Results obtained from a preliminary study suggested that rearranged RNA segments might overcome this limitation. Indeed, we first characterized human RV clones containing rearranged segment 7, 11, or both (8) and then compared the fitness levels of rearranged versus WT viruses. We found that viruses with rearranged segment 7 or 11 replicated less well than or equally to WT viruses, as judged by viral growth curve experiments. Surprisingly, in competition growth experiments, rearranged segment 7 or 11 was always selected into the viral progenies, even when mixed infections were performed with a ratio of 1 rearranged to 1,000 WT viruses (4). The absence of a growth advantage conferred on the virus by rearranged seg- ments, combined with their preferential segregation into the viral progenies, suggested that rearranged RNA segments might be packaged preferentially. These observations are in agreement with results of earlier studies showing that rear- ranged segment 5 or 11 segregated preferentially into viral progenies issued from mixed infections with WT virus (10, 19). We developed a reverse genetics system for RV on the basis of the preferential packaging of rearranged RNAs. We report here the rescue of recombinant viruses carrying cDNA-derived rearranged segment 7 (either unmodified or containing silent mutations introduced by site-directed mutagenesis to generate restriction enzyme sites as markers), with no selection pressure other than serial passage in cell culture. We also report the rescue of a recombinant virus expressing a double-sized recom- binant NSP3 protein encoded by an in vitro-modified cDNA- derived rearranged segment 7, showing for the first time that an in vitro-engineered gene encoding a modified nonstructural protein can be introduced into an infectious RV.
The role of the variable portion of the noncoding regions (NCRs) of the three Bunyaviridae RNA segments (L, M, S) in transcription, replication, and packaging was studied using the recently developed plasmid-driven RNA polymerase I minigenome system for Uukuniemi (UUK) virus, genus Phlebovirus (11), as a model. Comparison of the different segments showed that all NCRs were sufficient to mediate transcription/replication of a minigenome but demonstrated decreased promoter strength in the order M > L > S. Chimeric minige- nomes with flanking NCRs from different genome segments revealed that the number of total base pairs within the inverted, partially complementary ends was important for transcription and replication. Point mutations increasing the base-pairing potential produced increased reporter expression, indicating that complementarity between the 5 ⴕ and 3 ⴕ ends is crucial for promoter activity. The role of the intergenic region (IGR) located between the two open reading frames of the ambisense UUK virus S segment was analyzed by inserting this sequence element downstream of the reporter genes. The presence of the IGR was found to enhance reporter expression, demonstrating that efficient transcription termination, regulated by the IGR, is important for optimal minigenome mRNA translation. Finally, genome packaging efficacy varied for different NCRs and was strongest for L followed by M and S. Strong reporter gene activity was still observed after seven consecutive cell culture passages, indicating a selective rather than random genome-packaging mechanism. In summary, our results demonstrate that the NCRs from all three segments contain the necessary signals to initiate transcription and replication as well as packaging. Based on promoter strength, M-segment NCRs may be the preferred choice for the development of reverse genetics and minigenome rescue systems for bunyaviruses.
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To extend this analysis still further, we then tested the effects of mutating putative packaging signals in the context of the authentic PA, PB1, or PB2 segment, focusing only on sites where synonymous mutations could be used. As indicated in Table 1, we designed one additional mutant form of each of these vRNAs, in each case altering 5 to 8 bases to produce silent mutations in two to four consecutive codons. These silent mutations (designated PA-m15s, PB1-m12s, and PB2-m6s) roughly corresponded in location to the highly deleterious PA- m15, PB1-m12-m13, and PB2-m6 mutations described above and so targeted sites that our initial reporter-based studies had strongly implicated in packaging. When introduced into their respective reporters, each of these silent mutations reduced packaging to 2% or less of the wild-type efficiency (Table 1). We next introduced these same mutations into vectors encod- ing the authentic, full-length vRNAs and then used the 17- plasmid transfection system to create viruses carrying each mutant vRNA in place of its wild-type counterpart. When viral titers were determined for the supernatants at 48 h posttrans- fection, each of the mutants was found to impair virus produc- tion, yielding titers 4- to 67-fold lower than the wild-type level FIG. 2. Interstrain nucleotide variation within portions of the PA and PB1 packaging regions. Full-length sequences of PA and PB1 segments from diverse influenza A virus isolates (including all subtypes from various origins) were obtained from the NCBI influenza virus resource and aligned. Portions of the 5 ⬘ packaging regions from the PA vRNA (residues 2134 to 2183) and PB1 vRNA (residues 2255 to 2304) of strain WSN are shown, and the percentage of variation observed at each position in the alignment is indicated above, based on 609 PA sequences and 586 PB1 sequences. The locations of mutations tested in this study are indicated below, with those that reduced packaging efficiency to 2% or less indicated in red.
Here the kinetics of multipartite virus infection was re-exam- ined by using tripartite AMV and Nicotiana tabacum plants as a model system. A study design that allows a rigorous quantitative analysis of whether the genome segment number predicts the multipartite virus dose response and accounts for deviations from IAH model predictions was used. Three plants were used for dose- response experiments, Nicotiana tabacum L. cv. Samsun (referred to here as wild-type plants), a transgenic plant derived from N. tabacum cv. Samsun that expresses AMV genomic segment RNA2 under the control of the Cauliflower mosaic virus 35S promoter (P2 plants), and a transgenic plant expressing AMV genomic seg- ments RNA1 and RNA2 (P12 plants) (15). Note that uncoated AMV RNA segments can achieve cell-to-cell movement (16–18), whereas for systemic movement, the formation of virus particles, each again encapsidating a single RNA segment, is required (17). It has already been shown that the P2 and P12 transgenic plants can support full-blown AMV systemic infection in the absence of the expressed segment in the inoculum (15), and it was anticipated that the expressed RNA segments could therefore complement virus particles to generate primary or systemic infection. Here, we attempted to alter the infection kinetics of AMV from those of a tripartite virus to those of a bipartite or monopartite virus by inoculating AMV into transgenic plants expressing one or two viral genome segments. These results show that the underlying mechanisms are more complex than previously thought and sug- gest reasons why multipartition has evolved.
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The predominant rotavirus electropherotypes (e-types) during 17 epidemic seasons (1980 through 1997) in Finland were established, and representative virus isolates were studied by nucleotide sequencing and phylo- genetic analysis. The virus isolates were either PG1 or PG4 types. The G1 and G4 strains formed one G1 lineage (VP7-G1-1) and one G4 lineage, respectively. Otherwise, they belonged to two P lineages (VP4-P- 1 and -2) unrelated to their G types. Phylogenetic analysis of partial sequences of all 11 RNA segments obtained from the strains also revealed genetic diversity among gene segments other than those defining P and G types. With the exception of segments 1, 3, and 10, the sequences of the other segments could be assigned to 2 to 4 different genetic clusters. The results of this study suggest that, in addition to the RNA segments encoding VP4 and VP7, the other RNA segments may segregate independently as well. In total, the 9 predominant e-types represented 7 different RNA segment combinations when the phylogenetic clusters of their 11 genes were determined. The extensive genetic diversity and number of e-types among rotaviruses are best explained by frequent genetic reassortment.
The genome of influenza A viruses comprises eight negative-strand RNA segments. Although all eight segments must be present in cells for efficient viral replication, the mechanism(s) by which these viral RNA (vRNA) segments are incorporated into virions is not fully understood. We recently found that sequences at both ends of the coding regions of the HA, NA, and NS vRNA segments of A/WSN/33 play important roles in the incorporation of these vRNAs into virions. In order to similarly identify the regions of the PB2, PB1, and PA vRNAs of this strain that are critical for their incorporation, we generated a series of mutant vRNAs that possessed the green fluorescent protein gene flanked by portions of the coding and noncoding regions of the respective segments. For all three polymerase segments, deletions at the ends of their coding regions decreased their virion incorporation efficiencies. More importantly, these regions not only affected the incorporation of the segment in which they reside, but were also important for the incorporation of other segments. This effect was most prominent with the PB2 vRNA. These findings suggest a hierarchy among vRNA segments for virion incorporation and may imply intersegment association of vRNAs during virus assembly.
In contrast to RVFV, the terminal ⬃ 20-nt sequences of influ- enza A virus (FLUAV) RNA genome segments serve as minimal RNA replication signals (22), but the NCRs and the coding regions are both required for efficient genome packaging (5, 6, 13, 14, 16, 17, 20, 21, 27). Studies have shown that all eight RNA segments are required for efficient FLUAV RNA packaging (6, 18), which is similar, in principle, to the observations that efficient RVFV L RNA packaging occurs in the presence of M and S RNAs (26) and that a region within the 5= NCR of M RNA is necessary for the copackaging of M and S RNAs as well as L RNA packaging into VLPs (26). Because the mechanisms of FLUAV RNA genome co- packaging are largely unknown and the number of RNA genome segments packaged into RVFV is much lower than that packaged into FLUAV, these studies using RVFV could serve as a more FIG 3 Influence of the NCRs of L, M, and S RNAs on RNA packaging. (A) Schematic diagrams of LNCR-rLuc, MNCR-rLuc, and SNCR-rLuc, which carried the rLuc gene inserted between the 3= and 5= NCRs of L, M, and S RNAs, respectively. (B) Cell extracts and purified VLPs were prepared using the methods described for Fig. 1B. Western blot analysis using anti-RVFV mouse antibody shows the accumulation of Gn/Gc and N proteins in cells (IC) and in VLP (VLP) samples. (C) Intracellular RNAs (IC) and RNAs in VLPs (VLP) were subjected to Northern blot analysis using rLuc probe 2 that hybridizes with nt 1 to 702 of the rLuc gene and detects viral-sense RNAs of LNCR-rLuc, MNCR-rLuc, and SNCR-rLuc. The packaging efficiencies of the three RNAs were determined as described for Fig. 2 and reported as approximate percentages of the packaging efficiency of LNCR-rLuc RNA. Representative data from three independent experiments are shown.
Using a unique in vitro assembly of the BTV capsid, it was demonstrated that ssRNA copies of all 10 genomic segments were needed to generate an infectious viral core and that in absence of certain segments ssRNAs were not packaged [7, 24]. It was hypothesized that during genome assembly, nascent ssRNAs most likely interacted with each other through RNA-RNA interactions, thereby sorting the correct set and number of RNA segments for encapsidation. Moreover, studies carried out on the dsRNA synthesis of single-shelled insect cytoplasmic polyhedrosis virus (CPV) has shown that the ten segmented dsRNAs in CPV are organized with ten transcriptional enzyme complex of RNA-dependent RNA polymerase (RdRP) and NTPase VP4 (TEC) in a specific, non-symmetric manner, with each dsRNA segment attached directly to a TEC while in while in cypoviruses (NCPV and TCPV) each RdRP is anchored at the inner surface of the capsid and surrounded by multiple layers of dsRNA [25, 26] . In this study, we have shown evidences of intersegment interactions between medium and small size class initiation of stable RNA complexes. This is consistent with our previous findings [6, 7] and similar studies by others on other segmented viruses where some segments appeared to have preferential binding affinity to enhance formation of the RNA network [8, 27, 28].
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Thogoto virus (THOV) is the prototype tick-transmitted or- thomyxovirus (18). The genome of THOV consists of six sin- gle-stranded RNA segments of negative polarity that are en- capsidated by the viral nucleoprotein (NP) and associate with the viral RNA polymerase complex to form ribonucleoprotein complexes (vRNPs) (4, 17). Each individual segment codes for a single structural protein: the three subunits of the viral RNA polymerase complex (PB2, PB1, and PA) (11, 25), the viral surface glycoprotein (GP) (12), the NP (26), and the matrix protein (M) (10). Members of the genus Thogotovirus are structurally and genetically similar to the influenza viruses but are unique in their ability to infect mammalian as well as tick cells (15). The host change between vertebrates and arthropo- des requires specific adaptations to allow the virus to replicate in both cell types. Accordingly, THOV has unique features like the single GP that has no similarities to the influenza virus glycoproteins but has similarity with the surface glycoproteins of baculoviruses (12). In addition, THOV has a unique cap- snatching mechanism, using only the cap structure and one additional nucleotide from cellular mRNAs to initiate viral transcription (2, 26). Moreover, the genome of THOV does not encode additional proteins, like the NS2/NEP or the NS1 of influenza A virus (FLUAV). NS2/NEP is essential for the export of the newly synthesized vRNPs out of the nucleus (13, 16). The nonstructural protein NS1 has been shown to sup- press interferon production and the interferon-mediated anti- viral response of the infected host cell, most likely by seques- tration of double-stranded RNA molecules (7, 23). Since THOV lacks analogous proteins, it depends on the basic set of its six structural proteins to perform nuclear export of the vRNPs and to deal with the interferon-dependent suppression of viral replication. Specific manipulations of the THOV ge- nome should allow to assign such functions to defined viral genes.
La Crosse virus (LACV) belongs to the Bunyaviridae family and causes severe encephalitis in children. It has a negative-sense RNA genome which consists of the three segments L, M, and S. We successfully rescued LACV by transfection of just three plasmids, using a system which was previously established for Bunyamwera virus (Lowen et al., Virology 330:493–500, 2004). These cDNA plasmids represent the three viral RNA segments in the antigenomic orientation, transcribed intracellularly by the T7 RNA polymerase and with the 3 ⴕ ends trimmed by the hepatitis delta virus ribozyme. As has been shown for Bunyamwera virus, the antigenomic plasmids could serve both as donors for the antigenomic RNA and as support plasmids to provide small amounts of viral proteins for RNA encapsidation and particle formation. In contrast to other rescue systems, however, transfection of additional support plasmids completely abrogated the rescue, indicating that LACV is highly sensitive to overexpression of viral proteins. The BSR-T7/5 cell line, which constitutively expresses T7 RNA polymerase, allowed efficient rescue of LACV, generating approximately 10 8
phages such as MS2 and Q β ; picornaviruses, such as poliovirus; plant RNA vi- ruses, such as Tobacco mosaic virus; and others, such as flaviviruses, togaviruses, and coronaviruses. There are a few differences in the amino acid sequences among their respective RNA-replicases. This suggests that on the early Earth, at different locations, a variety of similar primitive “chicken” may have appeared which could have independently produced RNA-replicases having the same functions but slightly different amino acid sequences. Being isolated from each other for a long time may have caused the viruses in different places to develop into the different modern viruses, such as bacteriophages, protozoan viruses, plant viruses, and animal viruses, because of different hosts in different loca- tions.
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In our experience all stocks of influenza virus contain some level of a complex population of DI viruses, which makes examination of the activity of any individual transfected DI RNA in a cell culture infection model technically impossible. In such experiments it is neces- sary to use a high level of infectious virus which results in a prominent signal from the naturally occurring DI RNAs that obscures the signal from the transfected plas- mid expressing 1/244 DI RNA (unpublished data). We have therefore used an established in vitro RNP reconsti- tution approach [53–55] to examine the ability of DI RNAs to interfere with the expression of green fluores- cent protein (GFP) from a Seg 1-GFP construct. Here the coding region of GFP is inserted in-frame into a de- leted influenza segment 1 RNA (Fig. 1a). This system also distinguishes effects of DI RNA on RNA synthesis from any effect on RNA packaging as plasmids encoding key structural proteins (HA, NA, M1 and M2) are not included . The interfering ability of 1/244 and 1/244 AUG KO RNAs were determined by transfecting 293T cells with the relevant plasmids and plasmids expressing Seg 1-GFP and PB1, PB2, PA and NP proteins. 1/244 and 1/244 AUG KO RNAs both strongly inhibited fluor- escence in a dose-dependent manner (Fig. 3a) with over 90% inhibition at the dose of 0.5 μg for both 1/244 or 1/ 244 AUG KO plasmids (Fig. 3b).
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The poliovirus 3D RNA-dependent RNA polymerase contains two peptide segments previously shown to cross-link to nucleotide substrates via lysine residues. To determine which lysine residue(s) might be impli- cated in catalytic function, we engineered mutations to generate proteins with leucine residues substituted individually for each of the lysine residues in the NTP binding regions. These proteins were expressed in Escherichia coli and were examined for their abilities to bind nucleotides and to catalyze RNA chain elongation in vitro. Replacement of each lysine residue in the NTP binding segment located in the central portion of the 3D molecule (Lys-276, -278, or -283) with leucine produced no impairment of GTP binding or polymerase activity. Substitution of leucine for Lys-61 in the N-terminal portion of the protein, however, abolished the binding of protein to GTP-agarose and all detectable polymerase activity. A nearby lysine replacement with leucine at position 66 had no effect on enzyme activity. The three mutations in the central region of 3D were introduced into full-length viral cDNAs, and the infectivities of RNA transcripts were examined in transfected HeLa cells. Growth of virus containing 3D with a mutation at residue 278 (3D m 278) or 3D m 283 was indistin- guishable from that of the wild type; however, 3D m 276 generated extremely slow-growing, small-plaque virus. Polyprotein processing by 3CD m 276 was unaffected. Large-plaque variants, in which the Leu-276 codon had mutated again to an arginine codon, emerged at high frequency. The results suggest that a lysine residue at position 61 of 3D pol
Chemical synthesis and purification of RNA oligonucleotides have experienced great improvement since the 1990s. The general principles and methods of synthesis and crystallization of RNA molecules have been summarized and described previously (23-29). G-rich oligonucleotides have their own characteristic features and their conformations strongly depend on their interaction with metal ions (30). Our experience shows that oligonucleotides that contain more than four consecutive guanines may suffer some difficulty in purification. Also the present screening conditions of crystallization for oligonucleotides usually emphasize the effect of Mg 2+ ion (26,
medullary collecting ducts (IMCD), whereas it was negligible in proximal convoluted tubules (PCT) and medullary thick ascending limbs (MAL). In addition, the PCR product of ET-1 mRNA was also higher in glomeruli and IMCD, whereas it was undetectable in PCT and MAL. Furthermore, FCS and TGF-beta increased ET-1 mRNA in microdissected glomeruli and IMCD. These data clearly demonstrated that the production sites of ET-1 are glomeruli and IMCD among the nephron segments. ET-1 is an autocrine factor in these sites.