Mutation analysis of cis-elements in the 3'- and 5'-untranslated regions of satellite tobacco necrosis virus strain C RNA.

The putative, 3'-terminal stem-loop structure in satellite tobacco necrosis virus strain C (STNV-C) RNA constitutes an essential cis-acting structure for the promotion of negative-strand RNA synthesis and a single-stranded tail is also important. The putative, 5'-terminal stem-loop structure in STNV-C RNA is not essential for productive, plus-strand RNA accumulation but is required for optimal accumulation. Residues 2 and 3 are the minimal cis-acting sequences required for RNA synthesis. The RNA of chimeric mutants, which exchanged 3'- and 5'-untranslated regions between STNV-C and helper tobacco necrosis virus strain D RNAs, accumulated in protoplasts, implying similar replication mechanisms for both RNAs.     

3'-Terminal RNA secondary structures are important for accumulation of tomato bushy stunt virus DI RNAs

The plus-strand RNA genome of tomato bushy stunt virus (TBSV) contains a 351-nucleotide (nt)-long 3'-untranslated region. We investigated the role of the 3'-proximal 130 nt of this sequence in viral RNA accumulation within the context of a TBSV defective interfering (DI) RNA. Sequence comparisons between different tombusviruses revealed that the 3' portion of the 130-nt sequence is highly conserved and deletion analysis confirmed that this segment is required for accumulation of DI RNAs in protoplasts. Computer-aided sequence analysis and in vitro solution structure probing indicated that the conserved sequence consists of three stem-loop (SL) structures (5'-SL3-SL2-SL1-3'). The existence of SLs 1 and 3 was also supported by comparative secondary structure analysis of sequenced tombusvirus genomes. Formation of the stem regions in all three SLs was found to be very important, and modification of the terminal loop sequences of SL1 and SL2, but not SL3, decreased DI RNA accumulation in vivo. For SL3, alterations to an internal loop resulted in significantly reduced DI RNA levels. Collectively, these data indicate that all three SLs are functionally relevant and contribute substantially to DI RNA accumulation. In addition, secondary structure analysis of other tombusvirus replicons and related virus genera revealed that a TBSV satellite RNA and members of the closely related genus Aureusvirus (family Tombusviridae) share fundamental elements of this general structural arrangement. Thus, this secondary structure model appears to extend beyond tombusvirus genomes. These conserved 3'-terminal RNA elements likely function in vivo by promoting and/or regulating minus-strand synthesis.     

The primary nucleotide sequence of the bovine leukemia virus RNA packaging signal can influence efficient RNA packaging and virus replication

Two RNA stem-loop structures in the gag gene have been implicated as representing the primary encapsidation (packaging) signal for bovine leukemia virus (BLV), a member of the Delta retrovirus of the Retroviridae. In this study, we conducted an analysis of these RNA structures, stem loop 1 (SL1) and stem loop 2 (SL2), to determine if both the loop and the stem nucleotide bases are important for RNA encapsidation. We have found that the primary sequence of the unpaired bases located in the loop regions of both SL1 and SL2 are important for efficient RNA encapsidation and virus replication. The primary sequence of the bases that form the stems for both SL1 and SL2 was observed to aid in efficient encapsidation and replication. We also observed that the order of SL1 and SL2 is important for RNA encapsidation and virus replication efficiency. A viral RNA with two copies of either SL1 or SL2 was found to replicate and package RNA as efficiently as a viral RNA with only one copy of SL1 or SL2. This provides evidence that SL1 and SL2 are not functionally equivalent. Sequences from human T cell leukemia virus type 1 (HTLV-1) that are located in the same region of HTLV-1 as the SL1 and SL2 of BLV were used to replace the BLV SL1, SL2, or both in a BLV RNA. These BLV RNAs were still encapsidated and replicated, suggesting that these sequences may function as an encapsidation signal in HTLV-1. The chimeric RNAs did not replicate as well as the parental, indicating that the primary nucleotide sequence along with the secondary and tertiary structure of the RNA plays a role in efficient RNA encapsidation and replication.     

The structure and function of a cis-acting element located upstream of the IRES that influences Coxsackievirus B3 RNA translation

We have investigated the importance of a conserved hexa-nucleotide stretch in the apical loop within stem-loop C (SL C, nt 104-180), upstream of the ribosome landing site, on CVB3 IRES function. The deletion or substitution mutation at this apical loop resulted in significant decrease in IRES activity. Both the mutant IRES RNAs failed to interact with certain trans-acting factors. Furthermore, expression of CVB3 2A protease significantly enhanced IRES activity of the wild type, but the effect was not so pronounced on the mutant IRESs. It is possible that the mutant RNAs were unable to interact with some trans-acting factors critical for enhanced IRES function. Interestingly, the local structure of the IRES RNA was not significantly altered due to substitution mutation. Taken together, it appears that the SL C/c apical loop is critical for CVB3 IRES function.     

Cis-acting element (SL1) of Potato virus X controls viral movement by interacting with the NbMPB2Cb and viral proteins

A number of candidate tobacco proteins that bind to cis-acting elements (SL1 RNAs) of Potato virus X (PVX) have been identified in previous studies. We further characterized TMV-MP30 binding protein 2C (MPB2C) homologous protein. We isolated NbMPB2Cb from Nicotiana benthamiana and confirmed the interaction of NbMPB2Cb with SL1 RNAs in vitro. The mRNA level of NbMPB2Cb was increased upon infection by PVX and Tobacco mosaic virus. The movement of PVX was reduced by overexpression of NbMPB2Cb and increased by silenced of NbMPB2Cb. In contrast, PVX RNA accumulation was not significantly altered in protoplasts. Protein-protein interaction assays showed that NbMPB2Cb interacts with PVX movement-associated proteins. PVX infection altered the subcellular localization of NbMPB2Cb from microtubules to endoplasmic reticulum. These data suggest that the NbMPB2Cb negatively affects PVX movement by interacting with SL1 RNAs and movement-associated proteins of PVX and by re-localizing in response to PVX infection.     

Structure and sequence variation of the trypanosome spliced leader transcript

We have assessed the potential of using the spliced leader (SL) or mini-exon gene as a marker for molecular phylogenetic analysis of genus Trypanosoma. A total of 27 trypanosome sequences were compared, 18 of these being newly reported. In contrast to genus Leishmania, we found the non-transcribed spacer region of the SL locus in trypanosomes to be far too variable for informative comparison of all but the most closely related species. At the other extreme, the short (39 nt) SL exon was usually completely conserved and hence uninformative. The SL RNA showed variation in both length (97-152 nt) and sequence among different trypanosome species, with most variation occurring in stem-loop II. Consequently, this region could not be aligned with confidence in multiple sequence alignment, severely reducing the number of phylogenetically informative nucleotide positions. In computer simulation, most of the SL RNAs readily folded into the 3 stem-loop secondary structure predicted previously, but again stem-loop II was highly variable. No obvious correlation could be seen between the length of this stem-loop and trypanosome biology. We conclude that the SL repeat is not an informative phylogenetic marker for long range evolutionary studies of genus Trypanosoma.     

Altered balance of the synthesis of plus- and minus-strand RNAs induced by RNAs 1 and 2 of alfalfa mosaic virus in the absence of RNA 3

The synthesis of viral plus-strand and minus-strand RNAs in cowpea protoplasts inoculated with mixtures of alfalfa mosaic virus nucleoproteins (B, M, Tb, and Ta) was analyzed by the Northern blotting technique. A mixture of B, M, and Tb induced the synthesis of plus-strand RNAs 1, 2, 3, and 4 and three minus-strand RNAs corresponding to RNAs 1, 2, and 3, respectively. Compared to this complete infection, a mixture of B and M induced the synthesis of a reduced amount of plus-strand RNAs 1 and 2 and a greatly enhanced amount of minus-strand RNAs 1 and 2. No detectable viral RNA synthesis was induced by mixtures of B and Tb or M and Tb. It is concluded that expression of genomic RNAs 1 and 2 results in the formation of a replicase activity that produces roughly equal amounts of viral plus- and minus-strand RNAs and that an RNA 3-encoded product, possibly the coat protein, is responsible for a switch to an asymmetric production of viral plus-strand RNA. The observation that no minus-strand corresponding to the subgenomic RNA 4 is produced suggests that recognition of the genome segments by the viral replicase involves sequences outside the 3'-terminal regions that are homologous to RNA 4.     

Structural and mutational analyses of cis-acting sequences in the 5'-untranslated region of satellite RNA of bamboo mosaic potexvirus

The satellite RNA of Bamboo mosaic virus (satBaMV) contains on open reading frame for a 20-kDa protein that is flanked by a 5'-untranslated region (UTR) of 159 nucleotides (nt) and a 3'-UTR of 129 nt. A secondary structure was predicted for the 5'-UTR of satBaMV RNA, which folds into a large stem-loop (LSL) and a small stem-loop. Enzymatic probing confirmed the existence of LSL (nt 8-138) in the 5'-UTR. The essential cis-acting sequences in the 5'-UTR required for satBaMV RNA replication were determined by deletion and substitution mutagenesis. Their replication efficiencies were analyzed in Nicotiana benthamiana protoplasts and Chenopodium quinoa plants coinoculated with helper BaMV RNA. All deletion mutants abolished the replication of satBaMV RNA, whereas mutations introduced in most of the loop regions and stems showed either no replication or a decreased replication efficiency. Mutations that affected the positive-strand satBaMV RNA accumulation also affected the accumulation of negative-strand RNA; however, the accumulation of genomic and subgenomic RNAs of BaMV were not affected. Moreover, covariation analyses of natural satBaMV variants provide substantial evidence that the secondary structure in the 5'-UTR of satBaMV is necessary for efficient replication.     

Episomal expression of a hammerhead ribozyme directed against plum pox virus

Two related antisense RNAs directed against plum pox virus (PPV) were expressed episomally in Nicotiana clevelandii by infection with recombinant potato virus X (PVX). One recombinant PVX expressed an ordinary PPV antisense RNA of about 400 nucleotides, while the other expressed a related antisense RNA that carried the catalytic domain of a hammerhead ribozyme. Inoculation with the latter recombinant PVX resulted in the accumulation of ribozyme RNA that was catalytically active when tested in vitro with a PPV substrate RNA. Plants that had been inoculated with recombinant PVX viruses, expressing either PPV-directed antisense or ribozyme sequences or GUS RNA as a control, were challenged with PPV by a sequential second inoculation. In plants that expressed PPV antisense sequences, the appearance of PPV disease symptoms was delayed for 3-5 days. Quantification of PPV 1 week after inoculation showed that the protective effect by the episomally expressed catalytic antisense RNA was stronger than that of the ordinary antisense RNA. However, eventually all plants tested accumulated comparable titers of PPV.     

Cellular protein binds to sequences near the 5' terminus of potato virus X RNA that are important for virus replication

The sequences in the 5'-nontranslated region (NTR) of Potato virus X (PVX) genomic RNA were previously reported to contain several regulatory elements that are required for genomic and subgenomic RNA accumulation. To investigate whether cellular proteins bind to these elements, we conducted electrophoretic mobility shift assays (EMSA) with protoplast protein extracts and RNA sequences from the PVX 5'-NTR. These analyses showed that the 5' region of PVX positive-strand RNA formed complexes with cellular proteins. UV cross-linking studies of complexes formed with various deletions of the PVX RNA indicated that a 54-kDa cellular protein (p54) was bound to nt 1-46 at the 5' terminus of PVX RNA. Site-directed mutations introduced within this 46-nt region further indicated that an ACCA sequence element located at nt 10-13 was important for optimal binding. In addition, mutations that decreased the affinity of the template RNA for the cellular factor decreased PVX plus-strand RNA accumulation in protoplasts. These studies suggest that the p54 may function in PVX RNA replication by binding to the 5' terminus of the viral genomic RNA.     

Three discontinuous loop nucleotides in the 3′ terminal stem-loop are required for Red clover necrotic mosaic virus RNA-2 replication

The genome of Red clover necrotic mosaic virus (RCNMV) consists of positive-sense, single-stranded RNA-1 and RNA-2. The 29 nucleotides at the 3′ termini of both RNAs are nearly identical and are predicted to form a stable stem-loop (SL) structure, which is required for RCNMV RNA replication. Here we performed a systematic mutagenesis of the RNA-2 3′ SL to identify the nucleotides critical for replication. Infectivity and RNA replication assays indicated that the secondary structure of the 3′ SL and its loop sequence UAUAA were required for RNA replication. Single-nucleotide substitution analyses of the loop further pinpointed three discontinuous nucleotides (L1U, L2A, and L4A) that were vital for RNA replication. A 3-D model of the 3′ SL predicted the existence of a pocket formed by these three nucleotides that could be involved in RNA-protein interaction. The functional groups of the bases participating in this interaction at these positions are discussed.     

Tobacco mosaic virus replicase-mediated cross-protection: contributions of RNA and protein-derived mechanisms

Specific sequences of the tobacco mosaic virus (TMV) RNA-dependent RNA-polymerase (RdRp) gene were investigated for their ability to confer cross-protection. Nine overlapping segments ranging from 713 to 1070 nucleotides in length and covering the methyltransferase, helicase, and polymerase (POL) domains of the TMV RdRp open reading frame were systemically expressed in Nicotiana benthamiana using a potato X virus (PVX) vector [Chapman, S., Kavanagh, T., and Baulcombe, D. C. (1992). Plant J., 1, 549-557]. PVX-infected plants were subsequently challenge inoculated with 10 microg of wild-type TMV and monitored for TMV accumulation. Mock inoculated plants and plants preinfected with the unmodified PVX vector rapidly accumulated high levels of challenge virus. In contrast, plants preinfected with PVX vectors expressing segments of the TMV RdRp open reading frame displayed either high or low levels of protection. High protection levels were observed for PVX constructs expressing segments of the TMV POL domain, whereas low protection levels were observed for PVX constructs expressing segments covering the methyltransferase and helicase domains. Frameshift mutations that blocked protein expression from RdRp segments disrupted only the high levels of protection derived from POL segments and not the low levels derived from the other segments. However, all RdRp segments conferred similarly high levels of protection against a TMV construct with restricted local movement. Thus both RNA and protein sequences in conjunction with the speed of the infecting challenge virus can affect the protection derived from the TMV RdRp gene.     

The majority of the nucleotides in the top loop of the genomic 3' terminal stem loop structure are cis-acting in a West Nile virus infectious clone

The flavivirus genome RNA terminates with a conserved 3' stem loop (SL) structure that was shown to be essential for virus replication. A stretch of conserved nts is located in the top loop (TL) of this structure. Mutation of the TL nts (5' ACAGUGC 3') in a WNV infectious clone indicated that 3 of the 7 TL nts (5' ACAGUGC 3') are critical for virus replication. Mutation of 3 of the other nts reduced the efficiency of virus replication. The four 5' TL nts are conserved in both mosquito- and tick-borne flavivirus genomes, while the TL 3' C is conserved in mosquito-borne viruses. The conservation of two or three G-C base pairs in the TL flanking sequences suggests that a stable stem is necessary for precise presentation of the TL sequence. The TL may participate in RNA as well as protein interactions.     

Analysis ofCis-Acting Elements in the 5′ Leader Sequence of Alfalfa Mosaic Virus RNA 3

The leader sequence of RNA 3 of the Leiden isolate of alfalfa mosaic virus strain 425 consists of 345 nucleotides (nt) and contains four putative stem–loop structures each with a motif in the loop that resembles the internal control region 2 (ICR2) of tRNA genes. The sequence of the 5′ terminal 112 nt of this leader contains one of these stem–loop structures and is sufficient for a reduced accumulation of RNA 3 in protoplasts and a delayed accumulation in plants (E. A. G. van der Vossenet al., Nucleic Acids Res.21, 1361–1367 (1993). A number of mutations were made in this 112-nt leader sequence to investigate its role in RNA 3 accumulation. Deletion of nucleotides 23–43, 44–90, or 55–112 and inversion or duplication of nucleotides 44–90 all abolished RNA 3 accumulation. Similarly, two base substitutions in the ICR2 motif (nucleotides 60–77) abolished RNA 3 accumulation. Mutations in the stem sections of the putative stem–loop structure had various effects on RNA 3 accumulation and supported the notion that this structure is important for plus-strand promoter activity.     

Polypyrimidine tract-binding protein interacts with the 3' stem-loop region of Japanese encephalitis virus negative-strand RNA

The 3' stem-loop (SL) region of positive- and negative-strand RNA of Japanese encephalitis virus (JEV), like that of other flaviviruses, may function as cis-acting signals during RNA replication. In order to demonstrate the specific interaction between JEV 3' SL regions and BHK-21 cellular proteins, we performed gel mobility shift assay and UV-induced cross-linking assay. We identified seven cellular proteins of 110, 87, 67, 45, 38, 34, and 30 kDa that bound to the (+)3' SL RNA, and eight cellular proteins of 138, 110, 87, 67, 55, 52, 38, and 34 kDa that bound to the (-)3' SL RNA. The 55 kDa protein was identified as the polypyrimidine tract-binding (PTB) protein by immunoprecipitation assay. These data suggest that the 3' SL regions of JEV-RNA of both polarities may act as recruiting signals for the components of viral replication complexes including host cell-derived PTB protein.