (kb) and contained a single internal deletion. The absence of DI RNAs smaller than 2 kb suggested a size constraint for the survival of TSWV DI RNAs. This hypothesis was reinforced by the finding of a dimeric DI RNA formed by two 1.6-long monomers linked head to tail. Three types of junction sites were found, one type originating from a simple deletion; the second type contained a few extra nucleotides of unknown origin; and the third type contained a stretch of three to five nucleotides, originally occurring at both sides of the deletion and of which one was deleted. In 19 of the 25 DI RNAs studied, the original reading frame was maintained, suggesting a selective preference of DI RNAs with translational potency. Truncated proteins encoded by these DI RNAs were indeed detected in the nucleocapsid preparations. Folding studies of the complete L RNA revealed that the calculated minimal energy of folding was at 16°C lower than at 23°, indicating a higher stability of this molecule at low temperatures. The results suggest an involvement of locally folded secondary structures in the process of deletion, rather than the requirement of certain sequences around the deletion point. The DI RNA generation in TSWV is essentially, as discussed, similar to the process of RNA recombination described in many viruses.

Characterization of Putative Defective Interfering (DI) A/WSN RNAs Isolated from the Lungs of Mice Protected from an Otherwise Lethal Respiratory Infection with Influenza Virus A/WSN (H1N1): A Subset of the Inoculum DI RNAs

Defective interfering (DI) influenza virus A/WSN (H1N1) grown in embryonated eggs protected adult mice from a lethal respiratory infection with A/WSN virus. Eighteen bands of putative DI RNA, ranging in size from about 230 to 1020 nt, were identified in this preparation by reverse transcription and polymerase chain reaction using a segment-specific 3′ primer and a segment-universal 5′ primer. Every virion RNA segment was represented by one to four bands of putative DI RNA. However, only five bands of putative DI RNA could be isolated, using the same conditions, from lungs of WSN-infected mice protected from death by co-inoculation with egg-grown DI WSN These five bands originated from PB1, PB2, PA, and M virion RNAs end were all about 350-450 nt in length. Four putative DI RNAs originating from PB1, PB2, and PA virion RNAs were sequenced, and three were identical (including deletion junctions and base substitutions) to putative DI RNAs from the inoculum. These data suggest that the mouse lung was highly selective for a subset of inoculum DI RNAs and that one or more of these DI RNAs was responsible for protection in vivo. All putative DI RNAs had a single internal deletion.

Defective interfering L RNA segments of tomato spotted wilt virus retain both virus genome termini and have extensive internal deletions.

Defective interfering (DI) RNA molecules derived from the genomic L RNA segment of tomato spotted wilt virus (TSWV) were generated during sequential passage of the virus at high multiplicity. Characterization of DI RNAs from four distinct isolates by Northern blot analysis and sequence determination revealed that both the 5' and 3' genomic termini were retained in these molecules. Each DI RNA contained a single internal deletion of approximately 60% to 80% of the L RNA segment. All DI RNAs studied maintain an open reading frame (ORF) which suggests that these defective molecules should be translatable by ribosomes. Detection of only defective molecules with ORFs indicates either that association with ribosomes or translation is a prerequisite for the selection and maintenance of replicating DI RNAs, or that the truncated proteins produced play a role in their selection or replication. Analysis of the junction sites in the DI RNAs showed that short nucleotide sequences are repeated, one at the release and another at the reinitiation point on the L RNA. One of these is lost during the generation of the DI molecules. The presence of repeated sequences at the junction sites seems to be unique for tospovirus DI L RNAs; they have not been described for other DI systems of either positive- or negative-strand RNA viruses. A model for TSWV DI RNA generation is proposed in which the viral polymerase can 'jump' across the internal sequences from one secondary structure to another containing the repeated sequences, during the replication of the viral complementary L RNA segment.

Characterization of Bunyamwera virus defective interfering particles.

In an attempt to isolate conditional lethal amber nonsense mutants of Bunyamwera virus, five variants were found which produced small plaques on BHK and mouse L cells. Characterization of these variants by Northern blotting showed that they synthesized defective (subgenomic) RNAs derived from the L RNA segment. No subgenomic M or S segment RNAs were detected. The defective L RNAs were shown to be packaged into virus particles, and four of five preparations caused interference with the multiplication of standard virus. When defective-containing preparations were mixed with standard virus and grown in doubly infected cells a reduction in titre of standard virus of up to 400-fold was observed. Hence these preparations most probably contained defective interfering (DI) particles. Novel DI-specific polypeptides were synthesized in DI virus-infected cells. These novel proteins could be precipitated by antisera raised against either the N or C terminus, or both, of the L protein. Nucleotide sequence analysis of cloned cDNA to prominent DI RNAs in three different defective virus preparations revealed that the DI RNA in each case had suffered a single internal deletion of the L segment while retaining the 5'- and 3'-terminal sequences. The extent of the deletion ranged between 72% and 77% of the L RNA segment. Our results suggest that these DI particles may have arisen during the attempted isolation of Bunyamwera virus amber mutants on mouse L cells, since defective/subgenomic RNAs derived from the L and M segments were readily generated in mouse L cells but not in BHK cells, following infection with wild-type virus.

Replication of adeno-associated virus DNA: Complementation of naturally occurring rep− mutants by a wild-type genome or an fori− mutant and correction of terminal palindrome deletions

When the entire adeno-associated virus (AAV) genome is inserted into a bacterial plasmid, infectious AAV genomes can be rescued and replicated when the recombinant AAV-plasmid DNA is transfected into human 293 cells together with helper adenovirus particles. We have taken advantage of this experimental system to analyze the effects of several classes of mutations on replication of AAV DNA. We obtained AAV mutants by molecular cloning in bacterial plasmids of naturally occurring AAV variant or defective-interfering genomes. Each of these mutants contains a single internal deletion of AAV coding sequences. Also, some of these mutant-AAV plasmids have additional deletions of one or both AAV terminal palindromes introduced during constructions in vitro. We show here that AAV mutants containing internal deletions were defective for replicative form DNA replication ( rep −) but could be complemented by intact wild-type AAV. This indicates that an AAV replication function, Rep, is required for normal AAV replication. Mutants in which both terminal palindromes were deleted ( ori −) were also replication defective but were not complementable by wild-type AAV. The cis-dominance of the ori − mutation shows that the replication origin is comprised in part of the terminal palindrome. Deletion of only one terminal palindrome was phenotypically wild-type and allowed rescue and replication of AAV genomes in which the deleted region was regenerated apparently by an intramolecular correction mechanism. One model for this correction mechanism is proposed. An AAV ori − mutant also complemented replication of AAV rep − mutants as efficiently as did wild-type AAV. These studies also revealed an unexpected additional property of the deletion mutants in that monomeric single-stranded DNA accumulated very inefficiently even though monomeric single-stranded DNA from the complementing wild-type AAV did accumulate.

Molecular cloning of adeno-associated virus variant genomes and generation of infectious virus by recombination in mammalian cells.

Continued passage of the human parvovirus, adeno-associated virus (AAV), at high multiplicity of infection in human cells results in the accumulation of AAV particles containing variant genomes. We have analyzed the structure of individual variant AAV genomes by molecular cloning in the Escherichia coli plasmid, pBR328. Each of the AAV inserts in six individual recombinant plasmids contained a single internal deletion but in contrast to a previous model, the locations of the deletions were nonrandom. The molecular cloning protocol also generated recombinant plasmids containing the entire AAV2 DNA sequence which yielded infectious AAV particles when transfected into human 293 cells in the presence of helper adenovirus using a DEAE-transfection procedure. Infectious AAV genomes were also generated by recombination when cells were jointly transfected with a mixture of plasmids containing two different mutant AAV genomes. The efficiency of this recombination appear to be influenced by the degree of homology between the mutant AAV genomes.

Defective interfering influenza RNAs of polymerase 3 gene contain single as well as multiple internal deletions

Defective interfering (DI) RNAs of influenza virus arise from polymerase genes by internal deletions. Utilizing the recombinant DNA cloning and sequencing techniques we have determined the nucleotide sequence of two DI RNAs of L clone of A/WSN/33 (L2a-7 and L2a-17) which are of polymerase 3 origin. L2a-7 DI RNA is 659 nucleotides long and contains a single internal deletion of 1682 nucleotides (nucleotide position 273 to 1954) of P3 gene. L2a-17 DI RNA (611 nucleotides long), on the other hand, contains two internal deletions: one of 1682 nucleotides at the identical position as that in L2a-7, the other 48 nucleotides at the nucleotide position 2032 to 2079 of P3 gene. Except for a few base mismatches the sequence of DI RNAs are identical to the corresponding portion of the P3 gene including the 5′ and the 3′ termini. Since these two DI RNAs contain one identical deletion but differ in the other deletion as well as in base mismatches, these two DI RNAs appear to originate from a progenitor DI RNA rather than independently from the progenitor P3 gene. The sequences around the deletion point do not reflect a consensus sequence for the origin of these deletions and suggest the role of multiple mechanisms in the generation and evolution of influenza DI RNAs.

Complete sequence analyses show that two defective interfering influenza viral RNAs contain a single internal deletion of a polymerase gene.

Defective interfering (DI) influenza viral RNAs arise by internal deletion of progenitor RNAs. By using recombinant DNA cloning and DNA sequence analysis techniques, we have deduced the complete sequence of two such RNAs (L2b and L3), both arising from the same polymerase (P1) gene of WSN influenza virus. We have also partially determined the sequence of the P1 polymerase gene, including the sequence at the point of deletion and the flanking regions. Our sequence study shows the following. (i) Both L2b and L3 arise by a simple deletion in the P1 gene. (ii) L2b and L3 are 683 and 441 nucleotides long, respectively. (iii) The first 413 and 244 nucleotides of the 5' ends of L2b and L3, respectively, are identical to those of the 5' end of the P1 gene. (iv) The last 270 nucleotides of L2b and 197 nucleotides of L3 are the same as those of the 3' end of the P1 gene. (v) The entire sequence of L3 is present in the sequence of L2b. (vi) Both the 5' and the 3' termini, including the transcription stop and poly(A) addition signals of the progenitor P1 gene, are present in both L2b and L3. (vii) The sequences at the deletion point and the flanking region of the P1 gene do not resemble the consensus splicing sequence of spliced mRNA suggesting that a replicational event rather than splicing is involved in the formation of influenza defective interfering RNAs.