The fate of the motor upon reaching the synapse is not known. We found that wild-type UNC-104 is degraded at synaptic regions through the ubiquitin pathway and is not retrogradely transported back to the cell body. As a possible means to regulate the motor, we tested the effect of cargo binding on UNC-104 levels. The unc-104(e1265) allele carries a point mutation (D1497N) in the PIP2 binding pocket of the PH domain, resulting in greatly reduced preferential binding to PIP2in vitro and presence of very few motors on pre-synaptic vesicles in vivo. unc-104(e1265) animals have poor locomotion irrespective of in vivo PIP2 levels due to reduced anterograde transport. Moreover, they show highly reduced levels of UNC-104 in vivo. To confirm that loss of cargo binding specificity reduces motor levels, we isolated two intragenic suppressors with compensatory mutations within the PH domain. These show partial restoration of in vitro preferential PIP2 binding and presence of more motors on pre-synaptic vesicles in vivo. These animals show improved locomotion dependent on in vivo PIP2 levels, increased anterograde transport, and partial restoration of UNC-104 protein levels in vivo. For further proof, we mutated a conserved residue in one suppressor background. The PH domain in this triple mutant lacked in vitro PIP2 binding specificity, and the animals again showed locomotory defects and reduced motor levels. All allelic variants show increased UNC-104 levels upon blocking the ubiquitin pathway. These data show that inability to bind cargo can target motors for degradation. In view of the observed degradation of the motor in synaptic regions, this further suggests that UNC-104 may get degraded at synapses upon release of cargo.UNC-104/KIF1A is a Kinesin-3 motor that transports synaptic vesicles from the cell body towards the synapse by binding to PIP Caenorhabditis elegans model system, we have demonstrated for the first time the fate of a motor after it carries its cargo to the synapse from the cell body. We show that the UNC-104 motor, which carries pre-synaptic vesicles to the synapse, is degraded once it gets there. Moreover, our genetic studies show evidence that loss of cargo binding targets the motor for degradation, suggesting an attractive mechanism for the regulation of motors at the synapse. Our study opens up several further questions, such as the mechanism of motor degradation, and has significant implications for regulation of cargo transport.The cell body and the synapse in a neuron are often separated by significant distance, which is spanned by the axon connecting the two. Transport of various cargoes along the axonal highway is very important for neuronal function. The regulation of this complex process is not well understood. Using the  C. elegans, Drosophila, mouse and humans C. elegans, mutants in unc-104 have locomotory defects that arise from the absence of transport of synaptic vesicles, leading to reduced synaptic transmission at neuromuscular junction synapses Transport of pre-synaptic vesicles from the neuronal cell body to the synapse is an essential process to ensure that the nerve terminals can effectively participate in synaptic transmission 2 present on the carrier vesicle via its PH domain Molecular motors in neurons such as UNC-104 are thought to bind to their cargoes in the cell body of the neuron, get transported along microtubule tracks to synapses and release their cargo upon reaching the synapse 2 efficiently have also been suggested to affect processivity of the motor Several effects of cargo binding on the Kinesin-3 family motors have been shown. Cargo binding by a chimeric Kinesin-3 leads to aggregation of the motor on the cargo surface and improved processivity of the chimera Cargo release has also been postulated to play important roles in motor regulation C. elegans UNC-104 motor protein. To do so, it is necessary to address the following two questions. 1) Does the motor get degraded at the synapse? 2) Does the motor get degraded once there is no binding to the cargo? To answer the first question, we established that the wild type motor is degraded in synaptic regions and that it does not return to the cell body from the synapse. We further showed that the degradation near the synapse takes place through the ubiquitin pathway. To address the second question, we studied the effect of lack of cargo binding on the C. elegans UNC -104 motor protein. For this we used a mutant UNC-104 motor and showed that it has greatly reduced ability to preferentially bind PIP2in vitro as well as greatly reduced presence on pre-synaptic vesicles in vivo. We found that this leads to almost total loss of the motor in vivo, even though the motor still retains the ability to bind other lipids. The relationship between ability to bind cargo and motor levels was verified by analyzing two intragenic suppressors of the original mutation in the PH domain. The suppressors only moderately reduce the ability to preferentially bind PIP2 and we see that UNC-104 levels are partially restored. All three PH domain variants of the motor are degraded via the ubiquitin pathway in synapse rich regions of the animal. A triple mutant reversing the effect of one of the suppressor mutations again does not preferentially bind PIP2in vitro, does not provide behavioural rescue and does not show expression of UNC-104 in vivo. These findings, together with the observed degradation of wild type UNC-104 in synaptic regions, suggest that the synaptic vesicle motor UNC-104 is degraded upon release from pre-synaptic vesicles near the synapse.We sought to test this hypothesis for the C. elegans. We examined the posterior neurons (PLM) whose morphology and synaptic locations are very well defined in vivo function or localization. In a wild type background the UNC-104::GFP is present in the cell body, neuronal process and at synaptic regions (To determine whether the UNC-104/KIF1A motor is degraded at synapses we used a transgenic line over-expressing UNC-104::GFP in the six mechanosensory neurons of  regions  b1-b3.uba-1(it129ts) mutant. uba-1 encodes the only C. elegans E1 ubiquitin activating enzyme uba-1 animals ubiquitin-mediated degradation is reduced. At the lower growth temperature of 16°C the expression of UNC-104::GFP in uba-1 animals is not significantly different from wild type in the cell body, neuronal process or at synaptic regions  while there are significant effects on the levels of expression of the synaptic vesicle motor UNC-104 at synaptic regions.To confirm that the morphology of the mechanosensory neuron (including its synapses) is relatively unaffected in  animals . CompareC. elegans extract. Western blot analysis of immunoprecipitated UNC-104 motor showed that the same band of about 200 kDa was recognized by both the anti-UNC-104 and the anti-ubiquitin antibodies  molecules to the motor. To test if UNC-104 is ubiquitinated we immunoprecipitated the endogenous UNC-104 motor (approximately 200 kDa) from a mixed-stage tibodies . Furthertibodies . Howeveruba-1 animals and also did bleach recovery experiments to assess UNC-104 motor flow.Our observation that the motor reaching the synapse gets degraded predicts that there would be little retrograde transport of UNC-104 from the synapse back to the cell body. To test this hypothesis we carried out a transport assay by laser microsurgery of the mechanosensory neuron. We had observed that 1 hour after axotomy, cargoes such as GFP::RAB-3 and SNB-1::GFP accumulate on both sides of the cut site uba-1 animals, one hour after laser axotomy of mechanosensory neurons expressing UNC-104::GFP, showed robust levels of UNC-104::GFP at the proximal regions , a canonical allele, showed a single amino acid change D1497 to N in the PH domain P2 on to the homology model using GRAMM 2 interactions.We first attempted to identify a pre-existing allele affecting cargo binding by sequencing several  alleles . Of thesH domain . To testP1/PHISH  [32], , animals  [38]. In animals .unc-104(e1265) the marker GFP::RAB-3 accumulates in the cell body with nearly no protein present at synapses. In both intragenic suppressors more GFP::RAB-3 is present in synaptic regions with lower accumulations in the cell body (unc-104(e1265) (unc-104(e1265) animals (unc-104(e1265) but only moderately in the suppressors (Another synaptic vesicle marker GFP::RAB-3 ell body . Consist4(e1265) , S3, but4(e1265) . There i animals , suggestpressors . The retpressors , suggestThus both intragenic suppressors that map to the PH domain improve behaviour and cholinergic synaptic transmission by increasing the transport of cargo in the axon and the number of synaptic vesicles at the synapse.unc-104(e1265). The R1501Q mutation in unc-104(e1265tb107) is also on the surface of the PH domain (2 binding pocket. The R1501Q may reverse the loss of charge in the PH domain variant encoded by unc-104(e1265) through compensatory local short range interactions. The compensatory change M1540I in the unc-104(e1265tb120) suppressor lies ∼20 Å from D1497N and is likely to mediate any possible effect on PIP2 through long-range interactions P2 binding P2 resulting from neuronal over-expression of ppk-12. The previously described 2 levels in vivo P in vivo , suggestin vitro and in vivo data, we observe that pre-synaptic vesicles prepared from unc-104(e1265tb120) animals have larger amounts of UNC-104 present on them than those prepared from unc-104(e1265) animals (unc-104(e1265), the proteins encoded by the intragenic suppressors (1) partially restore preferential PIP2 binding in vitro, (2) are sensitive to PIP2 levels in vivo (3) have more UNC-104 molecules on pre-synaptic vesicles and (4) facilitate transport of synaptic vesicles to synapses through an improved ability to bind cargo vesicles, leading to improved behaviour.Consistent with the  animals . Both ge animals . Thus, cunc-104(e1265) are greatly reduced compared to wild type animals and residual protein is still localized in the synapse rich regions of the nerve ring and ventral cord (unc-104(rh43), which encodes the motor UNC-104(G96E G314E) with a mutation in the ATP binding pocket of the motor domain P2 in intragenic suppressors, along with improved behaviour Ppressors : d,e,i,jpressors : d,e,i,jehaviour , we see ehaviour . Again tehaviour . This isehaviour in vivo levels of the UNC-104 motor are directly related to its ability to bind pre-synaptic vesicles through PIP2, suggesting a link between specific binding of a motor to its cargo and levels of the motor in neurons.Taken together, our observations show that the unc-104 variants are due to its degradation. To rule out reduction in transcripts, we measured RNA levels of UNC-104 using real-time PCR. We saw no change in UNC-104 RNA levels between wild type and unc-104(e1265) animals  animals.We wished to test if the reduced UNC-104 levels in the  animals . To studovements . Thus miunc-104 allelic variants we built double mutants between these variants and the temperature sensitive allele of the E1 Ubiquitin ligase uba-1(it129ts)unc-104(e1265); uba-1 animals grown at 22°C compared to unc-104(e1265) animals grown at the same temperature  and unc-104(e1265tb120) in the uba-1 background P2 binding, we made low copy number transgenic lines of several UNC-104 variants by bombardment into unc-104(e1265) animals. The UNC-104 motor variant transgenic lines were made using wild type UNC-104, UNC-104(D1497N), UNC-104(D1497N R1501Q), UNC-104(D1497N M1540I) with and without a GFP fused to the C-terminus expressed under the control of the unc-104 promoter. The GFP containing and GFP lacking transgenic lines behaved identically in both locomotion and aldicarb resistance assays, suggesting that addition of GFP did not alter the function of the UNC-104 variants Pvariants . All varvariants . All travariants . All travariants . Howevervariants : c1,c2. variants . We thinvariants . However2 binding is co-related to in vivo motor levels, we mutated the W1549 to A. The intragenic suppressor M1540I carries out its suppression indirectly. This residue is ∼2.4 Å away from the highly conserved Trptophan at 1549. In the homology model, I1540 orients its β carbon methyl group towards W1549, which in turn lies close to KK1463/4 (∼7.8 Å/3.2 Å respectively) P2To further confirm that the ability to maintain preferential PIPctively) . We predin vitro lipid binding specificity of an UNC-104 PH domain carrying (D1497N M1540I W1549A) mutations and observed that this triple mutation abolishes the preferential PIP2 binding in vitro while increasing binding to PC and PI, and behaves similarly to the D1497N mutation alone . The UNC-104(W1549A)::GFP lines exhibit wild type locomotory behaviour, sensitivity to aldicarb and motor expression levels and localization (in vivo (unc-104(e1265) animals carrying this transgene continue to be resistant to aldicarb ::GFP and UNC-104(D1497N M1540I W1549A)::GFP into lization : j1,j2, P2 and hence pre-synaptic vesicles, leading to corresponding failure of pre-synaptic vesicles to reach synaptic regions. In these mutants, levels of the UNC-104 motor depend on its ability to bind cargo and moreover the motor is degraded in synapse rich regions of the nervous system. These data together provide support to the hypothesis that the UNC-104 motor is degraded at synaptic regions upon releasing its synaptic vesicle cargo.We provide the first evidence that the C. elegans neurons  and its two intragenic suppressors, the ability to bind PIP2 determines the levels of motors on pre-synaptic vesicles in vivo, the extent of transport of synaptic vesicle proteins to the synapse, and hence the extent of locomotion and of synaptic transmission. We think that the primary defect in these unc-104 mutants is differential abrogation of cargo binding ability, rather than other effects such as altered localization, motility, folding or stability of the mutant motor. The fact that over-expressed UNC-104(D1497N)::GFP and over-expressed UNC-104::GFP localize and move similarly in vivo  in UNC-104 expression in these regions after blocking ubiquitin-mediated degradation . The D14uba-1 is a mild temperature sensitive mutant providing sufficient function for viability of the uba-1 animals. This is also the probable reason behind the apparent lack of change seen in endogenous UNC-104 levels in uba-1 mutants alone in these assays  as the closest homolog. The two sequences were then aligned using CLUSTAL W (EBI server) and carefully adjusted using manual intervention, to ensure maximum conservation of motifs and minimal gap regions. A homology model was then generated using MODELLER v7.0 2 using GRAMM A BLAST search of the UNC-104 PH domain sequence against the RSCB protein data bank identifies DAPP1/PHISH , purified by Ni-NTA chromatography (QIAGEN) and kept frozen in 10mM Tris pH 8.0, 4mM EGTA, 5% sucrose.The starting constructs for all was an UNC-104 PH domain fused in frame to GFP 2 (Cat. no. 840046), PA (Cat. no. 840101), PI (Cat. no. 840044) and BL (Cat. no. 131101). Composition of brain lipids (BL) contains Phosphatidylethanolamine (16.7%), Phosphatidylserine (10.6%), Phosphatidylcholine (9.6%), Phosphatidic acid (2.8%), Phosphatidylinositol (1.6%) and others (58.7%).The followings lipids were purchased from Avanti Polar lipids. Egg PC (Cat. no. 840051), PI(4)P (Cat. no. 840045), PIP2 or brain lipid (BL) in chloroform. After mixing the desired and carrier lipids, chloroform was evaporated under a constant Nitrogen gas stream. Once the lipid film was dried completely, lipids were rehydrated by the addition of LB buffer . These lipids were sonicated (ultrasonic bath) for 30 seconds to break up the lipid aggregates and were then extruded through a 100 nm pore polycarbonate filter  using a miniextruder from Avanti polar lipids. The liposomes were stored in the dark at 4°C and used within a week of preparation.5 µM concentration of the desired lipids was used to prepare liposomes in the following ratio: 10% desired lipid and 90% carrier lipids. Phosphatidylcholine (PC) was used as a carrier lipid and the remaining 10% of the lipids used were either Phosphatidylinositol (PI), Phosphatidylinositol-4-phosphate PI(4)P, Phosphatidylinositol-4,5-bisphosphate PIPav (4°C) for 45 min in a TLS-120 rotor (Beckman). After centrifugation, fractions from the pellet that contains liposome bound protein and supernatant that contains unbound protein were collected. Samples were dissolved in 20 µl LB buffer and analyzed by SDS-PAGE followed by Coomassie staining. Gels were digitized on a flatbed scanner and protein bands were quantified using ImageJ . Binding specificity was determined by normalizing binding observed with PIP2 and brain lipid compared to binding observed using PC alone carrier liposomes.Liposomes were prepared as previously described unc-104(e1265) worms were washed with M9 buffer, using sterile glass pipettes. Washed worms were transferred into a tube of 1x PBS containing ethyl methanesulfonate (Sigma) at a final concentration of 50mM. Tubes were kept in a rotary shaker at 20°C for 4 hours. After mutagenesis, 3-4 worms were transferred each 60 mm Petri plate. F1 and F2 progeny were regularly examined under a Nikon SMZ645 dissecting microscope for improved locomotion in a non-clonal screen of approximately 60,000 haploid genomes. Intragenic suppressors were identified in genetic crosses that mapped them close to the unc-104 locus. Intragenic suppressors of unc-104(e1265) isolated were unc-104(e1265tb107) and unc-104(e1265tb120). Throughout the paper, proteins encoded by these alleles are referred to as UNC-104(D1497N), UNC-104(D1497N R1501Q) and UNC-104(D1497N M1540I) respectively.L4 1 day adult hermaphrodites were transferred on to a fresh NGM agar plate, allowing them to acclimatize for 1 hour. Movement was recorded on a Nikon SMZ800 dissecting microscope at 1 to 1.3 frames per sec (1000×1000 pixels) for 2-3 min with a cooled monochrome camera . Movement was tracked manually using ImageJ  software. Worms that moved for a minimum of 10 frames were tracked. Worm velocities were obtained by calculating the straight line distance between the centroid positions of the worm in a given interval.Aldicarb plates were prepared by adding aldicarb  solution (in 70% ethanol) to NGM agar. These plates were seeded with OP50 bacteria. All assays were performed on 1 day old adult hermaphrodites at room temperature (21-23°C). 30 individuals were incubated for 6-8 hr on aldicarb plates of defined concentration. At 30 min intervals each worm was touched with a platinum wire and was checked for paralysis unc-104 promoter driving the combination of intronless and genomic region of unc-104 and provides the entire open reading frame of the protein. Mutations were introduced using site directed mutagenesis using the Stratagene QuickChange protocol with TaKaRa Ex Taq. Various point mutations  were generated. All constructs were verified by DNA sequencing. GFP was deleted from UNC-104::GFP, UNC-104::GFP(D1497N), UNC-104::GFP(D1497N R1501Q) and UNC-104::GFP(D1497N M1540I) using the restriction enzymes Apa1 and Kpn1. After T4 DNA polymerase treatment, ligation was done using T4 DNA ligase.A wild type UNC-104::GFP construct was provided by Jon Scholey E.coli Strain OP50 under standard laboratory conditions . Strains used in the study, provided by the Caenorhabditis Genetics Center (CGC), are as follows: wild type N2, unc-104(e1265), unc-104(rh43), unc-104(rh142).Worms were grown at 20°C on NGM agar plates seeded with juIs1(punc-25-SNB-1::GFP) a transgenic strain expressing green fluorescent protein (GFP)-tagged synaptobrevin-1 in GABA motor neurons psnb-1::snb-1::GFP)jsIs1  a transgeneic strain expressing soluble GFP in mechanosensory neurons jsIs821(pmec7::GFP::RAB-3) a transgenic strain expressing GFP tagged RAB-3 in mechanosensory neurons trIs25  has Membrane–anchored yellow-fluorescent protein expressed in body wall muscles prab-3::gfp::rab-3)jsIs682P2 biosynthetic enzyme Type I PIP kinase ppk-1 in all neurons uba-1(it129ts) is a temperature sensitive mutant allele in the E1 ubiquitin activating enzyme pmec4::UNC-104::GFP)js1111 ( a transgenic strain that expresses UNC-104::GFP only in mechanosensory neurons.tbIs183UNC-104- 5 transgenic lines- tbIs147UNC-104::GFP- 5 transgenic lines- tbIs188. This transgene rescues unc-104(rh142), the lethal null alleleUNC-104(D1497N)- 3 transgenic lines- tbIs149. This transgene provides viability to unc-104(rh142), the lethal null alleleUNC-104(D1497N)::GFP- 3 transgenic lines- tbIs194UNC-104(D1497N R1501Q) - 3 transgenic lines- tbIs152UNC-104(D1497N R1501Q)::GFP- 3 transgenic lines- tbIs191UNC-104(D1497N M1501I)- 4 transgenic lines- tbIs156UNC-104(D1497N M1501I)::GFP- 3 transgenic lines- tbIs157UNC-104(M1540I)::GFP-13 transgenic lines- tbIs170UNC-104(R1501Q)::GFP- 4 transgenic lines- tbIs199UNC-104(D1497N M1540I W1549A)::GFP- 5 transgenic lines- tbIs181UNC-104(W1549A)::GFP- 2 transgenic lines – Underlined strain was most commonly used, at least one other transgenic strain was assayed in all assays and no co-injection marker was used to make the above transgenic animals.C. elegans unc-104(e1265) hermaphrodites was carried out using a BioRad Biolistic PDS-1000/HE particle delivery system  -104(e1265) L4. Worms were allowed to recover for 0.5 to 1 hr after bombardment and were then transferred on to 100mm seeded Na22 plates and grown at 20°C. After 8-12 days worms were screened for improved movement and/or GFP expression as examined using a Zeiss fluorescence microscope. Individual animals were cloned. Homozygous stable lines were identified by the complete absence of unc-104(e1265) mutant progeny over several generations unc-104(e1265) as the background for bombardment since this was the healthiest hypomorphic allele of unc-104 available.Micro particle bombardment of For quantitation of SNB-1::GFP puncta at motor neuron synapses synaptic, unsaturated images of immobilized worms were taken in the linear range of exposure and quantified using ImageJ (NIH) similar to what has been described in in vivo live imaging, young adult hermaphrodites were immobilized with 3-5mM levamisole (Sigma-Aldrich) in M9 and mounted on a 2% agarose pad. Time-lapse images of anterior mechanosensory neurons expressing GFP::RAB-3 were obtained with OLYMPUS IX81 using 100X/1.4 NA plain Apochromat objective attached with spinning disk confocal head (YOKOGAWA CSU22) equipped with EMCCD camera (ANDORiXon-897EMCCD). Time-lapse images (512×512 pixels) were taken at a constant frame rate of 6-7 frames per second. Image analysis was done using Image J . Kymographs were obtained from lines that were drawn along the axon from cell body towards synapse. Flux analysis was carried out within a range of 15-20 µm along the axon length, at a distance of 15-25 µm away from the cell body. Flux was calculated as number of anterogradely moving particles in a movie. Any particle static for 3 frames or with velocity less than 0.3 µm/s was considered as stationary. Pause frequency was calculated as the number of pauses taken by a particle for unit distance traveled .For All significance was calculated using pair-wise comparisons using the Student's T-test with unequal variance. p values less than 0.05 were considered as significant.unc-104(rh142), a null allele. All monoclonal antibodies tested showed pan-neural staining in wild type animals and no staining in the unc-104(rh142) animals (The protein region of UNC-104 (amino acid 740-1117) was cloned into pRSETA vector (Invitrogen) using standard techniques. Protein was expressed in BL21 cells (Invitrogen), and purified using Ni-NTA chromatography (QIAGEN). Purified protein was given to Bioklone, Chennai, India to generate monoclonal antibodies. Specificity of the antibodies was tested by immunostaining  animals .oC and freeze-thawed using liquid nitrogen and fixed for an additional 10 minutes at 4° C. Following this 4-5 washes with 0.5% BT buffer  and then 5-6 washes (1 hour each) with 0.5%BTB (BT with 2% mercaptoethanol) were carried out. Blocking was done with PBST . Samples were incubated two overnights with monoclonal anti-UNC-104 antibody (1:5), washed for 4-5 times with PBST (each of 15 minutes) before mounting. Rabbit anti-syntaxin was used at 1:10,000 Animals were fixed with 2% paraformaldehyde for 10 minutes at 4Western sample of worms were prepared by sonication. After sonication, worm lysates were boiled with SDS lysis buffer and proteins were separated on SDS PAGE (8% acrylamide). Proteins were transferred to a nitrocellulose membrane (Amersham), probed with a mouse serum or a mouse monoclonal antibody of anti-UNC-104 (1:60), rabbit anti-tubulin  (Thermo-scientific), rabbit anti-synaptobrevin (1:5000) Worms of respective genotypes were anesthesized in 5mM levamisole. Photobleaching experiments were done on confocal Zeiss LSM-5 Live (line scanner) equipped with a 63X objective  with a 488 nm solid state laser. Images were acquired on a CCD camera at the frame rate of 4 Hz. 35-40 µm of the axon was bleached across the synaptic branch. Fluorescence recovery was quantified from the distance covered by the UNC-104::GFP signal in bleached axons at fixed times after bleaching. The fluorescent recovery along the anterograde and retrograde directions was represented as velocity in both anterograde (recovery from cell body) and retrograde (recovery from synapse) directions. All the analysis was done using ImageJ version1.41 (NIH).jsIs1 and various unc-104 mutants in the jsIs1 background. The worms and various mutants were grown on 10-15 large plates until food was exhausted. Worms were mechanically homogenized in homogenization buffer  and mildly sonicated at 4°C. The final supernatant was centrifuged at 50,000g for 40 min in a TLA 100.3 rotor to clear debris and heavy membrane fractions. The supernatant was collected again and centrifuged at 175,000g in TLA100.3 rotor for 150 min. The final pellet was resuspended in homogenization buffer or IP buffer  as needed.N2 worms were used for immunoprecipitation. For sedimentation assays we used For immunoprecipitation the high speed re-suspended pellet was incubated with specific antibody for 5-6 hrs at 4°C. Final concentration of UNC-104 antibody used was 1:10 and ubiquitin antibody (Sigma-Aldrich) used was 1:10. Protein A agarose beads were added to the antigen-antibody mixture and incubated for 3-4 hours at 4°C. The beads were centrigufed, washed with IP buffer then analyzed by western blotting. A Western analysis was carried out on immunoprecipitated material using the anti-UNC-104 antibody and anti-ubiquitin antibody. The blot was first probed for UNC-104 and then stripped  and re-probed for ubiquitin (1:500) (Sigma-Aldrich). The anti-ubiquitin antibody recognized the same band detected by anti-UNC-104. A Western analysis was carried out on immunoprecipitate obtained using the anti-ubiquitin antibody. This blot was probed using anti-UNC-104 and a band that migrates at the same size as endogenous UNC-104 was observed.For sucrose gradient density, the resuspended high speed pellets were loaded on a discontinuous sucrose gradient centrifugation  and centrifuged in a SW41 rotor at 60,000g for 120 min. Fractions were collected from top of the gradient up to the first layer (between 0.05M-0.6M). The last two fractions collected were below the formed layer where no synaptic vesicle proteins were detected. Western blot analysis with exposure maintained in the linear range was carried out on the fractions collected.Figure S1C.elegans (CeUNC-104). The different domains of C. elegans UNC-104 (as indicated from left to right in figure) are: Motor domain (aa 1-354), fork head-homology (FHA) domain (aa 463-592), homologous to liprin binding (LBD) region (aa 589-1267) and pleckstrin homology (PH) domain (aa 1460-1558). Details of mutations in the various alleles of unc-104 are shown in the table below and their relative positions have been marked in the schematic representation. The intragenic suppressor that encodes UNC-104(D1497N M1540I) was isolated three independent times and named sup1, tb101 and tb120. Of these the nucleotide change in tb120 differs from those in sup1 and tb101 although the aa change is identical. (B) Primary sequence alignment of the PH domains of the following UNC-104 family members C. elegans (CeUNC-104), Drosophila melanogaster (DmUNC-104/imac), H. sapiens (HsATSV), Mus musculus (MmKIF1A) and Dictostylium discoidum (DdUNC-104). The D1497N residue mutated in unc-104(e1265) is highly conserved. The two intragenic suppressors unc-104(e1265tb107) and unc-104(e1265tb120) have two compensatory mutations M1540I and R1501Q respectively. The R1501 is well conserved while the M1540 varies but is still maintained as an acidic/neutral residue. Other residues demonstrated to be important for PIP2 binding, KK1463/4, R1496 are also highlighted. In addition, another highly conserved residue W1549 that mediates the suppression of M1540I on N1497 has also been marked. (C) The RSCB protein data bank identifies DAPP1/PHISH  as the closest homolog with an E-value of 3.4 (A) A schematic domain representation  of (4.41 MB TIF)Click here for additional data file.Figure S2trIs25. Muscle arm number is altered in unc-104(e1265) as well as its suppressors. Muscle arm number is significantly decreased in unc-104(e1265) shown in (b) as compared to wild type (a) and partially restored in intragenic suppressors unc-104(e1265tb107) (c) and unc-104(e1265tb120) (d). The 9th to 11th muscles in the dorsal right quadrant are shown in all panels. Arrow points to muscles arms. Scale bar: 20 µm. (B) Quantitation of muscle arm numbers. Muscle arms are significantly reduced in unc-104(e1265), but are partially restored in intragenic suppressors unc-104(e1265tb107) and unc-104(e1265tb120). Data represented as mean ± SEM. *p<0.05 (C) Aldicarb paralysis assays of wild type, unc-104(e1265), unc-104(e1265tb107) and unc-104(e1265tb120) showing all time points assayed. (D) GFP::RAB-3 distribution in mechanosensory neurons using the transgenic line jsIs821. GFP::RAB-3 (pre-synaptic vesicle marker) distribution in NR and process of posterior lateral mechanosensory neuron (PLM process) shown respectively in wild type , unc-104(e1265) , unc-104(e1265tb107) , unc-104(e1265tb120) . When compared to unc-104(e1265) animals, increased signal resulting from greater transport was observed both in the NR and PLM processes of the suppressors. In  arrow points to the nerve ring and in PLM axon, the arrowhead and arrow mark the cell body and axon respectively. Scale bar: 10 μm. The alleles unc-104(e1265), unc-104(e1265tb107) and unc-104(e1265tb120) are labeled in the figure by the respective protein changes they encode, namely D1497N, D1497N R1501Q and D1497N M1540I.(A) Muscle arms are visualized using (3.08 MB TIF)Click here for additional data file.Figure S3ppk-1 in neurons resulting in 40% increase in in vivo PIP2 levels. (B1 and B2) Different transgenic variants of UNC-104::GFP  in an unc-104(e1265) background were tested for aldicarb analysis. We have shown data for two independently isolated transgenic lines for each UNC-104::GFP variant construct.  Different transgenic variants of UNC-104 lacking GFP  in an unc-104(e1265) background were tested for aldicarb analysis and locomotion . UNC-104 transgenes with and without GFP behave identically in these assays. (D) Quantitation of real time unc-104 RNA levels in wild type and unc-104(e1265). (n = 3 in duplicate). (E) Over expression of ppk-1 using gqIs125 also decreases the paralysis time in UNC-104(R1496A) and UNC-104(KK1463/4AA) transgenic lines. This demonstrates that the motors encoded by these transgenes are responsive to changes in PIP2 levels in vivo like unc-104(e1265tb107) and unc-104(e1265tb120). Data represented as (mean ± SEM) time taken to paralyze the 50% of the worms. (n = 30). (F) Western blot analysis using anti-UNC-104 antibody of intragenic suppressors with and without gqIs25 over expressing PIP2 in neurons. Control for protein loading is done using an anti-tubulin antibody. The alleles unc-104(e1265), unc-104(e1265tb107) and unc-104(e1265tb120) are labeled in the figure by the respective protein changes they encode, namely D1497N, D1497N R1501Q and D1497N M1540I. Aldicarb paralysis/resistance assays in different mutant backgrounds that over-express (1.17 MB TIF)Click here for additional data file.Figure S4unc-104(rh43) in nerve ring (shown by arrow) as compared to (c) unc-104(e1265). Arrow in B points to the cell body. Scale bar, 10µm. (B) Distribution of UNC-104 in wild type worms is pan-neurally expressed in synapse rich regions of the ventral cord and nerve ring (arrows), some commissural process and a few cell bodies near the nerve ring (arrowhead). Scale bar 10µm. (C) Expression of UNC-104::GFP with various PH domain mutations in the ventral cord, sub-lateral cords, commisures and dorsal cord. The UNC-104 motor with deletion of the PH domain sometimes lacks signal in the commisures (arrowhead). (D) In unc-104(rh142); jsIs682  worms on which specificity of 25H11MAb against UNC-104 was tested. UNC-104 immunoreactivity was absent in (a) whereas immunoreactivity for GFP from GFP::RAB-3 was present (b) in worms of the same background. Scale bar 15µm. (E) Distribution of cargo (tagged with GFP::RAB-3) in the nerve ring and neuronal process of the PLM expressing UNC-104 PH domain variants lacking GFP in transgenic lines made in unc-104(e1265); jsIs821 background. UNC-104 wild type protein . Intragenic mutant UNC-104 protein versions restore transport (e-h) while the UNC-104(D1497N) expressing transgene does not . Arrow indicates nerve ring  and PLM neuronal process . Arrowhead marks cell body of PLM neurons . Scale bar: 25µm. (F) Ratio of Mean fluorescence intensity (cell body/ synapse) of UNC-104::GFP with and without uba-1(it129ts) grown at 16 °C (permissive) and 22 °C (restrictive).  Ratio of fluorescent intensities in various parts of the PLM neuron in animals expressing UNC-104::GFP (jsIs1111), soluble GFP (zdIs5) in mechanosensory neurons. (I) anti-UNC-104 immunoreactivity in UNC-104 PH domain variant alleles over-expressing PIP2 using the gqIs25 transgene. Upon over-expressing PIP2 UNC-104 levels consistently increase in the synapse-rich regions of the ventral cord of unc-104(e1265tb107) and unc-104(e1265tb120). An occasional inconsistent increase was observed in unc-104(e1265) and no gross changes in a wild type UNC-104 background were observed. The alleles unc-104(e1265), unc-104(e1265tb107) and unc-104(e1265tb120) are labeled in the figure by the respective protein changes they encode, namely D1497N, D1497N R1501Q and D1497N M1540I.Immunostaining of UNC-104 in wild type as well as different UNC-104 mutant alleles . (A) Immunostaining with anti-UNC-104 polyclonal antibody shows high immunoreactivity in (a) wild type as well as (b) (1.99 MB TIF)Click here for additional data file.Video S1Transport of GFP::RAB-3 in the anterior mechanosensory neurons of different genotypes.Movement of GFP::RAB-3 in wild type neurons.(3.09 MB MOV)Click here for additional data file.Video S2unc-104(e1265).Movement of GFP::RAB-3 marked vesicles in (1.58 MB MOV)Click here for additional data file.Video S3unc-104(e1265tb120).Movement of GFP::RAB-3 in (1.08 MB MOV)Click here for additional data file.


BMC Biology article by Taylor and Bruenn. The integrated virus-specific sequences are at least partially expressed and seem to evolve under pressure of purifying selection, indicating that these are functional genes. Together with similar reports on integrated copies of some animal RNA viruses, these results suggest that integration of DNA copies of non-reverse-transcribing RNA viruses might be much more common than previously thought. The integrated copies could contribute to acquired immunity to the respective viruses.Genomes of several yeast species contain integrated DNA copies of complete genomes or individual genes of non-retroviral double-strand RNA viruses as reported in a recent  BMC Biology article Taylor and Bruenn [In a recent d Bruenn  for the d Bruenn  and, strd Bruenn ; in the d Bruenn .Aedes albopictus and Aedes aegypti mosquitoes [So far, NIRV have been a completely different story: reliable reports of integration of DNA copies of non-retroviral RNA virus genes into host genomes can be counted on the fingers of one hand. The idea that reverse transcriptase (RT) present in eukaryotic cells could produce NIRV was first championed by Zhdanov soon after RT was discovered , followesquitoes ,11. The squitoes , in 5 fusquitoes .Saccharomyces cerevisiae) in 5 fungal genomes, some of which carry complete copies of viral genomes whereas others possess only individual viral genes , retains the catalytic residues , suggesting that it could be enzymatically active. An intriguing speculation is that the NIRV RdRp could contribute to antiviral immunity by amplifying virus-specific transcripts that might protect the cell via a RNA interference mechanism. Of course, it is impossible to rule out that NIRVs and their protein products possess other, 'normal' cellular functions in addition to or even instead of their putative roles in antivirus immunity. However, the sporadic distribution of NIRV in fungal genomes and, even more importantly, their likely spread via HGT appear to be better compatible with the immunity hypothesis.Taken together, these results indicate that the NIRV in fungal genomes encode functional proteins. So what could that function(s) be? A rather obvious possibility is protection against infection with exogenous viruses and the results of Taylor and Bruenn provide some indirect support for this hypothesis. In addition to its function as the CP protein, the totivirus CP is also an enzyme that inactivates host mRNAs by removing their 5' cap (decapping enzyme) . CompariThe possibility that fungal NIRVs provide resistance against exogenous viruses puts them into the context of the burgeoning research area of 'integration-based immunity'. A novel form of acquired, adaptive antiviral immunity that has been recently discovered in bacteria and archaea is mediated by the so-called CRISPR  system. This system functions by integrating small segments of virus (bacteriophage) DNA into specific loci in the host genome and utilizing them to abrogate the cognate virus replication, most probably via an RNA interference-like mechanism ,18. The The second important message of the NIRV story is the apparent relatively recent HGT between fungi . The preCP: capsid protein; CRISPR: cluster regularly interspersed short palindromic repeats; ds: double stranded; HGT: horizontal gene transfer; NIRV: non-retroviral integrated Rna viruses; LCMV: lymphocytic choriomeningitis virus; RdRp: RNA-dependant RNA polymerase; RT: reverse transcriptase.


Positive-strand RNA viruses, a large group including human pathogens such as SARS-coronavirus (SARS-CoV), replicate in the cytoplasm of infected host cells. Their replication complexes are commonly associated with modified host cell membranes. Membrane structures supporting viral RNA synthesis range from distinct spherular membrane invaginations to more elaborate webs of packed membranes and vesicles. Generally, their ultrastructure, morphogenesis, and exact role in viral replication remain to be defined. Poorly characterized double-membrane vesicles (DMVs) were previously implicated in SARS-CoV RNA synthesis. We have now applied electron tomography of cryofixed infected cells for the three-dimensional imaging of coronavirus-induced membrane alterations at high resolution. Our analysis defines a unique reticulovesicular network of modified endoplasmic reticulum that integrates convoluted membranes, numerous interconnected DMVs (diameter 200–300 nm), and “vesicle packets” apparently arising from DMV merger. The convoluted membranes were most abundantly immunolabeled for viral replicase subunits. However, double-stranded RNA, presumably revealing the site of viral RNA synthesis, mainly localized to the DMV interior. Since we could not discern a connection between DMV interior and cytosol, our analysis raises several questions about the mechanism of DMV formation and the actual site of SARS-CoV RNA synthesis. Our data document the extensive virus-induced reorganization of host cell membranes into a network that is used to organize viral replication and possibly hide replicating RNA from antiviral defense mechanisms. Together with biochemical studies of the viral enzyme complex, our ultrastructural description of this “replication network” will aid to further dissect the early stages of the coronavirus life cycle and its virus-host interactions. Viruses with a positive-stranded RNA genome replicate in the cytoplasm of infected host cells. Their replication is driven by a membrane-bound viral enzyme complex that is commonly associated with modified intracellular membranes. Little is understood about the formation and architecture of these replication structures and their exact role in viral RNA synthesis. We used electron microscopy and tomography for the three-dimensional imaging of the membrane alterations induced by severe acute respiratory syndrome (SARS)-coronavirus, a member of the virus group with the largest RNA genome known to date. Previously, coronaviruses were reported to induce large numbers of isolated “double-membrane vesicles” (DMVs). However, our present studies reveal an elaborate reticulovesicular network of modified endoplasmic reticulum membranes with which SARS-coronavirus replicative proteins are associated. The lumen of this unique membrane network contains numerous large (diameter 250–300 nm) “inner vesicles,” which were formerly thought to reside in isolated DMVs. Intriguingly, although the interior of these vesicles does not appear to be connected to the cytosol, it labels abundantly for double-stranded RNA, which presumably is present at the site of viral RNA synthesis. The ultrastructural dissection of this elaborate “replication network” shows how coronaviruses extensively reorganize the host cell's membrane infrastructure, to coordinate their replication cycle, and possibly also hide replicating RNA from antiviral defense mechanisms. Positive-strand RNA virus replication is associated with membranes in the host cell's cytoplasm. Here, advanced 3D electron microscopy reveals that SARS-coronavirus induces an elaborate reticulovesicular network of modified ER membranes that supports viral RNA synthesis. Viruses rely on the host cell's infrastructure and metabolism during essentially all stages of their replication cycle and have therefore adopted strategies to coordinate a variety of molecular interactions in both time and intracellular space. The fact that the replication complexes of positive-strand RNA (+RNA) viruses of eukaryotes are invariably associated with (modified) intracellular membranes appears to be a striking example of such a strategy –8. SpeciFollowing the 2003 outbreak of severe acute respiratory syndrome , the coThe nidovirus replicase includes several (presumed) multispanning transmembrane proteins that are thought to physically anchor the replication/transcription complex (RTC) to intracellular membranes. In the case of coronaviruses, these domains reside in nsp3, nsp4, and nsp6  23,24].,24.23,24To develop a 3-D ultrastructural model for the RTC-related membrane alterations in SARS-CoV–infected cells, we have now employed electron tomography . ThiPreviously, we experienced that, compared to more traditional chemical fixation protocols, the preservation of the fragile coronavirus DMV structures could be significantly improved by using a combination of cryofixation and freeze substitution (FS) . We now Using these optimized conditions to prepare thin sections (100 nm) of SARS-CoV–infected Vero E6 cells, we could detect the first DMVs at 2 h postinfection (h p.i.) and were able to monitor the subsequent development of virus-induced membrane alterations. Early DMVs had sizes ranging from 150 to 300 nm, were distributed throughout the cytoplasm, and were sometimes located in the proximity of small reticular membranes with which, occasionally, they appeared to be connected A. From 4From 3 h p.i. on, we also observed large assemblies of convoluted membranes (CM), often in close proximity to DMV clusters D. These n = 99), whereas later in infection, their average diameter (DMVs and VPs combined) increased to about 300 nm .Especially at later stages of SARS-CoV infection , we observed packets of single-membrane vesicles surrounded by a common outer membrane, as previously described by Goldsmith et al. . The diaOur observations define VPs as a third distinct modification of intracellular membranes that is induced by SARS-CoV infection. By 10 h p.i., VPs appeared to have merged into even larger cytoplasmic vacuoles, containing both vesicles as well as significant numbers of budding and completed virions E. DMVs, Although, occasionally, the analysis of “conventional” thin sections suggested CM and DMV outer membranes to be continuous and connected to ER cisternae, a more accurate assessment required an analysis in three dimensions. We therefore employed ET of semi-thick (200 nm) sections of cryofixed, SARS-CoV–infected Vero E6 cells. By using a specimen holder that could also be tilted around a second axis, perpendicular to both electron beam and first tilt axis, we obtained datasets, each consisting of 262 differently tilted 2-D images, which were used to produce a high-resolution reconstruction in three dimensions. Such “dual-axis” tomograms allowed us to visualize and analyze membrane continuities between the respective structures defined in the previous paragraph  that most or—likely—all coronavirus DMVs are interconnected by their outer membrane and (2) that they are part of an elaborate network that is continuous with the rough ER. As illustrated by the 3-D reconstruction in By 7 h p.i., in part of the cells, the formation of VPs had begun  and 5, fIn order to assess the association of replicase subunits with the various coronavirus-induced membrane structures, we performed IEM experiments on SARS-CoV–infected Vero E6 cells. In view of previously experienced problems to preserve DMV ultrastructure for IEM , the FS For samples fixed at 8 h pi, highly specific immunogold labeling results were obtained with antisera  recognizA critical step in the replication of +RNA viruses is the production of a negative-stranded copy of the genome, which is used as a template for genome replication by the viral RdRp. Coronaviruses also generate a set of subgenome-length negative-strand RNAs, which serve as templates for subgenomic mRNA synthesis ,20. It iFor a variety of +RNA viruses, the (presumed) dsRNA intermediates of replication have been visualized in situ by using antibodies recognizing dsRNA ,48–50. IIn subsequent IEM experiments, the J2 antibody was found to retain its reactivity for dsRNA in sections of cells that had been embedded in Lowicryl, following the FS procedure described above. An abundant and highly specific labeling for dsRNA was observed on the interior of SARS-CoV–induced DMVs , with soThe functional dissection of the multienzyme complexes that drive +RNA virus replication and transcription is essential for our understanding of the molecular biology of this important group of pathogens. Presumably, the membrane structures used to compartmentalize RTCs provide a suitable scaffold for viral RNA synthesis facilitate the organization of the viral replication cycle, and aid in evading or delaying antiviral host cell responses, including those that can be triggered by viral dsRNA ,33,52,53We have now employed ET to analyze the replication structures of SARS-CoV, a prominent member of the coronavirus group which—at about 7,000 amino acids—encodes the largest known +RNA virus replicase . DMVs haFinally, our analysis of cells at more advanced stages of SARS-CoV infection  opens thBy analogy with the replication-associated “membrane spherules” of several other +RNA viruses ,55, it wIn view of the resolution provided by our tomograms, we are confident that we would have readily detected connections to the cytosol with a diameter (8–10 nm) in the range previously described for other +RNA virus replication structures ,55. ThusIn addition to the recent data on MHV nsp4 , resultsPresumably, membrane pairing is followed by the wrapping of membrane cisternae around cytosolic constituents and leads to the membrane fission event that is needed to explain the sealed DMV inner membrane. However, despite the presence of several hundred DMVs in infected cells and despite the extensive EM analysis of hundreds of cells in the course of this study, we were unable to find morphological profiles that seemed obvious examples of an actual DMV-forming fission event. Although some smaller DMVs were sometimes observed , the aveThe observed narrow neck-like connections in the network  and the In infected cells, several other groups of +RNA viruses induce membrane alterations that differ from the spherular membrane invaginations described, e.g., for nodaviruses  and alphOur studies may in fact have uncovered a closer parallel to the membranes with which the replication complexes of flaviviruses are associated. Ultrastructural studies of cells infected with Kunjin virus have defined various characteristic membrane structures, which were implicated in viral RNA synthesis on the basis of immunolabeling and biochemical studies  was use2, 10 mM EGTA) at various time points after infection. For cryofixation, cell monolayers adhered to Thermanox coverslips (Nunc) were plunged into liquid ethane. Freeze substitution was performed at −90 °C in an automated freeze-substitution system (Leica) using an FS medium consisting of 90% acetone and 10% water, containing 1% osmium tetroxide and 0.5 % uranyl acetate. After washing with pure acetone at room temperature, the samples were embedded in epoxy LX-12 resin. Thin sections were contrasted with uranyl acetate and lead hydroxide, and subsequently viewed at 80 kV with a Philips CM-10 transmission electron microscope.For ultrastructural morphological investigations, SARS-CoV–infected Vero E6 cells were prefixed (for biosafety reasons) overnight with 3% paraformaldehyde in 0.1 M PHEM buffer .When quantifying DMVs per infected cell, thin sections were cut in the direction parallel to the substrate, and the slice producing the largest nuclear diameter was analyzed, since this plane was generally found to contain the largest number of DMVs. Electron micrographs (between 20 and 100) covering the entire cross-section of the cell were recorded, and to facilitate counting, these were digitally merged to produce a single image representing a 100-nm-thick plane through the center of the infected cell. Merged images were analyzed with Zoomify software. Only DMVs for which the surrounding bilayers could be readily distinguished were counted, and their diameter was measured using ImageJ software . To compute the electron tomogram, the dual-axis tilt series were aligned by means of the fiducial markers using the IMOD software package [http://ccdb.ucsd.edu; [Freeze-substituted infected cell samples, processed for morphological investigation as described above, were used to cut 200-nm-thick sections. To facilitate the image alignment that is required for the subsequent image reconstruction step, a suspension of 10-nm gold particles was layered on top of the sections as fiducial markers. For dual-axis tomography, two single-axis tilt series were recorded of the specimens with an FEI T12 transmission electron microscope operating at an acceleration voltage of 120 kV. Per single-axis tilt series, 131 images were recorded at 1° tilt increments between −65 °C and 65 °C. Automated tomography acquisition software was used . Images were acquired with a cooled slow-scan charge-coupled device (CCD) camera  with 4,096 × 4,096 pixels and were recorded by binning 2. The electron microscope magnification was 18,500×, corresponding to a pixel size of 1.2 nm at the specimen level. To enable dual-axis tomography, the specimens were rotated 90° around the  package . The sizcsd.edu; ) under aThe 3-D surface-rendered reconstructions of viral structures and adjacent cellular features were processed using AMIRA Visualization Package (TSG Europe) by surface rendering and thresholding. During this process, some volumes were denoised using the nonlinear anisotropic diffusion filtering . DenoiseInfected cells on glass coverslips were fixed with 3% paraformaldehyde in PBS at various time points after infection and were processed for IF microscopy essentially as described previously . FollowiSamples were examined with a Zeiss Axioskop 2 fluorescence microscope   or with a Leica SP5 confocal laser scanning microscope, using a pinhole size of 1 airy unit (for both channels) to give optical sections with a theoretical thickness of 236 nm. Images were minimally optimized for contrast and brightness using Adobe Photoshop CS2.Figure S1See the legend to (2.15 MB JPG)Click here for additional data file.Figure S2Anaglyph images were produced and superimposed to provide a stereoscopic 3-D effect when viewed with spectacles with red (left) and green (right) glasses.(6.68 MB JPG)Click here for additional data file.Video S1The video shows a group of interconnected DMVs and also shows the connections of DMV outer membranes with the ER. The tightly apposed double membranes and fibrous material inside the DMVs are clearly visible. To facilitate image alignment during image reconstruction, a suspension of 10-nm gold particles was layered on top of the sections as fiducial markers.(7.25 MB WMV)Click here for additional data file.Video S2(8.25 MB WMV)Click here for additional data file.Video S3The video shows a group of interconnected DMVs and also a type 1 VP. The tightly apposed double membranes and fibrous material inside the DMVs are clearly visible. To facilitate image alignment during image reconstruction, a suspension of 10-nm gold particles was layered on top of the sections as fiducial markers.(3.99 MB WMV)Click here for additional data file.Video S4The video illustrates a relatively late stage of SARS-CoV–induced membrane alterations, during which DMVs seem to merge into larger VPs (type 2). Also, various stages of virus budding can be observed at the outer membranes of these type 2 VPs. Note that clear discontinuities in the membranes of the inner vesicles can be observed, ostensibly resulting in fusion of DMV contents with each other and with the lumen of the VP. To facilitate image alignment during image reconstruction, a suspension of 10-nm gold particles was layered on top of the sections as fiducial markers.(3.31 MB WMV)Click here for additional data file.


A cis-acting RNA regulatory element, the Rev-responsive element (RRE), has essential roles in replication of lentiviruses, including human immunodeficiency virus (HIV-1) and equine infection anemia virus (EIAV). The RRE binds the viral trans-acting regulatory protein, Rev, to mediate nucleocytoplasmic transport of incompletely spliced mRNAs encoding viral structural genes and genomic RNA. Because of its potential as a clinical target, RRE-Rev interactions have been well studied in HIV-1; however, detailed molecular structures of Rev-RRE complexes in other lentiviruses are still lacking. In this study, we investigate the secondary structure of the EIAV RRE and interrogate regulatory protein-RNA interactions in EIAV Rev-RRE complexes. Computational prediction and detailed chemical probing and footprinting experiments were used to determine the RNA secondary structure of EIAV RRE-1, a 555 nt region that provides RRE function in vivo. Chemical probing experiments confirmed the presence of several predicted loop and stem-loop structures, which are conserved among 140 EIAV sequence variants. Footprinting experiments revealed that Rev binding induces significant structural rearrangement in two conserved domains characterized by stable stem-loop structures. Rev binding region-1 (RBR-1) corresponds to a genetically-defined Rev binding region that overlaps exon 1 of the EIAV rev gene and contains an exonic splicing enhancer (ESE). RBR-2, characterized for the first time in this study, is required for high affinity binding of EIAV Rev to the RRE. RBR-2 contains an RNA structural motif that is also found within the high affinity Rev binding site in HIV-1 (stem-loop IIB), and within or near mapped RRE regions of four additional lentiviruses. The powerful integration of computational and experimental approaches in this study has generated a validated RNA secondary structure for the EIAV RRE and provided provocative evidence that high affinity Rev binding sites of HIV-1 and EIAV share a conserved RNA structural motif. The presence of this motif in phylogenetically divergent lentiviruses suggests that it may play a role in highly conserved interactions that could be targeted in novel anti-lentiviral therapies. There is no conservation among the lentiviral RREs at the RNA sequence level; however, the RRE regions of several primate and non-primate lentiviral genomes map to the SU/TM junction in trans-activating protein, Tat  differs from other lentiviral RREs with respect to location and function. The ERRE is located in a 555 nt region near the 5′ end of in vitro footprinting analysis of EIAV Rev-RRE complexes, and identify two distinct domains within the essential RRE that undergo significant structural transitions upon Rev binding. Computational analyses identified an RNA secondary structural motif within the high affinity Rev-binding sites of both HIV-1 and EIAV that is present within the mapped RREs of four additional lentiviruses. The discovery of a conserved recognition element for lentiviral Rev-RRE interactions lays the groundwork for investigating highly conserved RNA-protein interactions that could be targeted in novel anti-lentiviral therapies.In this paper, we investigate the RNA structure of the EIAV RRE and its interactions with the Rev protein. We propose an RNA secondary structure model for the essential RRE in EIAV, based on a combination of secondary structure prediction and chemical probing experiments. We present detailed The secondary structure of the ERRE was analyzed using a combination of computational and experimental approaches. To further refine and validate the RRE secondary structure models, we used available sequence information from EIAV variants to perform covariation analyses, using RNAalifold 32P-labeled primer extension products visualized by denaturing polyacrylamide gel electrophoresis, sites of modification correspond to a “stop” or an enhanced band located one position downstream (3′) of the band corresponding to the modified ribonucleotide site.Previous experiments had implicated the purine-rich ESE within the ERRE as a primary binding site for EIAV Rev protein A summary of the footprinting results is shown in guanine-adenine-purine) motifs have been identified as binding sites for the host splicing factor, SF2/ASF Within RBR-1, corresponding to the 5′ terminal region of the ERRE, several purine-rich regions and GAR  and the RNA secondary structure of the RRE To explore the possibility that RNA secondary structural elements required for Rev binding in EIAV might also be found in other lentiviruses, we first asked whether RBR-1 and RBR-2, identified in this study, have any predicted RNA structural features in common with one another or with the previously identified high affinity Rev binding site in HIV-1 RRE (stem-loop IIB) The unexpected RNA secondary structural similarities detected through pairwise analysis of the Rev-binding domains in HIV-1 and EIAV prompted us to generate a computational RNA structural motif model based on shared features of the two regions using RNAMotif cis-acting RNA regulatory element recognized by the Rev proteins of lentiviruses, including HIV-1 and EIAV. Rev-RRE interactions regulate viral gene expression and genomic replication by mediating the export of incompletely spliced or unspliced lentiviral mRNAs from the nucleus to the cytoplasm of infected host cells The Rev-responsive element (RRE) is an essential Two important aims of this study were to determine the secondary structure of the EIAV RRE and to obtain high-resolution experimental data regarding Rev-RRE interactions, for comparison with available HIV-1 data. Using a combination of computational and experimental approaches, we generated an optimized RNA secondary structure model for the EIAV ERRE. In the context of this structure, individual ribonucleotides and secondary structural elements that are protected from chemical cleavage or undergo structural transitions upon Rev binding were identified using detailed RNA footprinting experiments.in vivo.One striking characteristic of our proposed ERRE structure is the relative paucity of stable stem structures and abundance of single-stranded loops, several of which are unusually large. The calculated free energy of the optimized ERRE secondary structure, based on a combination of thermodynamic calculations, chemical probing data and ribonucleotide covariation frequencies using RNAalifold is −77.9 kcal/mol , a valueThe ERRE footprinting experiments provided a detailed view of the interaction between EIAV Rev and RBR-1, which contains the ESE region previously implicated in Rev binding. Within loop B, changes in chemical reactivities of every purine residue in PuA and several As in PuB were detected, consistent with previous studies reporting decreased Rev binding when mutations converting several GAA motifs into GCAs were introduced within PuB in vitro binding assays demonstrated that RBR-2 is required for high affinity binding of EIAV Rev; ERRE fragments containing both RBR-1 and RBR-2 have much higher affinity for EIAV Rev than those lacking RBR-2 .RBR-2, a novel Rev-binding region within the ERRE, was identified as second Rev-mediated footprint, encompassing relatively long tracts of residues protected from hydroxyl radical cleavage and several individual ribonucleotides with enhanced reactivity to kethoxal and DMS. Quantitative E. coli.For RBR-2, all computational methods produced the same RNA secondary structure. The Rev-induced footprint in SL-Z (nt 439–462) in RBR-2 is especially interesting because the topology of this stem-loop structure is very similar to that of stem-loop IIB in HIV-I, which corresponds to the primary high-affinity Rev binding site in the HIV-1 RRE in vitro binding assays such as UV-crosslinking and EMSA (unpublished data), but we are not aware of any direct evidence for a functional role of EIAV Rev multimerization in vivo. Further investigation will be required to dissect the functional relationships among RBR-1, RBR-2 and the full length EIAV RRE.In HIV-1, an extended RRE sequence that includes structured regions surrounding the stem-loop IIB binding site has been shown to enhance Rev binding and promote its multimerization on HIV-1 RNAs in vitro and in vivo assays to assess the functional effects of mutations that alter the sequence, but preserve (or destroy) the conserved RNA structural motif, will be required to fully evaluate its potential biological significance.To investigate whether the RBR-2 domain might have RNA structural features in common with the high affinity Rev binding stem-loop IIB region of HIV-1, we performed pairwise comparisons using Dynalign vif protein tat and rev. Interestingly, we found the conserved RNA structural motif in RELIK sequence, also within the env gene region . Transcribed RNA was purified using MEGAClear kits , denatured at 90°C for 2 min and annealed by slow cooling. Ethanol precipitation was performed to remove salts and concentrate ERRE RNA. Concentrated RNA was stored at −80°C. EIAV Rev protein was expressed as an MBP-ERev fusion protein and purified as described previously The EIAV ERRE was amplified by PCR from pERRE-All 2, and 0.5 mM EDTA). To generate ERev-ERRE protein-RNA complexes for footprinting experiments, samples containing one to thirty-fold molar excess of purified MBP-ERev fusion protein were incubated with pre-folded ERRE RNA (35pmol) in binding buffer in a total volume of 87.5 µℓ for 20 min on ice. Samples containing folded ERRE RNA alone or RNA-protein complexes were incubated with 10.5 µℓ 880 mM dimethylsulfate (DMS)  or 7 µℓ 720 mM kethoxal  or 16 µℓ hydroxyl radical probing mixture (4 µℓ of 50 mM Fe(NH4)2(SO4)2·6H2O, 4 µℓ of 100 mM EDTA, 4 µℓ of 250 mM ascorbic acid, and 4 µℓ of 2.5% hydrogen peroxide) for 10 min at room temperature. DMS modification reactions were stopped by addition of 59.3 µℓ of DMS stop buffer . For the kethoxal probing, 8.2 µℓ of 0.5 M potassium borate was added for stabilizing kethoxal. Hydroxyl radical probing reactions were quenched by the addition of 92.8 µℓ of 1 M thiourea. After ethanol precipitation of RNA or RNA-protein complexes, 3 phenol and 2 chloroform extractions were performed to purify RNA from RNA-protein complexes. RNA was concentrated and washed using 100% and 70% ethanol precipitations. Finally, RNA was resuspended in 35 µℓ of water  or 35 µℓ of 40 mM potassium borate for kethoxal modification. Additional details are provided in Prior to chemical probing or footprinting experiments, the annealed ERRE in RNA storage buffer  was pre-incubated at 42°C for 15 min. To generate unmodified and modified unbound ERRE samples for RNA secondary structure probing experiments, two aliquots (each containing 35 pmol) of ERRE were added to RNA binding buffer  or subfragment of the ERRE  using standard procedures. Binding affinities were calculated using Graphpad Prism 5 software Nitrocellulose filter binding assays were carried out using purified env gene variant sequences, remained. The concept and methods for analysis of information content are described elsewhere http://www.ebi.ac.uk/clustalw/) iI is the information content for the nucleotide position i in the alignment, the index j sums over all possible nucleotides , ijq represents the observed frequency of nucleotide j at position i and jp represents the expected frequency value, which is 0.25. The calculated information content at each nucleotide position was plotted using Microsoft Excel.The EIAV genomic RNA sequences used in the present study were originally collected for analyzing sequence variants in the EIAV Env SU protein by Mealey, et al. http://frontend.bioinfo.rpi.edu/applications/mfold/cgi-bin/rna-form1.cgi) http://sfold.wadsworth.org) http://www.tbi.univie.ac.at/ivo/RNA/) http://rna.urmc.rochester.edu/rnastructure.html) http://www.tbi.univie.ac.at/ivo/RNA/) http://www.ebi.ac.uk/clustalw/) http://wilab.inha.ac.kr/pseudoviewer2/) Different methods for RNA secondary structure prediction have been reviewed recently http://rna.urmc.rochester.edu/rnastructure.html) http://www.scripps.edu/mb/case/casegr-sh-3.5.html) RNAstructure Dynalign software Click here for additional data file.Figure S1in vitro. Quantitative nitrocellulose filter binding assays were performed to measure binding affinity of RNAs that contain both RBR-1 and RBR-2  and those that contain only RBR-1 . Purified 32P-labeled RNAs were incubated with increasing concentrations of purified MBP-ERev, and RNA-protein complexes specifically bound to nitrocellulose were separated from unbound RNAs and quantified by scintillation counter. Binding isotherms based on experimental data were fit to models generated using Prism software (http://www.graphpad.com/prism/Prism.htm). Binding constants estimated from these models were: Kd = 19 nM for RNA containing both RBR- and RBR-2 (blue) and Kd = 5200 nM (5.2 µM) for RNA lacking RBR-2 (red).Deletion of RBR-2 from EIAV RRE results in loss of high affinity Rev binding (0.17 MB TIF)Click here for additional data file.


These findings provide unique insights into the process of RNA export in general, and more specifically, of HIV-1 genomic RNA trafficking.Once transcribed, the nascent full-length RNA of HIV-1 must travel to the appropriate host cell sites to be translated or to find a partner RNA for copackaging to form newly generated viruses. In this report, we sought to delineate the location where HIV-1 RNA initiates dimerization and the influence of the RNA transport pathway used by the virus on downstream events essential to viral replication. Using a cell-fusion-dependent recombination assay, we demonstrate that the two RNAs destined for copackaging into the same virion select each other mostly within the cytoplasm. Moreover, by manipulating the RNA export element in the viral genome, we show that the export pathway taken is important for the ability of RNA molecules derived from two viruses to interact and be copackaged. These results further illustrate that at the point of dimerization the two main cellular export pathways are partially distinct. Lastly, by providing Gag  High genetic diversity of HIV-1 presents a difficult barrier for drug treatment and vaccine development. HIV-1 efficiently reassorts mutations in its genome by packaging two copies of RNA into one virion and using portions of each RNA as the template for DNA synthesis. Thus, a key to HIV-1's genetic diversity is how it packages two RNAs into one virion. We have previously shown that HIV-1 packages RNA as a dimer, rather than two monomers. In this report, we determined that HIV-1 RNA finds its copackaged dimer partner mostly in the cytoplasm, not the nucleus, of the infected cells. We also found that the host cell machinery used to transport the HIV-1 RNA out of the nucleus affects the ability of two RNAs to copackage into one virion: HIV-1 RNAs transported using the CRM-1 pathway does not copackage efficiently with RNA transported by the NXF1 pathway. This is also the first demonstration that RNAs transported by the two major cellular pathways, CRM-1 and NXF1, are partially segregated at some point in the cytoplasm. Therefore, this work provides novel insights to both HIV replication mechanisms and cellular RNA transport pathways. HIV-1 full-length RNAs serve at least two functions: as a template for Gag/Gag-Pol translation, and as genetic material packaged in the virion. Many cellular factors ensure the correct macromolecular trafficking between nucleus and cytoplasm; specifically, mechanisms exist to prevent the export of intron-containing transcripts, such as the full-length HIV-1 RNA Many studies have shown that it is possible to alter the retroviral RNA transport pathway by manipulating the cis- and trans-acting viral elements. Interestingly, a recent study indicates that the transport pathway the RNA takes can influence the function of the protein it encodes. By altering the transport pathway of the HIV-1 Gag-encoding RNA from CRM-1 to NXF1, one can relieve the HIV-1 assembly block in murine cells GCGCGC to GGGGGG) but can form perfect base pairs with the DIS of a partner virus (such as a virus with CCCCCC at the DIS). We reasoned that in the coinfected cells if dimeric RNAs are packaged, then the GGGGGG viral RNA would preferentially pair with CCCCCC viral RNA, and we would therefore observe an increase in the formation of heterozygous virions. In contrast, if two monomeric RNAs are packaged, then we would not observe an increase in heterozygous viruses. Our results revealed that most of the virions from coinfected cells were heterozygous, indicating that copackaged RNA partner selection, i.e. dimerization, occurred prior to the packaging of virion RNA Retroviruses package two copies of full-length RNA in one virion. One of the consequences of packaging two RNAs is frequent recombination during DNA synthesis when reverse transcriptase uses parts of both RNAs as templates Many questions remain concerning how HIV-1 full-length RNA traffics through the cell to fulfill its various roles. For example, it has been unclear at which point along the RNA trafficking pathway, from the site of transcription to the site of viral budding, HIV-1 RNA selects its copackaged RNA to initiate the dimerization process. It remained unclear whether HIV-1 RNAs are dimerized in the nucleus and traffic out of the nucleus as a dimer, or exit the nucleus as a monomer and dimerize in the cytoplasm. Furthermore, it is not known whether the RNA transport pathway used by the viral RNA affects its interactions with other viral RNA or Gag proteins, and thereby influences the abilities of the RNA to dimerize and be packaged into a nascent virion. In this current work, we addressed these questions by using three systems: a cell-fusion-dependent recombination assay to establish the cellular location of the dimerization event, a Rev-independent HIV-1 to investigate the influence of the NXF1 and CRM-1 export pathways on these processes, and a single virion visualization assay to directly analyze the RNA content of each viral particle. These studies resulted in three novel findings. We demonstrate that the majority of HIV-1 RNA dimerization occurs within the cytoplasm, not within the nucleus. Moreover, the RNA transport pathway taken by the viral RNA has a major effect on the ability of the RNA to be copackaged; these results revealed that RNAs trafficking through the CRM-1 and NXF1 transport pathways are partially separated at least at the stage of RNA commitment to dimerization. Finally, we demonstrate that although the RNA pathways are partially distinct at the point when initial dimerization occurs, selection of packaged RNA by Gag does not discriminate between RNA in these two pathways. These findings provide insights into the mechanisms of HIV replication including RNA-RNA and RNA-Gag interactions, and demonstrate a new approach to the unraveling of the detailed mechanisms of the cell's nuclear-cytoplasmic trafficking pathways.nef, an inactivated green fluorescent protein gene (gfp) and a second gene that is either a mouse heat-stable antigen gene (hsa) or a mouse thy1.2 gene (thy) ; recombination between which can occur during reverse transcription to generate a recombinant progeny expressing a functional gfp. The multiplicity of infection (MOI) of the GFP+ events, as a percentage of the total infection MOI (determined using the expression of HSA and Thy), provides a measure of the recombination efficiency between the two viruses. As retroviral recombination occurs between two copackaged RNAs, the rate of recombination is dependent on the formation of heterozygous virions; therefore, recombination can be used as a measurement of the dimerization efficiency between two viral RNAs, as we have previously shown Our previous experiment revealed that the selection of the copackaged RNA occurs prior to encapsidation into the virion ne (thy) . The twogag mutant virus that has a severe replication defect. When the two cell lines are fused using conditions that facilitate cytoplasmic but not nuclear fusion, the Gag proteins from these two viruses can coassemble and complement each other's function to rescue virus replication. Our previous study demonstrated that HIV-1 RNA dimerization occurs prior to Gag packaging gfp mutation. Therefore, very few GFP+ phenotypes will be formed by recombination. In contrast, if the selection of the copackaged RNA mostly occurs in the cytoplasm, fusing the cytoplasm of the two cell lines will allow the RNAs derived from the two nuclei to interact and dimerize prior to packaging by Gag. In this scenario, heterozygous virions can be formed, which can generate numerous GFP+ recombinants. Therefore, in the fusion experiment, the location of the RNA dimerization event, in the nucleus or in the cytoplasm, predicts different levels of the recombinant GFP+ phenotype.We modified the recombination system to examine whether we could detect cytoplasmic RNA dimerization events . In this–). The NC* mutation reduces RNA packaging to 5% of wild-type and abolishes infectivity – mutation reduces viral budding and viral titer drops to ∼2% of wild type We generated cell lines containing either a nucleocapsid (NC) mutant virus (T6-NC*) or a late domain mutant virus , two CTE-dependent proviruses (H0-CTE.B6-CTE), or one RRE- and one CTE-dependent provirus . Flow cytometry analyses of the markers encoded by the two viruses indicated that most of the cells (>90%) in each cell line express both viruses (data not shown). These cell lines were transiently transfected with a plasmid expressing HIV-1 Env and the released viruses were used to infect target cells; the titers generated from these infections are shown in E. coli antitermination protein BglG, respectively . Similarly, when both HIV-1 genomes used the CTE element for RNA export, these RNA species were also copackaged together efficiently; ∼42% of the CeFP+ particles have both RNA signals , the NXF1 pathway (CTE-Gag), or both pathways (RRE-Gag and CTE-Gag). We then compared the relative viral titers generated from these experiments with those from the H0-CTE.T6-RRE cell line, in which both viruses expressed functional Gag and the relative viral titers were 44%:56%  were detected, irrespective of the RNA export pathway used by the gag mRNA . Similargag mRNA , indicatin trans can efficiently package HIV-1RNAs exported using either the CRM-1 or the NXF1 pathway. In addition to gaining an increased understanding of the HIV-1 replication cycle, our demonstration that RNAs from the NXF1 and CRM-1 pathways are partially segregated in specific cellular locations also provides insights into the nuclear trafficking pathways themselves.The diverse roles played by the full-length HIV-1 RNA necessitate strict control over each aspect of its biogenesis and fate. Initially, newly synthesized RNA must be spliced for expression of early gene products such as Rev and Tat. Once accumulated to sufficient levels, Rev directs the export of the RRE-containing transcripts, including the unspliced RNA. Once in the cytoplasm, full-length viral RNA can be used for Gag/Gag-Pol translation, or can be packaged into nascent particles as the template for DNA synthesis in the next replication cycle. At least two viral proteins, Rev and Gag, are involved in the trafficking of the full-length RNA from the nucleus to the viral particle. Additionally, interactions between the viral components and the cellular pathways play a crucial role in the process, the extent and specifics of which are not yet completely understood. Our findings in this work provide an outline of events that lead to packaging of the HIV-1 full-length RNAs. Following export from the nucleus, HIV-1 full-length RNA selects its copackaged RNA partner by forming base-pairing at the DIS. Our data demonstrated that the initial dimerization event occurs in the cytoplasm, at a point where RNA molecules exported from NXF1 and CRM-1 pathways are not fully mixed. Although HIV-1 normally uses the CRM-1 transport pathway, this is not a prerequisite for efficient copackaging of RNAs from two viruses; HIV-1 RNAs derived from two viruses both using the NXF1 pathway for RNA transport can also be efficiently copackaged. The dimeric RNAs then interact with and are packaged by the Gag polyproteins; furthermore, Gag expressed Our finding that selection of HIV-1 copackaged RNAs and initial dimerization events mainly occur in the cytoplasm provide a contrast to the suggested nuclear MLV dimerization events In order to transport the full-length or singly spliced RNAs from the nucleus to the cytoplasm, HIV-1 uses the cellular protein-shuttling CRM-1 pathway instead of the NXF1-dependent pathway. To determine the amount of overlap between these two pathways and investigate the reliance of HIV-1 on its particular pathway to assort RNAs transcribed from different proviruses, we generated an RRE-independent HIV-1 that uses the MPMV CTE and the NXF1 pathway for RNA export from the nucleus. Using these viruses, we showed that the ability of RNA from different HIV-1 proviruses to fully assort does not depend on using the CRM-1 pathway. Furthermore, we showed that the two pathways (CRM-1 and NXF1) are distinct at the point of HIV-1 dimerization, although some recombination was detected, indicating a partial overlap. To our knowledge, this is the first time that an interaction between RNAs in these two pathways has been addressed. The partial overlap between the CRM-1 and NXF1 pathways also raises the interesting possibility of RNA-RNA interactions between two differentially regulated viruses, such as simian immunodeficiency virus and MPMV, during the nuclear export of their full-length RNAs within dual-infected cells.Although the results presented here, which were generated from three different cell lines, clearly and consistently indicate that the recombination rates between an RRE- and a CTE-dependent virus are lower than those from two viruses using the same RNA transport pathway, it is worth noting that this lower recombination rate was not consistently observed when viruses were produced by cotransfection of an RRE- and a CTE-dependent viral DNA. Viral titers generated by our transfection experiments are routinely much higher than those from cell lines containing integrated proviruses, most likely because high expression from multiple copies of transfected DNA. We hypothesize that overexpression in the transiently transfected cells can overload the system and blur the boundaries between the two transport pathways. We believe the results from the cell lines containing integrated proviruses most closely mimic the events during HIV-1 replication in the human population and are more biologically relevant than any transient transfection data.Using an HIV-1-based minimal vector and a helper construct, it was recently shown that in the absence of the Rev-RRE interaction, the level of vector RNA in the cytoplasm was slightly reduced, but the virion RNA detected in the supernatant was severely affected Why some retroviruses, such as HIV-1, have evolved a dependence on the CRM-1 pathway whereas other retroviruses, such as MPMV, use the NXF1 pathway remains a mystery. However, the use of two differentially exported HIV-1 viruses within the same coinfected cell line provides a unique tool to analyze this and many other questions. Moreover, the two pathways could be further dissected to define novel cellular factors involved in RNA trafficking, which may lead to the identification of novel targets for the development of antiviral compounds specific for HIV-1 RNA export. This cell line might also provide a basis for a high-throughput screen to identify antiviral compounds that specifically target the interplay of HIV-1 RNA with the CRM-1 pathway.– contain a CCHC/CCHC-to-CCHH/CCCC mutation in NC and a PTAP-to-LIRL mutation in p6, respectively. H0-ΔRRE was derived from H0-RRE by deleting the entire RRE (nt 7644–8330 in NL4-3). H0-CTE was generated by inserting a DNA fragment containing four copies of the MPMV CTE (4XCTE) obtained from pGPV-CTEx4 thy with mouse B7 gene; B6-CTE was generated by replacing the NotI-to-XhoI fragment of H0-CTE with that of B6-RRE. T6-RRE-Spe and H0-CTE-Spe each contains a premature stop in gag located in the middle of the CA domain. RNA derived from H0-Spe, which has identical structure as H0-RRE except containing the aforementioned premature gag stop codon mutation were packaged at a similar efficiency to RNA derived from H0-RRE For clarity, previously described pON-H0 and pON-T6 gag, tat and rev, and cis-acting elements such as LTRs, 5′ untranslated regions, and RRE; however, they have inactivating deletions in pol, vif, vpr, vpu, and env. To generate GagCeFP-MS2SL-CTE and GagCeFP-BglSL-CTE, a CTE-containing DNA fragment from B6-CTE was used to replace the corresponding RRE-containing DNA fragment in GagCeFP-MS2SL-RRE and GagCeFP-BglSL-RRE, respectively, using a two-step cloning process. The general structures of newly generated vectors were characterized by restriction enzyme mapping; inserted fragments that were amplified by PCR were verified by DNA sequencing.Plasmid MS2-YFP was a gift from Robert Singer The cell line 293T.CC, a kind gift from Dr. Robert Doms, is a derivative of the 293T cell line that expresses CD4 and CCR5. Hut/CCR5 and 293T cells were maintained as described previously + phenotype using flow cytometry. The numbers of infected cells or cells infected with recombinants in the population were then converted to MOI using the Poison distribution. Recombination rates were calculated as the MOI of GFP+ cells divided by the MOI of infected cells. Additionally, infections were carried out at relatively low MOI when each parental viral titer was less than 0.5 and a minimum of 750 GFP+ cells were sampled in each measured rate.Cell lines containing two proviruses were produced as previously described – genomes were produced by cotransfection with helper plasmids expressing functional Gag. These viruses were used to infect 293T or 293T.CC cells separately; provirus-containing cells, identified by their expression of HSA or Thy marker, were enriched by two to three rounds of cell sorting. To perform the fusion assay, the provirus-containing 293T-based cell line was transfected with pIIINL(AD8). The provirus-containing 293T.CC-based cell line were cocultured with equal numbers of successfully transfected 293T cells at 24 h posttransfection. Viruses were harvested from the coculture 16 h after mixing the two cell lines, clarified through a 0.45-µm-pore-size filter, and used to infect Hut/CCR5 cells by spinnoculation (1200×g for 1 h at 15°C) in the presence of 10 µg/ml polybrene. Where indicated, the mixed cells were cultured in the presence of roscovitine, added at the time of mixing, at a final concentration of 10 µM.To generate the cell lines used for the fusion assay, pseuodotyped viruses containing T6-NC* or H0-PTAP2 before image acquisition.Human 293T cells were maintained as described above; transient transfection was performed with FuGENE HD (Roche) according to the manufacturer's recommendation. For all visualization experiments, 293T cells were cotransfected with a pair of modified HIV-1 plasmid DNA, one expressing GagCeFP and the other wild-type Gag at equal weight ratios; although only the plasmid with GagCeFP was mentioned for concise description. Virus particles were harvested 17 h posttransfection, and clarified by filtration as described above. To visualize particles, polybrene was added to the culture supernatant ; this mixture was placed in a glass-bottom dish and incubated for 2 h at 37°C with 5% COEpifluorescence microscopy was performed with an inverted Nikon TE2000E2 microscope and a 100×1.40 numerical aperture oil objective, using an X-Cite 120 system (EXFO Photonic Solution Inc.) for illumination. Digital images were acquired using a Hammamatsu ORCA-ER camera and Openlab software (Improvision) with the excitation and emission filter sets 427/10 nm and 472/30 nm for CeFP, 504/12 nm and 542/27 nm for YFP, and 577/25 nm and 632/60 nm for mCherry, respectively. Virus particle and RNA signal analysis and colocalization were performed by using custom software developed with Matlab™ and dipimage as previously described Figure S1Cell fusion without nuclear fusion. The cell line 293T.CC, expressing CD4 and CCR5, was mixed with 293T cells, which were transiently transfected with a plasmid encoding a CCR5 tropic HIV-1 Env. These two types of cells were mixed at equal ratios and were incubated for 16 hours in the presence of roscovitine prior to fixing. Prior to mixing the two cell types, the 293T cells were stained green with SYTO 11 (shown in panel A) and the 293TCC cells were stained red with SYTO 64 (shown in panel B). Nuclei were stained blue using Hoechst 33342 (panel C). Large, fused multinucleated cells appeared positive for both red and green  and distinct nuclei  were observed in these fusion experiments demonstrating that these cells were fused but each nucleus remained intact at 16 hours.(2.33 MB PDF)Click here for additional data file.Figure S2gfp gene. The two marker genes, B7 and gfp, are located in the nef reading frame; the translation of gfp is directed by an IRES.Representative flow cytometry analysis of cells infected with the HIV-1 vector BIG reveals efficient co-expression of B7 and GFP markers. BIG is identical to B6-RRE shown in  except i(0.29 MB PDF)Click here for additional data file.Figure S3Representative images of RNAs in single virions. In these images, Gag is detected with the CeFP signal, BglSL-containing HIV-1 RNA is detected with the mCherry signal, and MS2SL-containing HIV-1 RNA is detected with the YFP signal. Viral particles generated from cotransfection of GagCeFP-MS2SL-CTE and MS2-YFP (A), GagCeFP-MS2SL-CTE, GagCeFP-BglSL-CTE, MS2-YFP and Bgl-mCherry (B), and GagCeFP-MS2SL-CTE, GagCeFP-BglSL-RRE, MS2-YFP, and Bgl-mCherry (C). The channels used to detect these images are shown on top of each panel. To better demonstrate colocalization of signals, a merged and shifted panel was generated for each set of the images; in this panel, images captured from different channels were merged and the signals from the YFP channel were shifted by 4 pixels and signals from the mCherry channel were shifted by 8 pixels.(0.19 MB PDF)Click here for additional data file.


RNA interference (RNAi) mediated by siRNAs has proved to be a highly effective gene silencing mechanism with great potential for gene therapeutic applications. However, siRNA agents have been shown to exert non-target-related biological effects and toxicities, including immune stimulation. Specifically, siRNA synthesized from a T7 RNA polymerase system can trigger the potent induction of type I IFN in a variety of cells. The single-stranded RNA can also stimulate innate cytokine responses in mammals. We found that pppGn (n = 1–3), associated with the 5′ end of the shRNA produced from the T7 RNA polymerase system, did not induce detectable levels of IFN. The residual amount of G associated with the 5′-end of the transcript was proportional to the reduction of the interferon response. We describe a T7 pppGn (n = 1–3) shRNA synthesis system that alleviates the IFN response, which will facilitate the design of siRNAs while maintaining their full efficacy. Both of these studies documented significant nonspecific changes in gene expression as a consequence of the delivery of siRNAs. Two recent studies have indicated that the mechanism of the IFN response might include the recognition of the siRNAs by TLR3. In addition, long dsRNA and single-stranded (ss) RNA of ssRNA viruses are detected through TLR7 and TLR8, located in the endosomal membrane. Recently, Kim et al. showed that siRNAs synthesized using the T7 RNA polymerase system can trigger the potent induction of IFN-α and -ß in a variety of cells. Themediators of this response revealed that an initiating 5′-triphosphate was required for IFN induction.RNAi is a newly described natural biological phenomenon mediated by siRNA molecules, which target specific mRNAs for degradation by cellular enzymes. Surprisingly, recent studies have indicated that siRNAs can induce global upregulation of the expression of IFN-stimulated genes.–5 This e by TLR3.,6 In addmembrane. Recentlyim et al. showed tIn this study, we describe the absence of interferon induction by an new type of shRNAs, pppGG-shRNA, synthesized by bacteriophage polymerase.+ cells were transfected with the pppGG-luc-shRNAs corresponding to the luciferase gene, and then were further transfected with pGL3-control (Firefly) and phRG-TK (Renilla). The six pppGG-luc-shRNAs (luc-1-6) all inhibited the luciferase activities (data not shown), with luc-3 and 4 showing the most inhibitory activity.To investigate the RNAi-mediated silencing of luciferase activity, we initially synthesized six shRNAs targeting the luciferase gene transcript, using T7 RNA polymerase. The locations of these targets are provided in  showed that siRNAs synthesized using the T7 RNA polymerase system can trigger the potent induction of IFN-α and -ß in a variety of cells. The mediators of this response revealed that an initiating 5′-triphosphate was required for IFN induction. To verify the induction of INF by pppGG-shRNA, we designed pppGn (n = 0–3) associated with the 5′ end of shRNA-luc-3, which was transcribed by T7 RNA polymerase . Interferon assays from the pppGn  associated with 5′ end of shRNA revealed no interferon induction in HeLa CD4+cells (+ cells was elicited by the 5′-HOGn (n = 1–3)  . Furthern = 1–3)  could be