Lys3 annealing to the viral RNA of human immunodeficiency virus type-1 (HIV-1) is an essential step in the virus life cycle, because this tRNA serves as the primer for initiating reverse transcription. tRNALys3 annealing to viral RNA occurs in two steps. First, Gag promotes annealing of tRNALys3 to the viral RNA during cytoplasmic HIV-1 assembly. Second, mature nucleocapsid (NCp7), produced from the processing of Gag by viral protease during viral budding from the cell, remodels the annealed complex to form a more stable interaction between the viral RNA and tRNALys3, resulting in a more tightly bound and efficient primer for reverse transcription.tRNAin virio SHAPE analysis of both the 5´-untranslated region in HIV-1 RNA and the annealed tRNALys3 to determine structural differences of the annealed complex that occur between protease-negative (Pr-) and wild type viruses. Our results indicate that the weaker binding of tRNALys3 annealed by Gag in Pr- virions reflects both missing interactions of tRNALys3 with viral RNA regions in the upper PBS stem, and a weaker interaction with the internal stem-loop found within the unannealed primer binding site in viral RNA.In this report, we have used Lys3/viral RNA annealed complexes in PR- and wild type viruses that support the two-step annealing model by showing that Gag promotes a partial annealing of tRNALys3 to HIV-1 viral RNA, followed by a more complete annealing by NCp7.We propose secondary structure models for the tRNAThe online version of this article (doi:10.1186/s12977-015-0171-7) contains supplementary material, which is available to authorized users. Lys3 that was previously annealed during viral assembly to an 18 nt sequence in the viral RNA termed the primer binding site (PBS). The 18 nt PBS (nt 182–199) is found immediately downstream of the unique 5´ (U5) region in viral RNA, and is complementary to the 3´ terminal 18 nt of tRNALys3. Annealing to the PBS sequence is required for tRNALys3 annealing [Lys3. They were first proposed in the case of an HIV-1 MAL isolate that belongs to sub-type A by Marquet group [Lys3, but this interaction appears to occur only during the initiation of reverse transcription [Following infection of a host cell by HIV-1, the viral RNA genome (vRNA) is reverse transcribed into a double-stranded DNA, which then integrates into the host cell’s genome. Reverse transcription is initiated from a primer tRNAet group , 5. Whilet group , the Weeet group . An addi nt 182–19 is founLys3 to viral RNA is a multi-step process, promoted by both the viral Gag precursor, and later, after viral protein processing, by mature nucleocapsid (NCp7). tRNALys3 annealing to viral RNA in vitro at 37 °C does not occur at a wide range of RNA concentrations unless either Gag or NCp7 is present [Lys3 annealing in vivo in protease-negative viruses up to 80 %, independently of any effect such mutations might have on viral genomic RNA packaging [Lys3 [Lys relative to non-lysyl tRNAs is also increased 8–10 fold over that in the cytoplasm due to a specific interaction of Gag with the major tRNALys binding protein, lysyl-tRNA synthetase [The annealing of tRNA present , 11. Furackaging . Gag prong [Lys3 . The binng [Lys3 , 15. Hownthetase .Lys3, Gag-annealed tRNALys3 has a 2/3 reduced ability to initiate reverse transcription [Lys3 was shown by the ability of an in vitro reverse transcript from a primer upstream of the primer binding site to displace tRNALys3 annealed by Gag in a Pr- virus, but unable to displace tRNALys3 annealed by NCp7 in a Pr+ virus. Furthermore, in that report, we showed that weaker binding to viral RNA, and the reduced ability of Gag-annealed tRNALys3 to function as a primer for reverse transcription, can be rescued by a transient exposure to purified NCp7 of total viral RNA isolated from Pr(−) virions [Lys3 to the viral RNA, after viral protein processing, which may occur during or after viral budding [Lys3 to produce an annealed tRNALys3 that will function more efficiently as a primer.Compared to NCp7-annealed tRNAcription , and bincription . Thus, u virions . This su budding , exposurLys3 annealing within the viral particle. To examine how tRNALys3 anneals to HIV-1 RNA, we compared the structural interactions that occur between tRNALys3 and viral RNA in protease-negative (Pr-) and wild type (Pr+) virions, using in virio SHAPE . This method was developed by the group of K. Weeks [Lys3/viral RNA complex in wild type virions, based upon the reactivity of individual nucleotides (nts) in the viral RNA with the acylating agent NMIA (N-methylisotoic anhydride). NMIA reacts with the ribose 2´-hydroxyl group in flexible, unpaired, nucleotides. In this work, we have used SHAPE to measure the reactivity toward NMIA of nts in both viral RNA and annealed tRNALys3, within both Pr- and wild type virions. Our results indicate that the weaker binding of tRNALys3 annealed by Gag in Pr- virions reflects missing interactions of tRNALys3 with the upper PBS stem, and an altered interaction of tRNALys3 with the internal stem loop found within the unannealed PBS sequence.The aim of this study is then to elucidate the structural differences between Gag- and NCp7-mediated tRNAK. Weeks , and usein virio SHAPE with the proposed best thermodynamically stable structures, as described in Lys3 with the viral RNA shown in Fig. Lys3 interacts with the viral RNA [Lys3 will also bind to two other regions in the upper PBS stem of viral RNA, termed the C-rich and poly(A) loop regions. SHAPE reactivities and the predicted secondary structure model of the 5´-UTR within Pr- viruses are similar to those of wild type viruses, except in the regions that include the upper PBS stem and the PBS loop  and protease-negative BH10-Pr- (Pr-) were produced from transfected 293 T cells, and purified on a sucrose-gradient as described in iral RNA . In addisee Fig. . This isLys3 annealing, we compared in virio SHAPE reactivities in these regions for wild type and Pr- virions with the SHAPE reactivities of an unannealed in vitro transcribed 5´-UTR of HIV-1. We previously described the production and characteristics of this construct [in vitro transcribed 5´-UTR, and viral RNAs found in Pr- and wild type virions. The unannealed PBS sequence itself contains an internal stem loop, and other nucleotides with low reactivities within the larger PBS loop suggest some tertiary interactions that constrain the flexibility of these nucleotides. As seen in Figs. Lys3 to these sequences by Gag. Nucleotides 193 and 194 show low reactivities in unannealed, Pr-, and Pr + RNAs, and the differences between all three samples are not significant. These nucleotides are probably stabilized in a conformation unfavorable for modification by NMIA, even if they are not involved in a Watson-Crick base pair. In Fig. Lys3 annealing in the absence of Gag, we incubated in vitro transcribed 5´-UTR viral RNA and tRNALys3 (1 to 10 ratio) in the absence of Gag for 30 min at 37 °C, and performed SHAPE experiments. The reactivities within the PBS sequence remained similar to that of unannealed 5´-UTR RNA  and performed SHAPE analysis. The complete data sets are shown in Additional file ex virio and in virio shape reactivities are similar for the nucleotides we analyzed to establish our hypothesis, and support our assumption that the observed changes between Pr + and Pr- are due to an altered tRNALys3 annealing to viral RNA.As shown in Figs. on Figs.  and 4. Iys3 Fig. . Howeverrus Fig. . These oLys3 are based solely on SHAPE analysis of viral RNA. To investigate the validity of these conclusions, we also determined the SHAPE reactivities of the annealed tRNALys3 within wild type and Pr- viral particles. In this procedure, we eliminated non-annealed tRNAs present in the virion by using columns that retain only RNAs larger than 200 nts after extracting the NMIA-modified RNA from the viral particles. The annealed tRNAs were released from the viral genomic RNA by heating to 70 °C for 5 min, and poly(A) tails were added to the tRNA as described in Lys3 at the 3´ end. As shown in Fig. Lys3, the sequence that binds to the PBS, could not be calculated. For comparative studies we also determined SHAPE reactivities of an in vitro transcribed tRNALys3. Several stops were noticed in the modified and the unmodified reverse transcription reactions of the in virio tRNAs from wt and Pr- viral particles, but not in the in vitro transcribed tRNA. These stops correspond to modified nucleotides 10, 16, 20, 37, 47, and 53. These nucleotides were marked as not analyzed. They are shown in grey in the figures, and given a −999 value in the reactivities datasets , while U33 and U36 have high reactivities  has in fact low reactivity, presumably because it forms a base pair within the viral stem. Similarly, the low reactivity of tRNALys3(U34) coupled with the high reactivity of A168 in viral RNA also suggests that these two nts are not base- paired, and that the low reactivity of tRNALys3(U34 and U35) in the tRNA is a result of a failure of NMIA modification as discussed above. Low reactivities for both tRNALys3(U35) and vRNA(A169) suggest a possible base pair, but the high reactivities of both A170 in viral RNA and U36 in tRNALys3 suggest the absence of base pairing. Thus, 3 of the 4 potential base pairs in this region of interaction between viral RNA and tRNALys3 are absent in the Pr- virus.The region including nucleotides U33-U36, which is part of the anticodon loop in annealed tRNAons Fig. . HoweverLys3, referred to in Fig. Lys3 (41–46)). U145 is the only nucleotides that would be free if the annealing is weakened or does not occur  could result from maintaining the upper PBS stem, or could result from annealing with a complementary region in tRNAsee Fig. . As expesee Fig. . In suppLys3 annealed either by Gag in vitro, or within a Pr- virus, is less tightly bound to the viral genomic RNA, and is a less efficient primer for reverse transcriptase, than tRNALys3 annealed either in vitro with mature nucleocapsid (NCp7) or in wild type viruses [in virio SHAPE analysis to investigate these differences while inducing minimal changes in the RNA structure during isolation of RNA from viruses. The results reported here show structural differences within the tRNALys3/viral RNA complex isolated from wild type or Pr- viruses that correlate with these findings. Although some positions show high standard deviations, all our experiments were repeated three times independently, and showed a similar trend in all cases. The proposed models are further supported by the changes in NMIA reactivities occurring only in the interacting regions of viral RNA and the tRNALys3  regions in viral RNA upstream of the PBS that are also involved in annealing in wild type viruses. SHAPE reactivities obtained for both the annealed regions in viral RNA and the C-rich and poly(A) regions of annealed tRNALys3 were found to be complementary, but for technical reasons, the SHAPE reactivities of the 3´-terminal 18 nts of tRNALys3 could not be obtained and compared with the PBS SHAPE reactivities.Previous studies have indicated that tRNA viruses , 18. We s3 Figs. , 3 and 4in virio and ex virio [in virio and protein-free ex virio states were very similar for the major length of the viral RNA. They also examined more specifically the effect of NCp7 binding on SHAPE reactivities in virio by exposing virions to a reagent that inhibited NCp7 binding to the viral RNA. They found distinct regions in the viral RNA that bound to NCp7, which depending upon the nature of the binding, could cause increases or decreases in NMIA reactivity of individual nts. However, none of the nts demonstrated here to be involved in tRNALys3 annealing showed binding to NCp7 in their study, and in fact, these nts showed similar reactivities to NMIA both in virio and ex virio. Furthermore, to support our interpretation of the changes observed, we have shown that changes in NMIA reactivities noticed in the annealing areas on the viral RNA are accompanied by similar changes in the complementary nucleotides on the tRNALys3 molecule.In this work, the differences in acylation sensitivity found in Pr + and Pr- samples is assumed to reflect differences in RNA backbone structure, and not different abilities of protein bound to the RNA to inhibit or promote access to the acylation reagent. To support this assumption, we performed SHAPE analysis on de-proteinized Pr + and Pr- viral RNA, and found that the nucleotides we analyzed did not show any significant difference in SHAPE reactivity in absence or presence of the proteins. In fact, the effect of proteins on NMIA modification was previously studied by Kevin Weeks’s group for the first 5´- 990 nts of the HIV-1 genome, and done both ex virio , SHAPE rLys3 to regions in viral RNA upstream of the PBS may be due to a limited ability of nucleocapsid sequence within Gag to denature RNA duplexes. It has been observed previously that Gag and mature NCp7 destabilize RNA structures in different propensities [The inability of Gag to anneal tRNAensities , 22. NMRensities , and thaensities . In abseLys3 with the PBS in viral RNA, it appears that only mature NCp7 promotes the interactions of two regions within the anticodon stem-loop of tRNALys3 with nucleotides 142–147 and 167–170 of the upper PBS stem. The in vitro interaction of NCp7 with tRNALys3 has been shown to destabilize the acceptor stem, the T-stem-loop, and the anticodon stem-loop [While nucleocapsid sequences within Gag can promote a relatively weak interaction of the terminal 18 nts of tRNAtem-loop , 26, andLys3 to an 18 nt PBS sequence within a structurally complex >9 kb viral genomic RNA may explain the need for a multistep annealing mechanism, even if the initial cytoplasmic binding of the tRNALy3 to viral RNA is weak relative to annealing by mature NCp7. An early cytoplasmic HIV-1 assembly intermediate composed of unprocessed Gag and GagPol and viral RNA will promote this initial annealing through its molecular organization [Lys3 at the cytoplasmic site of viral assembly through a specific Gag/LysRS interaction [The difficulty of bringing cytoplasmic tRNAnization , its abieraction , and itseraction , 28. TheLys3 to viral RNA by Gag appears to serve another function in addition to preparing tRNALys3 to act as a primer for reverse transcription. Thus, it has been reported that annealing of tRNALys3 to the PBS aids in promoting a conformation change in the 5´-untranslated region of viral RNA that promotes viral RNA dimerization [The cytoplasmic annealing of tRNArization , 20, a prization , 30.Lys3 and the upper PBS stem. We also found major differences in the interactions between viral RNA and annealed tRNALys3 from Pr- and wild type virions. These differences reveal the reasons why annealed tRNALys3 is less tightly bound in Pr- virions. These findings are consistent with the multi step annealing mechanism proposed previously.In conclusion, our study confirms the findings that in wild type HIV-1 subtype B virions, interactions occur between tRNASVC21.BH10 is a simian virus 40-based vector that codes for the wild type sequence of HIV-1 proviral DNA . The proex virio analysis, the viral particles were treated with proteinase K for 30 min at 37 °C prior to treatment with NMIA.For each SHAPE reading, 24 cell culture plates (15 cm diameter) were inoculated with 6 million HEK 293 T cells per plate and grown in complete Dulbecco’s modified Eagles’s medium (DMEM). After overnight incubation, the cells were transiently transfected by wild type BH10 or BH10 Pr- plasmids using lipofectamine 2000 from Invitrogen according to the manufacturer’s recommendations. The supernatant was collected 48 h post-transfection, and filtered through 0.2 μm filters. The viral particles were pelleted in a Beckman Ti-45 at 35 000 rpm for 1 h. The pellets were then purified by 15 % sucrose onto a 65 % sucrose cushion by centrifugation in SW41 rotor at 26 500 rpm for 1 h. The layer of purified viral particles was collected and the virus was pelleted using Beckman SW41 rotor at 35 000 rpm for 1 h, in 1X TNE buffer . The viral particles were resuspended in 600 μl of NMIA reaction buffer , and modified using NMIA for 50 min at 37 °C as described in . For ex Prior to modification with NMIA, 2 % of the viral particles were mixed with protein gel loading dye , lysed by boiling, and run on a 10 % SDS-PAGE gel. Following gel transfer by electroblotting, the membrane was probed with mouse anti-CAp24 antibody (NIH AIDS Research and Reference Reagent Program).The modified and control (unmodified) viral particles were lysed with 1 % SDS and Proteinase K treatment at room temperature for 20 min, followed by a phenol extraction as proposed by . The RNAin vitro transcription from a BH10 template, and was analyzed by SHAPE as described previously [The products were resolved by capillary electrophoresis at the IRIC (Institut de Recherche en Immunologie et Cancérologie) genomic center. The data were analyzed and normalized as described in , 33 usineviously .Lys3 was reverse transcribed as described for viral RNA in presence of VIC fluorophore labeled oligomer that includes 6 nucleotides specific to tRNALys3 and a poly U tail, 5´ VIC-TTTTTTTTTTTTTTTTGGCGCCCG 3´. The reverse transcription reaction was purified on Sephadex G25 column, ethanol precipitated, resuspended in water, and analyzed by capillary electrophoresis as described for viral RNA. The tRNALys3 ladder used for performing SHAPE analysis, was produced by sequencing a DNA template coding for tRNALys3 with a poly(A) tail, the same oligomer used for SHAPE analysis, and Sequenase™ version 2.0 DNA sequencing kit from USB (cat # 70770 1kt) according to the manufactuer’s recommendations for short readings.The viral particles were produced and treated with NMIA as described for viral RNA. Following viral lysis, the RNA was treated with acidic phenol, and the aqueous phase was purified using Clean and Concentrate kit in conditions that retain RNAs larger than 200 nucleotides. The RNAs were heated to 70 °C for 5 min and then chilled on ice. Following a short spin at 12 000 rpm, the RNAs were treated with poly(A) polymerase using poly(A) tailing kit from Ambion (Cat# AM1350). The mix was treated with phenol, and the aqueous phase was purified on RNA clean and concentrate columns in conditions were RNAs larger than 17 nucleotides are retained. tRNAin vitro transcribed 5´-UTR were incubated with thirty pmoles of tRNALys3, without heat denaturation, at 37 °C for 30 min, in 10 mM HEPES pH 8.0, 1 mM MgCl2, 10 mM NaCl, 150 mM KCl. SHAPE experiments were performed on the 5´-UTR RNA as described above.Three pmoles of


Detection of bacterial gene expression in soil emerged in the early 1990s and provided information on bacterial responses in their original soil environments. As a key procedure in the detection, extraction of bacterial RNA from soil has attracted much interest, and many methods of soil RNA extraction have been reported in the past 20 years. In addition to various RT-PCR-based technologies, new technologies for gene expression analysis, such as microarrays and high-throughput sequencing technologies, have recently been applied to examine bacterial gene expression in soil. These technologies are driving improvements in RNA extraction protocols. In this mini-review, progress in the extraction of bacterial RNA from soil is summarized with emphasis on the major difficulties in the development of methodologies and corresponding strategies to overcome them. After cin, USA) , 139 andin, USA) , 76 chroin, USA) .Impurities are extracted from soil along with RNA, and the majority of these impurities are humic substances, which are dark-colored, heterogeneous organic compounds in soil . Based oet al., unpublished data), although this commercial kit has been proven to extract RNA from diverse soils successfully  can be found all over the world. In Japan, Andosols cover about 16.4% of land surface and 46.5% of arable upland fields ; thus, iessfully , 145. Itessfully ; howeveressfully , 30. Alsessfully . Thus, tessfully , and 85%essfully . It is kessfully , 47, 64.essfully , it seemessfully . Second,essfully , which messfully . A recenessfully ; howeverUnlike eukaryotic mRNA, bacterial mRNA does not normally possess a poly(A) tail, which makes the purification of bacterial mRNA difficult. Although recent studies revealed that some mRNA molecules in bacteria possess a poly(A) tail, those mRNA molecules only occupy a small portion of the whole transcriptome and will be subject to fast degradation ; thus, oIt is easy to recover large quantities of total RNA from a pure culture of bacteria (tens of micrograms per extraction), but it is rather difficult in soil. According to reports, the yields of RNA extracted from soil range from tens of nanograms to several micrograms per gram of soil , 139. Su2(SO4)3 under alkaline conditions prior to cell disruption , showed a similar tendency with RNA; in particular, the adsorption of 5′-AMP at pH 4 and pH 6 was about 60 times higher than at pH 8  but no Mg2+ or Ca+, has been successfully applied to extract high-purity RNA from a sterilized brown forest soil inoculated with either Gram-positive or -negative bacteria . This result suggests that controlling the pH and divalent cations in an extraction buffer is not sufficient to inhibit the adsorption of RNA by soil; therefore, the optimal buffer composition for RNA release from Andosols should be explored in the future. In successful extractions of DNA from Andosols, an appropriate additive is often required. Some additives have been tested and shown to be helpful in assisting the release of DNA from Andosols to recover DNA from soil  and high fidelity (r2 between 0.85 and 0.92 for the comparison between amplified RNA and unamplified RNA)  . Successied RNA)  and highied RNA) , 72, 116) , soil texture, pH, water content, moist soil color (the Munsell color system is preferred), organic and inorganic components. In addition to information about soil characteristics, information about the extracted RNA, such as the UV spectrum, and the ratio of OD260 nm/OD280 nm and OD260 nm/OD230 nm, and the absorbance at 320 nm or other evaluated wavelengths for measurement of humic substances , “The beauty of science is to make things simple.” Thus, we believe that such a universal method should be as simple as possible so that it can be mastered by regular researchers without much experience with RNA experiments. To reach this goal, first, the removal of humic substances should be as simple as possible without loss of purification power. Because biologists have already spent two decades improving the methodology of RNA extraction from soil, it is apparently difficult to simplify the purification procedures based on the current technologies. This may require contributions from chemists or physicists to develop new technologies. Second, RNA extraction from diverse soil types collected worldwide is required. Because it is difficult to conduct such a systematic test for technical, economic, and political reasons, a sub-universal method could be developed, Gene expression as an important tool in the study of soil microbial ecology and physiology can be expected to be more popular and more important with the development of methods of bacterial RNA extraction from soil.


Analysis of multiple analytes from biological samples can be challenging as different analytes require different preservation measures. Heat induced enzymatic inactivation is an efficient way to preserve proteins and their modifications in biological samples but RNA quality, as measured by RIN value, has been a concern in such samples. Here, we investigate the effect of heat stabilization compared with standard snap freezing on RNA quality using two RNA extraction protocols, QiaZol with and without urea pre-solubilization, and two RNA quality measurements: RIN value, as defined by the Agilent Bioanalyzer, and an alternative qPCR based method. DNA extraction from heat stabilized brain samples was also examined. The snap frozen samples had RIN values about 1 unit higher than heat stabilized samples for the direct QiaZol extraction but equal with stabilized samples using urea pre-solubilization. qPCR based RNA quality measurement showed no difference in quality between snap frozen and heat inactivated samples. The probable explanation for this discrepancy is that the RIN value is an indirect measure based on rRNA, while the qPCR score is based on actual measurement of mRNA quality. The DNA yield from heat stabilized brain tissue samples was significantly increased, compared to the snap frozen tissue, without any effects on purity or quality. Hence, heat stabilization of tissues opens up the possibility for a two step preservation protocol, where proteins and their modifications can be preserved in the first heat based step, while in a second step, using standard RNA preservation strategies, mRNA be preserved. This collection strategy will enable biobanking of samples where the ultimate analysis is not determined without loss of sample quality. This is particularly challenging when selecting collection strategies for biobanking sample where the ultimate analysis is not known at the time of sampling. Classical RNA preservation strategies, e.g., RNA Later, while preserving RNA quality do not prevent the more rapid changes to protein phosphorylation that occur post-sampling. Heat induced enzymatic inactivation on the other hand have been shown to be an efficient way to preserve protein phosphorylation but RNA quality has been a concern in such samples e.g., RIN value using an Agilent Bioanalyzer assay, has indicated a reduction in quality in samples after heat denaturation and a further reduction in RIN value when heat stabilized samples are incubated at room temperature indicating that RNases remain active after heat denaturation e.g., the use of a ratio Cq from 2 shorter qPCR assays The removal of a biological sample from its e.g., protein phosphorylation, as well as mRNA as a secondary analyte both with high quality. DNA extraction from heat stabilized brain samples was also examined to investigate the possibility to also analyze DNA from heat stabilized samples.The aim of the present study was to investigate the effect of heat based sample stabilization through protein denaturation done using the Stabilizor system  on RNA quality using Bioanalyzer  and TATAAs RT-qPCR (mRNA based) quality scoring. If mRNA can be shown not to be degraded by heat stabilization in the Stabilizor system it would enable analysis of the primary analyte, 22.1ad libitum. Animal rooms were temperature- and humidity-controlled and kept on a reversed 12 h light/dark cycle with lights off at 6:00 a.m. The animal experiments were approved by the regional ethics committee and were in accordance with the Swedish Animal Protection Legislation.Male adolescent Wistar rats, 36 in total,  were acclimatized one week prior to the experiments. They were housed three per cage with food and water 2 and decapitated when euthanized. The brain was removed from the scull right after decapitation and two adjacent sagital sections of 1 mm thickness was cut from the posterior part of the cerebrum for the RNA experiments. From eight animals, one section was snap frozen directly while the other was heat stabilized with the Stabilizor™ System  using the Auto Fresh mode prior to snap freezing. Four additional animals were dissected in a similar way but both sections were heat stabilized individually and then incubated for 12 h at room temperature prior to being frozen, see n = 4 per protocol). Two groups were either instantly dissected from fresh tissue, or dissected after heat stabilization of the intact brain. The brains from the third and fourth groups of animals were kept at room temperature for 60 and 300 min, respectively, to allow for post-sampling changes to appear before the samples were snap frozen. The brains from the fifth and sixth group were removal from the scull and heat stabilization as whole brains of the brain instantly dissected after and left at room temperature for 60 min and 300 min, respectively, before the samples were snap frozen. All tissue samples were stored at −80 °C prior to tissue extraction.The rats were sacrificed one at a time using CO2.2i.e., 10 μl per 1 mg of tissue, of urea buffer . This was followed by the transfer of 400 μl urea buffer sample extract to a new tube and 600 μl of QiaZol was added to lyse the samples. The other half set of samples was extracted directly in 1 ml QiaZol. An extra step of centrifugation  after homogenization was added to all samples due to foaming of the samples.Total RNA was extracted from the samples as follows: The samples were homogenized while frozen using the Tissuelyser II (Qiagen) and extraction buffer added directly to the frozen powder. Extraction was done using QiaZol and Qiagen RNeasy Mini Kit either according to Qiagen manual for RNeasy Mini Kit or by including a urea pre-solubilization step. One set, constituting an identical half of the samples were pre-solubilization with 10 volumes, 2.3The concentration and purity (A260nm/A280nm and A260nm/A230nm) of the 24 extracted RNA samples was measured on DropSense96 (Trinean). The quality of the RNA was measured on BioAnalyzer (Agilent Technologies) to assign RIN-values to each RNA extract. For further analysis the samples were normalized to 100 ng/μl using the DropSense96 value.2.4® GrandMaster Mix # TA01 (TATAA Biocenter AB). Reverse transcription was performed in single measurements in 10 μl reaction volume on CFX96  with the following program, 5 min at 22 °C, 30 min at 42 °C, 5 min at 85 °C, hold at 4 °C. Negative controls were included during extraction and a NoRT control was added to check for DNA contamination. RNA quality was checked using three qPCR based long and short RNA quality assays: two based on mRNA transcripts (B2 M and mtCO1) and one based on the 18S rRNA transcript . All samples including extraction negatives, NoRT, gDNA and NTC were run in duplicates in 10 μl reactions on LightCycler480 platform . All pipetting was performed by a pipetting robot . The temperature program, melting for 60 s at 95 °C followed by 45 cycles of 20 s at 60 °C alternating with 20 s at 72 °C, was used and detection was performed in the SYBR channel.cDNA was synthesized from each sample using TATAA Grandscript cDNA synthesis kit #A103 (TATAA Biocenter AB) with a mix of random and poly-dT primers with 500 ng of total RNA as starting material. qPCR was performed with TATAA SYBR2.5DNA extraction was done using GeneElute™ Mammalian Genomic DNA miniprep kit (Sigma–Aldrich). In brief, brain samples were homogenized manually with a micropestle in 1.5 ml Eppendorf tubes together with lysis buffer containing Proteinase K. The samples were incubated at 55 °C for 4 h to allow for tissue digestion. RNase was added to allow for extraction of RNA-free genomic DNA. DNA isolation was done using spin columns according to the manual for the Kit. The DNA was eluted in 100 μl of elution solution. To increase the elution efficiency, the tubes were incubated for 5 min with the elution solution prior to centrifugation to ensure equilibration. After centrifugation the collected elution solution was pipetted back on the binding column and the elution procedure was repeated once.2.6The concentration and purity (A260nm/A280nm and A260nm/A230nm) of the 24 extracted DNA samples was measured on a Nanodrop (Thermo Scientific). The quality of the DNA was further evaluated with gel electrophoresis. Equal amounts of DNA (100 ng) were loaded on a 1% agarose gel and subjected to electrophoresis at 70 V for 90 min. DNA bands were visualized with UV light after ethidium bromide staining.3The RNA yield, purity and quality has been measured in RNA extracts from heat stabilized and snap frozen brain tissue samples with the aim of investigating the effect of heat denaturation on two different RNA quality measurements, either Bioanalyzer (primarily rRNA based) or qPCR (mRNA based).3.1RNA was extracted using two different protocols and yield and purity as measured using A260nm/A280nm ratio, see 3.2e.g., RIN value, for heat stabilized versus snap frozen samples could be attributed to the association of rRNA with proteins, enzymes and extensive chemical reactants and that rapid heating may cause rRNA-protein complexes to cleave and degrade the rRNA The RIN value, as determined using the Bioanalyzer, is primarily based on rRNA and functions as a surrogate quality marker for mRNA. Snap frozen samples pre-solubilized in urea prior to QiaZol had significantly lower RIN values compared to snap frozen samples directly extracted in QiaZol . The RIN3.3As most RNA analyses are based on mRNA, it would be more correct to base the RNA quality assessment on mRNA rather than rRNA. qPCR amplification is quantitative and well suited to measure levels of individual mRNA transcripts that can be used to asses RNA quality. Various approaches for using qPCR to measure mRNA quality have been proposed, e.g., the use of a ratio Cq from 2 shorter qPCR assays e.g., RNAlater, to enable extraction of both high quality proteins and RNA.The qRCR mRNA quality score was very close for all three qPCR markers tested for both snap frozen and heat stabilized samples regardless of extraction method . This sh3.4The DNA yield or the A260nm/A280nm and A260nm/A230nm ratios were not affected in samples that were kept at room temperature for 60 or 300 min, to allow for post-sampling changes to appear, compared to samples that were directly snap frozen . The DNA4, e.g., freezing or RNA Later should be added in a second step. This will enable multiple analyses of DNA, mRNA and protein phosphorylation with high quality from the same sample.Analysis of multiple analytes from precious biological samples can be challenging as different analytes require different preservation measures. Although heat inactivation does not preserve RNA from further post-sampling degradation the results in this study show that heat stabilization does not directly affect mRNA quality when using the mRNA based qPCR quality measure and confirms the previously reported decreased in quality when using the primarily rRNA based RIN quality value. Further studies with other RNA quality scores and more samples would be required to understand the apparent difference in RNA quality between the two quality metrics and RNA sub-classes. However, this finding opens up the possibility of a combination protocol where proteins and their modifications can be preserved by heat stabilization while retaining high quality DNA and mRNA. To ensure long-term stability of mRNA, standard RNA preservation strategiesM.B. works for Denator, the manufacturer of the Stabilizor system. R.S. works for TATAA Biocenter, the CRO that invented and performed the qPCR based mRNA quality scoring used in this study.


A majority of viruses are composed of long single-stranded genomic RNA molecules encapsulated by protein shells with diameters of just a few tens of nanometers. We examine the extent to which these viral RNAs have evolved to be physically compact molecules to facilitate encapsulation. Measurements of equal-length viral, non-viral, coding and non-coding RNAs show viral RNAs to have among the smallest sizes in solution, i.e., the highest gel-electrophoretic mobilities and the smallest hydrodynamic radii. Using graph-theoretical analyses we demonstrate that their sizes correlate with the compactness of branching patterns in predicted secondary structure ensembles. The density of branching is determined by the number and relative positions of 3-helix junctions, and is highly sensitive to the presence of rare higher-order junctions with 4 or more helices. Compact branching arises from a preponderance of base pairing between nucleotides close to each other in the primary sequence. The density of branching represents a degree of freedom optimized by viral RNA genomes in response to the evolutionary pressure to be packaged reliably. Several families of viruses are analyzed to delineate the effects of capsid geometry, size and charge stabilization on the selective pressure for RNA compactness. Compact branching has important implications for RNA folding and viral assembly. Single-stranded (ss) RNA molecules are typically branched, with physical properties that depend on the secondary and tertiary structures determined by their primary nucleotide (nt) sequence On the other hand, the statistics of model branched molecules and aggregates are well studied ssRNA viral genomes are special RNA molecules in several significant ways. First, because they involve two or more genes, they are necessarily thousands of nt long. Also, unlike other long RNAs, such as edited messenger RNA transcripts or ribosomal RNA, they are constrained to have physical sizes compatible with being packaged spontaneously by viral coat proteins into small volumes corresponding to the inside of a rigid viral capsid. As proposed in earlier theoretical work In this paper, we study the correlation between the nt sequence and the physical size of large RNA molecules – in particular, whether the sequence of a viral RNA codes not just for required protein products but also for its own physical size. We compare the experimentally determined size of a 2117-nt viral RNA with those of non-viral sequences of identical length and find the viral sequence to be the most compact. The relative sizes of these sequences are explained by analyzing the nature of branching in predicted ensembles of secondary structures. We show that compactness originates from an increased density of branching defined by the number, degree and organization of multi-helix junctions in secondary structures. We compare several families of ssRNA viruses and find that genomes with propensity for denser branching typically belong to families where other means of RNA compaction  may not exist. Finally, we outline how compactness improves the robustness of RNA folding and enhances the ability of a viral genome to package in a capsid.in vivo and in vitroTo test the relationship between the primary nt sequence of an RNA and its physical size, we study nine RNAs of identical lengths (2117 nt), but different nt compositions and biological functions. The first is genomic RNA3 of Brome Mosaic Virus (BMV) To compare the four B3-based RNAs with those not expected to have evolved with a selective pressure to be compact, the remaining five RNAs were transcribed from arbitrarily chosen non-overlapping sections of the yeast genome (see Methods). Labeled Y1 through Y5, three  contain both non-coding and coding regions, one (Y3) is a subset of a large gene and therefore fully coding, and one (Y4) is from a region with no known genes.2+-free buffers, are consistent with their predicted secondary structures, and that the relative compactness of viral-sequence RNAs observed in these buffers is preserved in higher-ionic-strength Mg2+-containing  buffers. More explicitly, the presence of Mg2+ and higher ionic strength will of course decrease the absolute sizes of the RNA molecules, but the radii of gyration for viral-sequence molecules are shown both buffers. Therefore, as in our previous studies, we choose in the present work a 10 mM 10∶1 Tris:EDTA (TE) buffer (pH 7.4) as the appropriate solvent for measuring the relative sizes of the RNAs listed above, again accentuating the role of secondary structures under conditions where tertiary interactions are minimal. We are not suggesting that secondary structure is the only important factor in determining the compactness of RNA; tertiary folding effects can of course contribute substantially as well. Rather, we are suggesting that the extent and nature of branching in the secondary structure is a dominant factor. Accordingly, our predictions and conclusions relate exclusively to differences in these properties between viral and non-viral sequences.To study correlations between nt sequence and physical size, measurements are best made under solution conditions where the morphology of secondary structures is most evident. We recently showed Sizes are first investigated by electrophoresis  through The RNA band positions in rμ) are calculated with respect to the fastest migrating RNA, in this case B3 in lane 1 . Relative mobilities (hR) are measured. FCS  is used to determine the characteristic time  are significant outliers. Knowledge of the maximum extents of secondary structures of sequences is therefore not sufficient to reliably predict their relative mobilities. For large RNAs, the b, which represents the mean helix length (≈5 base pairs).The average length of a base-paired helical segment in large RNAs is independent of the length of the sequence rR. As with previous measurements, RNAs that differ significantly in rR can have similar values of rRs can have significantly different rR, making it a less sensitive measure of differences between sequences. These limitations indicate a need for deeper analysis of the differences in branching patterns.The data and linear regression line in d) of all the vertices in a graph is twice the total number of edges (E). First shown by Euler Because the degree of a vertex is the number of edges connected to it, the sum of the degrees . In Eq. 2 shows that knowing where the expression in parentheses represents the surplus of Ratios of the maximum numbers of junctions to In Thus, knowledge of the number of stem-loops and 3-helix junctions in a secondary structure is sufficient to predict higher-order branching and therefore the compactness of an RNA. These differences in higher-order branching propensities reveal useful details about the patterns of base pairing in the primary sequence. To illustrate this, we analyze the relative proximities of vertices in secondary structure trees and their implications on the information content of the RNA sequence.To verify that higher-order branching significantly increases the compactness of trees within an ensemble, we compute a graph-distance distribution function In order for a few stograms , where nThis work establishes that differences in the shapes and sizes of long equal-length RNAs in solution can be determined using standard experimental techniques, and explained by the coarse-grained properties of secondary structure ensembles predicted from their sequences. Using graph-theoretical arguments we have shown that knowledge of the number of stem-loops and three-helix junctions in experimentally determined base-pairings – when the sequence lengths begin to exceed several hundred nts. But this failure is not relevant to predicting coarse-grained properties of the secondary structures such as vertex order distributions and the extent of higher-order branching, etc., much as we had explicitly shown earlier 2+ and high ionic strength Because of the central role played in our analyses by the ensembles of secondary structures associated with different primary sequences, and because the sequences involved are thousands of nts long, it is important to comment on the robustness of our predictions. It is well-known, of course, that all secondary-structure computational algorithms begin to degrade significantly – in their prediction of We showed recently Bacteriophage MS2 provides a compelling example of the relevance of this kind of analysis to understanding the role of RNA branching statistics in viral assembly. The packaging propensity of MS2 RNA is known Differences in branching propensities between viral families  of their capsids sufficiently small.By demonstrating i) that experimentally determined RNA sizes are related to the compactness of branching patterns in secondary structure ensembles, and ii) that the compactness of several families of viral genomes are consistent with the selective pressures imposed by capsid size and electrostatics, we have shown that the density of secondary-structure branching is a degree of freedom available for optimization in viral RNA genomes. When other means of condensing the genome are absent, viral RNAs are unusually compact.in vitro transcribed from PCR templates using T7-polymerase , followed by DNAse digestion of the template. Protein impurities were removed by phenol-chloroform extraction and the RNA isolate was rid of shorter polynucleotides and unreacted ribonucleotides by repetitive additions of TE (pH 7.4) buffer and filtration through a 100 kDa MWCO Centricon device. RNA samples were equilibrated in TE buffer at 4°C for 24 hours to obtain uniform ensembles Saccharomyces cerevisiaeThe RNA molecules were About 1 The Advanced Light Microscopy shared facility at the California NanoSystems Institute (UCLA) was used for fluorescence correlation. The setup contains a custom-made confocal configuration built with an Axiovert 100  inverted microscope as its base. The 488-nm line from a continuous-wave Argon Laser  was used with excitation power ranging from 5–90 where RNA primary sequences were obtained from the NCBI Viral Genome project File S1Combined Supporting Information. Single PDF file containing Figures S1-S5 and Tables S1 & S2. Legends are provided within the file below each Figure or Table.(PDF)Click here for additional data file.


RNA isolation with RNA in a high quantity is a basic analytical method in plant genetics, molecular biology and related physiological investigations. To understand the genetic and molecular biology of Chinese fir, sufficient high-quality total RNA must be obtained for cDNA library construction and other downstream molecular applications. However, extracting RNA from Chinese fir is difficult and often requires the modification of existing protocols. Chinese fir tissues containing large amounts of polysaccharides and polyphenol compounds and are one of the most difficult plant tissues for RNA isolation. Therefore, we developed a simple method for extracting high-quality RNA from Chinese fir tissues. RNA isolations were performed within two hours, RNA quality was measured for yield and purity. Total RNA obtained from this procedure was successfully used for cDNA library construction, RT-PCR and transcriptome sequencing. It was proven that extracted RNA was intact and suitable for downstream molecular applications, including RT-PCR and qPCR, and other downstream molecular applications. Thus, this protocol represents a simple, efficient, and low-cost method. Obtaining high-quality RNA in a large quantity is a preliminary step for many investigations in plant molecular biology such as reverse transcription polymerase chain reaction (RT-PCR), real-time fluorescent quantitation polymerase chain reaction (qPCR), cDNA synthesis and cDNA library construction, gene isolation, gene expression analysis, and next generation sequencing . HoweverTo date, many published conventional RNA extraction methods, such as cetyltrimethylammonium bromide (CTAB), TRIzol Plus RNA Purification kit , RNeasy plant Mini kit  and other RNA reagent kits are currently available for extraction of RNA from plant tissues ,5. Each However, extracting high-quality RNA from Chinese fir tissues is extremely difficult because Chinese fir is often rich in polyphenols, polysaccharides, proteins, lipids, and several other secondary metabolites that interfere with RNA extraction by degrading or coprecipitating with RNA. In addition, using an RNA isolation kit to extract the total RNA of Chinese fir tissues is expensive. Therefore, it is necessary to find an efficient, rapid, and reproducible method for isolating high-quality RNA from Chinese fir tissues.In this study, we developed a simple, less expensive, and time-saving RNA extraction procedure for Chinese fir tissues (root and leaf) that efficiently yields high-quality RNA, modifying the method described by Chan et al . High-quCunninghamia Lanceolata (Lamb.) Hook) plants grown in greenhouses and frozen immediately in liquid nitrogen . All samples were stored at -80°C before RNA extraction.Fully expanded roots and leaves were sampled from Chinese fir  ; 0.02 M, EDTA; 1%, SDS) was modified from the protocol proposed by Chan et al  with theOne of the critical points for obtaining a high yield in the extraction of genetic material is the grinding. It is essential to grind the tissue as finely as possible, maintaining samples as cold as possible during grinding to avoid degradation.A high-throughput organization grinding machine  was used for grinding roots and leaves. Approximately 0.1 g of frozen roots and leaves were placed in 2 ml RNase/DNase-free tubes; then, two 5 mm sterilization steel balls were added into the same tube. Subsequently, the tubes containing the frozen tissue plus steel balls were put in the adapter to pre-cool with liquid nitrogen for 2 min and then shaken in the high-throughput organization grinding machine (Scientz-192) for 3 min at frequency 20 (1200 times/min) without buffer at low temperature.1 ml SEB and 50μl 2-mercaptoethanol (SEBM) were mixed sufficiently in a 1.5 ml RNase-free microcentrifuge tube.1 ml SEBM was added to pulverized tissue, which was mixed by brief vortexing or flicking the bottom of the tube until the sample was thoroughly resuspended. Then, the sample was incubated for 10 min at room temperature.The mixture was centrifuged for 15 min at 13000×g in a microcentrifuge at 4°C. The supernatant was transferred (~600μl) to a new 1.5 ml RNase-free microcentrifuge tube.300μl 5 M NaCl and 500μl chloroform were added, and the sample was mixed gently by inverting the tubes six to eight times and centrifuge the sample at 4°C for 5 min at 13000×g to separate the phases. The aqueous phase was transferred (~600μl) to a new 1.5 ml RNase-free microcentrifuge tube.An equal volume of a mix of phenol:chloroform (1:1) was added to the aqueous phase, which was then mixed gently by inverting the tubes six to eight times. Then, the sample was centrifuged at 4°C for 5 min at 13000×g to separate the phases.The upper aqueous layer (~400μl) was carefully transferred to a new 1.5 ml RNase-free microcentrifuge tube, and an equal amount of isopropyl alcohol was added to the tube; then, the mixture was mixed gently by inversion with a swing type vortex mixer  at room temperature for 10 min.The mixture was transferred to an RNase-free spin column and centrifuged at 4°C for 1 min at 13000×g.The flow-through was discarded, a 500μl 75% (v/v) ethanol was added, and then the mixture was centrifuged at 4°C for 1 min at 13000×g.Step 8 was repeated  ethanol and then centrifuging at 4°C for 1 min at 13000×g).The flow-through was discarded, and the mixture was centrifuged at 4°C for 1 min at 13000×g to remove the traces of ethanol.The bottom tube was changed with a new 1.5 ml RNase-free microcentrifuge tube, and 30–50μl DEPC-treated water was added to the spin column.After incubating at room temperature for 2 min, the sample was centrifuged at 4°C for 1 min at 13000×g.To increase the RNA yield, the RNA solution was retransferred to the spin column and incubated at room temperature for 2 min.After centrifuging at 4°C for 1 min at 13000×g, the RNA samples were stored at −80°C.RNA isolation was carried out based on a protocol proposed by Ni  for RNA 260/A280 ratio was calculated to determine the purity of RNA sample. RIN values were determined on an Agilent 2100 Bioanalyzer using Expert software (Rev. B.02.08.SI648) for plant RNA [In order to verify the RNA integrity, 1μg total RNA was loaded on a standard 1.0% agarose gel, stained with ethidium bromide, and visualized using UV illuminator Red Alpha Innotech  . The quaALMT1 gene. The sequences were 5'- ATTGATCACGGCAGAGAG-3' for the forward and 5'-GGTGTACTCCATGACGACG -3' for the reverse primer.The SuperScriptIIReverse Transcriptase  was used to convert total RNA transcripts into 1st-strand cDNA as per the manufacturer’s instructions, utilizing RNase Inhibitor . A PCR primer pair that is universally applicable to plants was used for the amplification of the 18 (500μg/ml) and a reaction volume filled to 12μl with DEPC water. The contents were briefly centrifuged to collect all components at the bottom, and the tube was placed at 65°C for 5 min. The tube was removed and chilled immediately on ice. The contents of the tube were collected by brief centrifugation, and then 4μl 5×PrimeScript Buffer , 2μl 0.1 M DTT, 1μl RNase Inhibitor (Rasin), and 1μl SuperScript Reverse Transcriptase were added to the mixture. The reaction was performed at 42°C for 50 min. The reaction was inactivated by heating at 70°C for 15 min. Subsequently, the tube was removed and quickly chilled on ice. The cDNA could then be used as a template for amplification in PCR.A 20μl reaction volume was performed in a 0.2 ml DEPC-treated PCR tube by taking 5μl of RNA, 1μl of 10 mM dNTP Mix, 1μl of anchored oligo-(dT)2, 1μl of 10 mM dNTP Mix, 1μl of 10μM forward primers, 1μl of 10μM reverse primers, 0.4μl of 5 U/μl Taq DNA polymerase, 2μl cDNA template, and a reaction volume filled to 50μl with DEPC water. The PCR cycles were programmed as follows: initial denaturation at 94°C for 2 min followed by identical 40 cycles with 1 min denaturing at94°C, 1 min annealing at 52°C, and 1 min elongation at 72°C followed by a single final cycle of 5 min at 72°C. After the reaction, 10μl of assay mixture was used for agarose electrophoresis using TAE buffer (1×) following the usual procedure. The remaining products were stored at -20°C.PCR amplification of first-strand cDNA was performed using Taq DNA polymerase. In this experiment, only 10% of the first-strand reaction products were used for PCR. Higher volumes may not increase amplification and may result in decreased amounts of PCR product. Therefore, the reaction was made by using 5μl of 10×PCR buffer , 1.5μl of 50 mM MgClMethods for RNA extraction are usually evaluated in terms of quantity, quality, and integrity for RT-PCR, qPCR, cDNA library construction, and expression analysis with a proper representation of each expressed gene. However, Chinese fir is often rich in polyphenolics, polysaccharides, proteins, lipids, and several other secondary metabolites that interfere with RNA extraction by degrading or coprecipitating with RNA. For this particular reason, RNA isolation from Chinese fir tissues containing high levels of extractable polyphenolics , proteinIn addition, using the RNA isolation kit to extract the total RNA of Chinese fir tissues is expensive. Therefore, it is necessary to find an efficient, rapid, and reproducible method for isolating high-quality RNA from Chinese fir tissues. The current study describes a rapid, efficient and reliable method that allows the isolation of high-quality total RNA from Chinese fir tissues. The RNA prepared by this protocol was verified for high quality and quantity, and it was successfully used for downstream applications such as RT-PCR, qPCR, cDNA library construction, gene expression analysis, and RNA-seq (unpublished data).The success of total RNA isolation should be verified by testing the quantity, quality and integrity of RNA. The yields of total RNA (μg/g fresh wt.) isolated from the roots and leaves of Chinese fir are shown in Firstly, 1.0% agarose gel electrophoresis showed that ribosomal bands of 28 S (b), 18 S (c) and 5 S (d) were intact and bright, demonstrating that the total RNA isolated with our detailed method were not degraded, and no high molecular weight gDNA contamination was detected , and the target gene was amplified from cDNA by PCR . We have successfully used isolated RNA to generate high-quality RNA-seq libraries for mRNA (unpublished data).In conclusion, the method reported here is useful for RNA extraction from Chinese fir tissues rich in lipids, polyphenolics and other secondary metabolites.S1 Fig(TIF)Click here for additional data file.S2 Fig(TIF)Click here for additional data file.S3 Fig(TIF)Click here for additional data file.S4 Fig(TIF)Click here for additional data file.S5 Fig(TIF)Click here for additional data file.S6 Fig(TIF)Click here for additional data file.S1 Table(XLSX)Click here for additional data file.


Plant viruses recruit cellular translation factors not only to translate their viral RNAs but also to regulate their replication and potentiate their local and systemic movement. Because of the virus dependence on cellular translation factors, it is perhaps not surprising that many natural plant recessive resistance genes have been mapped to mutations of translation initiation factors eIF4E and eIF4G or their isoforms, eIFiso4E and eIFiso4G. The partial functional redundancy of these isoforms allows specific mutation or knock-down of one isoform to provide virus resistance without hindering the general health of the plant. New possible targets for antiviral strategies have also been identified following the characterization of other plant translation factors  that specifically interact with viral RNAs and proteins and regulate various aspects of the infection cycle. Emerging evidence that translation repression operates as an alternative antiviral RNA silencing mechanism is also discussed. Understanding the mechanisms that control the development of natural viral resistance and the emergence of virulent isolates in response to these plant defense responses will provide the basis for the selection of new sources of resistance and for the intelligent design of engineered resistance that is broad-spectrum and durable. Although plant viruses encode a number of essential proteins  their coding capacity is limited and they must rely on host factors for every stage of the infection cycle ,2,3. An 7G cap structure and is also enhanced by interaction of the polyA-binding protein (PABP) with their 3′ polyA tail. The large eIF4G scaffold protein binds to both eIF4E and PABP promoting circularization of the mRNA. The tight association between eIF4E and eIF4G constitute the core of the eIF4F complex. eIF4G also interacts with eIF4A, a DEAD-box ATPase and ATP-dependent RNA helicase. DEAD-box helicases are so-named because they contain a conserved Asp-Glu-Ala-Asp (DEAD) motif. eIF4A unwinds the mRNA to assist ribosome scanning. RNAs with long structured 5′ untranslated regions (UTR) are particularly dependent on eIF4A or other DEAD-box helicases. The eIF4G-eIF4A interaction is looser than that between eIF4G and eIF4E and although eIF4A is considered a component of eIF4F, it is easily lost during purification. Other DEAD-box RNA helicases such as Dhx29 and Ded1/Ddx3 also participate in mRNA translation and may have distinct specificities for mRNAs with specific features in their 5′ UTR. eIF4E, eIF4G, eIF4A and other DEAD-box RNA helicases are key translation factors frequently usurped/manipulated by viruses and are attractive targets for antiviral strategies (Met ternary complex) to the mRNA. As discussed below, a subunit of eIF3 has been implicated in the translation reinitiation mechanism of cauliflower mosaic virus (CaMV) polycistronic mRNA.Eukaryotic mRNA translation has been reviewed by others ,14 and wrategies A. The eIArabidopsis thaliana) [A. thaliana cells, only cognate complexes (i.e. eIF4E-eIF4G and eIFiso4E-eIFiso4G) accumulate in detectable amounts [A. thaliana causes severe defects in plant health. In contrast, knock-out or down-regulation of eIF4E or eIFiso4E is tolerated and plants show little signs of impairment [in vitro, although with lower binding activity than that shown for the cognate complexes, and these hetero-complexes are active in translation [in vitro [A. thaliana cells, eIF4A1/2 is the predominant helicase found in association with both eIF4F and eIFiso4F complexes [In plants, two isoforms of eIF4F are present: eIF4F, which consists of eIF4E and eIF4G, and eIFiso4F, which includes eIFiso4E and eIFiso4G ,16,17,18 amounts  and in w amounts . Knock-opairment ,22. Intepairment . Indeed,nslation . In spitin vitro . As willomplexes . In quieMet) and delivers these tRNAs to the elongating ribosome. As discussed below, eEF1A is also frequently found in association with the replication complexes of positive-strand RNA viruses  allow continued synthesis of the nascent polypeptide chain  Figure B. Eukary viruses . eEF1A fA. thaliana cultured cells, eIF4E is predominantly cytoplasmic although its partition to the nucleus is regulated by the cell growth cycle [In addition to their roles in the canonical translation of cellular mRNAs, translation factors have other biological functions that are of interest for viruses. In mammalian cells, a sub-population of eIF4E accumulates in nuclear bodies and has been implicated in the regulated nuclear export of mRNAs that contain a specific structure (the 4E-sensitivity element) ,26,27. Ith cycle . eIFiso4th cycle ,29. The th cycle . eEF1A, th cycle . VirusesAs mentioned above, viruses depend on cellular translation factors to synthesize their proteins. The RNAs of positive-strand RNA viruses differ from plant mRNAs in that they lack the 5′ cap structure and/or the polyA tail. Viral RNAs often have highly structured 5′ UTRs with multiple stem-loops or pseudoknots that are necessary for other steps of the infection cycle  and that can interfere with normal scanning of ribosomes. Finally, some viruses have polycistronic RNAs that require translation reinitiation mechanisms or internal ribosome entry sites to facilitate translation at downstream open reading frames (ORFs). Thus, viruses have developed a variety of non-canonical mechanisms to translate their RNAs. The reader is referred to a collection of excellent recent reviews that describe these mechanisms in detail ,34,35,36cis-acting translation enhancing sequences (CITE) present on the viral RNAs. These cis-acting sequences include internal ribosome entry sites (IRES) in the 5′ untranslated region (UTR) or in intergenic regions, and other CITE sequences in the 3′ UTR or even within an ORF. Long-distance kissing loop interactions between cis-acting sequences in the 5′ and 3′ UTRs are often required for translation enhancement by promoting circularization of the RNA. A natural melon recessive resistance gene to melon necrotic spot virus  corresponds to a mutation of eIF4E that disrupts its interaction with 3′ CITE sequences [Nicotiana benthamiana was recently reported to be caused by an incompatibility between the viral RNA and eIF4E [One common strategy used for the translation of non-canonical viral RNAs is the recruitment of initiation factors  or ribosomal RNAs by equences  and willnd eIF4E . Potyviridae and Secoviridae and of the genus Sobemovirus are covalently linked with a small protein (termed VPg) at their 5′ end [A. thaliana and Chenopodium foetidum was attributed to the inability of this isolate to recruit translation initiation factors [in vitro, thereby facilitating their translation [in planta [A. thaliana T-DNA insertion lines revealed the dependence of potyviruses on specific eIF4G isoforms that work concomitantly with the corresponding eIF4E isoform [A. thaliana eIFiso4E deficient plants is not correlated with defects in viral RNA translation [in vitro, and although the eIFiso4E-binding domain was within the protease region, binding to eIFiso4E was enhanced by the presence of the VPg [The uncapped RNAs of some viruses, notably members of the families r 5′ end . Althougr 5′ end ,40,41,42r 5′ end , was latr 5′ end . As will factors . Potyvir factors . The VPgnslation ,46,47. En planta . In addin planta . In addi isoform , suggestnslation . Thus, anslation . This isnslation . As will the VPg . Howevercis-acting sequence present only in the 5′ UTR of RNA2. Thus the translation of RNA1 and RNA3 was not affected by DED1. Of interest, mutation of DED1 (ded1-18 mutant with two amino acid substitutions) hinders the accumulation of the 2a RdRp and reduces virus replication but does not affect yeast mRNAs translation or yeast growth. Although this is an attractive candidate for bromovirus resistance, it is not known whether mutation of an equivalent helicase in plant would have a similar effect on BMV accumulation. Knock-down of the yeast DED1 also impacted the translation of tomato bushy stunt virus  RNA [Using a yeast replication model, DED1, a DEAD-box helicase related to eIF4A and implicated in yeast mRNA translation, was shown to facilitate the translation of the RNA2-encoded viral RNA-dependent RNA polymerase (RdRp) from brome mosaic virus  . DED1 inrus) RNA . InteresThe RNAs of several plant viruses  have a tRNA-like structure at their 3′ end rather than a polyA tail . These sin vitro and in vivo. An A. thaliana T-DNA insertion line that prevent the expression of RISPa, the gene encoding RISP, shows only modest resistance to CaMV, probably due to the presence of a second highly homologous gene (RISPb) with overlapping function [RISPa and RISPb is not viable. The A. thaliana 35-7 line, which is silenced for TOR expression by RNA interference (RNAi), seemed more promising as it displayed strong resistance to CaMV [A. thaliana lines silenced for TOR expression (including the 35-7 line) are partially resistant to watermelon mosaic virus (a potyvirus) but fully susceptible to TuMV [A distinctive feature of some viral RNAs is that they are polycistronic or contain small ORFs in their 5′ UTRs. Leaky scanning, ribosome shunting or reinitiation mechanisms allow tight regulation of the translation rate of downstream ORFs . The CaMfunction . Unfortu to CaMV . Interes to TuMV . Treatme to TuMV . Thus, mcis-acting sequences on viral RNAs (see below) or with viral proteins other than the RdRp, for example the potyvirus VPg protein. The TuMV VPg, or rather the VPg domain in the larger membrane-targeted 6K-VPg-protease polyprotein, was initially shown to recruit eIFiso4E to the VRC suggesting an as yet-undefined role for this factor in viral RNA replication [A. thaliana T-DNA insertion lines that knock-out the expression of PABP show increased resistance to TuMV without visibly affecting plant growth or health [In addition to their obvious role in viral RNA translation, cellular translation factors are recruited to membrane-associated viral replication complexes (VRCs) and regulate viral RNA replication. In many viruses, translation and replication are tightly linked and viral RNAs or proteins are brought to the VRCs co-translationally, along with translation factors ,68,69. Slication . Later, lication ,75,76. Dr health , providiA. thaliana RH20 DEAD-box helicases that, similarly to DED1, bind to the 3′ end of TBSV (−) strand RNA to stimulate (+) strand RNA synthesis [A. thaliana) also participate in the selective enhancement of TBSV (+) strand RNA synthesis by binding to another cis-acting replication enhancing element in the 5′ terminal region of the (−) strand RNA [N. benthamiana results in enhanced viral accumulation and symptom severity [DEAD-box RNA helicases, related to eIF4A, are recruited to tombusvirus VRCs . Contrarynthesis . A seconrand RNA . This alseverity . AlthougA. thaliana eIF4A genes [A. thaliana lines with T-DNA insertion in eIF4A genes could not be generated, likely because of pleiotropic effects of these mutations. However, knock-out of another related DEAD-box helicases, AtRH8, resulted in increased resistance to two potyviruses (PPV and TuMV) without visibly impacting the plant [Using a yeast-two hybrid screen, the VPg protein from PPV was shown to interact with a peach DEAD-box helicase (PpDXL) closely related to 4A genes . Homozyghe plant . Interache plant . Of prache plant . This doPotyviruses, tobamoviruses and tombusviruses recruit eEF1A to their VRCs ,80,81. Icis-acting element in the 3′ UTR of the RNA. These interactions are thought to stimulate the recruitment of viral RNAs to the replication complex. eEF1A also helps stabilizing the p33 accessory protein and enhances (−) strand RNA synthesis. Screening of eEF1A mutants in yeast identified several mutants that could not stimulate viral replication, thereby resulting in partial resistance to TBSV [N. benthamiana plants resulted in severe reduction of TBSV RNA accumulation and symptom amelioration. The silenced plants also displayed partial resistance to TMV, suggesting a potential for the development of broad-spectrum resistance [The multiple functions of eEF1A in tombusvirus RNA replication have been studied in details although primarily in the yeast model system ,83. eEF1 to TBSV ,83. Howe to TBSV . eEF1Bγ sistance . cis-acting element in the viral RNA (a pseudoknot in the upstream region of the 3′ UTR) [N. benthamiana, eEF1A is encoded by at least six genes corresponding to two groups of related sequences [In the case of tobamoviruses such as TMV, eEF1A also interacts with both the RdRp and with a  3′ UTR) ,85. In Nequences . Using tequences . Silenciequences . PartialMost interactions described above regulate the translation or replication of viral RNAs. However, in some cases, and as already discussed for the TMV-eEF1A interaction, other steps of the infection cycle are apparently regulated by translation factors. Plants deficient for eIF4E show restricted cell-to-cell movement of some potyviruses, possibly related to the microtubule-binding affinity of the eIF4G binding partner of eIF4E ,89. In aN. benthamiana plants infected with tomato ringspot virus [RNA silencing is a ubiquitous gene regulation mechanism and a well-documented antiviral defense in plants ,90,91. Sot virus . In thisTranslation repression has been best studied in fly and mammalian cells and is mediated by imperfect base-pairing of miRNAs to target mRNAs . In animin vitro study revealed several distinct translation repression mechanisms in plants [The mechanism of translation repression in plant cells is less well understood. Translation repression of cellular mRNAs by miRNAs or siRNAs depends on AGO1 or AGO10 and usually requires perfect or near-perfect base pair complementarity . There an plants . Consistn plants . The ideAs discussed above, translation factors influence multiple steps of the virus infection cycle, as exemplified for eIF4E, eIF4A-like helicases and eEF1A. This presents welcome opportunities to develop multifaceted antiviral strategies. However, in many cases mutations of translation factors also have unwanted consequences to the plant physiology. In fact, only in a few instances have manipulation of translation factors resulted in demonstrated applications for virus resistance in the field. In the next two sections, I will discuss well-documented examples of field resistance based on mutations of eIF4E or eIF4G isoforms. Potyviridae is one of the largest families of plant viruses and includes many economically important pathogens of cultivated crops, especially members of the genus Potyvirus [The family otyvirus . Cultivaotyvirus ,107. An otyvirus . This inpvr1 allele that conferred resistance to TEV were not strictly correlated with reduced cap-binding activity [Trans-complementation studies of naturally occurring mutated eIF4E alleles (pea sbm1 and lettuce mo1 resistance genes) with overexpressed wild-type or mutated forms of eIF4E have also shown that mutants of eIF4E isoforms with reduced interactions with potyviruses are not necessarily hindered in their ability to bind to the cap structure or to other translation factors [Natural recessive resistance to potyviruses is generally associated with mutations of eIF4E or eIFiso4E that hinder their interaction with the VPg protein. Most of these mutations have been mapped to two surface-exposed regions of eIF4E near the cap-binding pocket ,107. Howactivity . Trans-c factors ,110,111. factors ,115,116. factors ,118,119. factors ,122,123. factors . in vivo bimolecular fluorescence complementation assays or in vitro co-immunoprecipitation methods to identify the specific plant eIF4E isoform-VPg interaction for the virus under study can be a useful preliminary step before engineering potyvirus resistance based on manipulation of eIF4E isoforms. However, some potyviruses have been shown to recruit either eIF4E or eIFiso4E and down-regulation or mutation of both isoforms may be necessary for durable resistance [Brassica rapa lines have three copies of eIFiso4E and TuMV was shown to use at least two of these genes [The potyvirus VPg protein interacts specifically with either eIF4E or eIFiso4E and this specificity is influenced by the specific host-virus combination and can even vary from one virus strain to another ,125,126.sistance ,128,129.se genes . mo1 resistance gene was shown to be overcome by mutations in the CI protein in virulent lettuce mosaic virus (LMV) isolates [cyv2 resistance gene, although the mechanism is not known [Virulent potyvirus isolates have been described that overcome recessive resistance genes corresponding to eIF4E/iso4E alleles. In most cases, virulence has been mapped to mutations in the VPg protein ,131,132.isolates ,135. Theisolates . The CI-isolates ,137. Finot known . Taken tCapsicum species did not reveal clear trends, possibly because of pleiotropic effects of mutations in the VPg protein, making the prediction of virulence emergence difficult [Brassica rapa was shown to provide broad-spectrum resistance to TuMV isolates that may also be more durable.Can this information be used towards the generation of durable resistance? Analysis of the co-evolution between potato virus Y (PVY) and eIF4E recessive resistance alleles in ifficult ,139. Howifficult . OverexpPotyviridae but can also target a variety of (+) strand RNA viruses including carmoviruses, cucumoviruses, sobemoviruses and waikaviruses. This information is summarized in Natural or induced resistance conferred by mutations/knock-out of eIF4F isoforms (eIF4E or eIF4G) is not restricted to members of the family rymv1 naturally found in Oryza sativa (rymv1-2) or O. glaberrima  [rymv1-2 and rymv1-3 [rymv1-2 or rymv1-3 are distinct and very few isolates are able to overcome both alleles [rymv1-2-breaking isolate identified mutations in a region of p2a that is distinct from the VPg domain, although the biological function of this protein domain is not known [The natural resistance to a sobemovirus conferred by a mutation in eIFiso4G is an interesting case. Sobemoviruses although unrelated to potyvirids also have a VPg protein linked to the 5′ end of their RNAs see . Resistarymv1-5) ,161. The rymv1-3 ,164,165. alleles . In fact alleles . It was  alleles . In sobeot known .nsv1 eIF4E allele against melon necrotic spot virus  [in trans [N. benthamiana, a plant normally not susceptible to MNSV. Thus, N. benthamiana non-host resistance to avirulent MNSV was correlated with an incompatibility between the 3′ CITE and NbeIF4E [Luteoviridae [Another well-characterized natural recessive resistance is that of the melon viridae) . The MNSviridae) ,169. Thein trans . In cont NbeIF4E . Of partoviridae . Artificoviridae . In the oviridae . InterfaCharacterization of natural recessive virus resistance has provided new insights on the interactions between plant viruses and translation factors and on the selection pressures controlling the emergence of virulent isolates. In addition, the systematic characterization of interactions between viral RNAs or proteins and translation factors has identified new potential targets for antiviral resistance. As discussed in


T=3 capsid. This is the most compelling demonstration to date that this ssRNA virus, and by implications potentially very many of them, assemble via a PS-mediated assembly mechanism.Using RNA-coat protein crosslinking we have shown that the principal RNA recognition surface on the interior of infectious MS2 virions overlaps with the known peptides that bind the high affinity translational operator, TR, within the phage genome. The data also reveal the sequences of genomic fragments in contact with the coat protein shell. These show remarkable overlap with previous predictions based on the hypothesis that virion assembly is mediated by multiple sequences-specific contacts at RNA sites termed Packaging Signals (PSs). These PSs are variations on the TR stem-loop sequence and secondary structure. They act co-operatively to regulate the dominant assembly pathway and ensure cognate RNA encapsidation. In MS2, they also trigger conformational change in the dimeric capsomere creating the A/B quasi-conformer, 60 of which are needed to complete the  We have now provided mechanistic explanations for both these events.T=3 shellSingle-stranded, positive-sense RNA phages co-assemble their protective coat protein shells around copies of their genomic RNA. This is one of the 2 common virus assembly mechanisms, the other being formation of a procapsid and the subsequent introduction of the viral genome, most commonly in the form of dsDNA, via specialized protein machinery. Spontaneous co-assembly is an essential feature of the lifecycles of a very wide range of important pathogens in every kingdom of life.PSs facilitate virion assembly: The plant satellite virus, Satellite Tobacco Necrosis Virus (STNV) also assembles using a PS-mediated mechanism. In that case the capsid is a T = 1 shell that encompasses a genome encoding only the CP gene, flanked by untranslated regions that enhance the efficiency of its replication and translation. STNV relies on a helper virus, Tobacco Necrosis Virus, for production of a replicase. In a T = 1 shell all the capsomeres, coat protein subunits, in the shell are in identical conformations. However, the STNV CP is similar to very many plant virus coat proteins, with a highly basic N-terminal region that is on the interior of the capsid and expected to interact with the RNA genome. Such electrostatic interactions were thought to provide the driving force for assembly, with the positively charged amino acids in the CP overcoming mutual repulsions between segments of the RNA chain. Using single molecule fluorescence correlation spectroscopy (smFCS) assays of STNV reassembly  that both serves as the attachment point to the initial bacterial receptor, the pilus  and guidThis is a startling prediction, but there is independent evidence that this is true. An asymmetric tomographicT = 3 icosahedral surface lattice except in the one capsomere position taken up by the MP. Such asymmetric capsid features have implications for capsid stabilityThe occurrence of such genome un-coating intermediates highlights the fact that all virions are asymmetric objects despite the highly symmetrical coat protein shells they assemble. The asymmetry comes in many forms: The genome is an asymmetric object, used in PS-mediated assembly to facilitate the construction of a symmetrical shell. PS-CP contacts formed impart asymmetric properties to each CP or capsomere contacted. Since PSs are sequence/structure degenerate their affinities for CP vary and hence they have differing impacts on CP dynamics. Another explicit form of asymmetry arises when the capsid shells contain unique structural features. For the RNA phages this comes in the form of a unique copy of a maturation protein (MP). The MS2 MP also has specific RNA binding sites on the viral genome located toward each end of the RNA, in effect making it circular and thus reducing the complexity of the potential conformations of the packaged genome, which has significant impact on virus assembly kinetics.The new cross-linking studiesPSs must be appropriately presented for CP recognition, and in the examples studied to date this is in the form of an RNA stem-loop. Such structures are transient. Indeed, in isolated oligos their free energy of folding may be positive. They are also in competition with other RNA folds required to promote replication or translation. A question therefore arises as to the mechanism of PS formation. We tested this directly using lead-ion RNA structure probing of CP-free RNAs and the genomic RNA within virions. The data suggest that some PSs are present before encapsidation while others form during the assembly process. Since the RNA exists as an ensemble of folded states, the functional folds of the PSs must flicker in and out of existence until CP binding and initiation of assembly.The assembly of ssRNA viruses, one of the largest classes of viral pathogens that infect organisms in all kingdoms of life, is regulated by multiple, sequence-specific RNA-CP contacts making the process highly co-operative, accurate and efficient. This mechanism is probably also vital for the earliest stages of new infections. These principles have largely been worked out using the RNA phage MS2 which has already provided many firsts in the history of molecular biology, e.g. the first complete genome sequence.