Towards an understanding of the poliovirus replication complex: the solution structure of the soluble domain of the poliovirus 3A protein. Richards O. A Gly1 to Ala substitution in poliovirus capsid protein VP0 blocks its myristoylation and prevents viral assembly.
Marc D. Reticulon 3 binds the 2C protein of enterovirus 71 and is required for viral replication. Tang W.
The twenty-nine amino acid C-terminal cytoplasmic domain of poliovirus 3AB is critical for nucleic acid chaperone activity. Gangaramani D. Membrane integration of poliovirus 2B viroporin. Martinez-Gil L. Schein C. Cell Sci. RNA nuclear export is blocked by poliovirus 2A protease and is concomitant with nucleoporin cleavage.
Castello A. Functional analysis of picornavirus 2B proteins: effects on calcium homeostasis and intracellular protein trafficking. Cleavage of poly A -binding protein by poliovirus 3C proteinase inhibits viral internal ribosome entry site-mediated translation. Bonderoff J. Ventoso I. Hijacking components of the cellular secretory pathway for replication of poliovirus RNA. Belov G.
Interaction of poliovirus polypeptide 3CDpro with the 5' and 3' termini of the poliovirus genome. Identification of viral and cellular cofactors needed for efficient binding.
Harris K. Myristoylation is important at multiple stages in poliovirus assembly. Moscufo N. Fujita K. The structure of the poliovirus S cell entry intermediate at angstrom resolution reveals the location of an externalized polypeptide that binds to membranes. Bubeck D. Complexes of poliovirus serotypes with their common cellular receptor, CD Rodriguez P. Structures of poliovirus complexes with anti-viral drugs: implications for viral stability and drug design. Grant R. Protein is linked to the 5' end of poliovirus RNA by a phosphodiester linkage to tyrosine. Ambros V.
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Imaging poliovirus entry in live cells. Brandenburg B. Crystal structure of CD and electron microscopic studies of its complexes with polioviruses. For active states, a Gaussian fit obtained a mean velocity of 6. For the temporal duration of active states, the data were fitted to an exponential decay with a median time of 6. These measurements indicated that the motion exhibited by a virion undergoing fusion was largely diffusive, with short, nonlinear durations of active transport interspersed during the course of observation.
We confirmed that the motion and shape changes of the filaments were most likely due to fusion and not endocytosis by immunostaining for markers of early EEA1 or late CD63 endosomes and lysosomes LAMP1. We also recorded live-cell videos of filamentous virion dynamics in cells transduced with a baculovirus adapted to mammalian cell culture expressing Rab5-GFP, showing that Rab5 did not accumulate around a filament as it underwent fusion Supporting Figure 8, Supporting Movie 2.
Taken together, these results indicate that the changes in morphology and motion of a filament during fusion to cells were not due to endocytosis, but rather due to the viral fusion process with the cell membrane. It should be noted that along with imaging hRSV viral filament RNA entry we did attempt to combine this strategy with membrane labeling using the carbocyanine lipophilic dye DiO, which is typically used in this sort of assay.
Cells decorated with high numbers of filaments were largely unlabeled, while neighboring cells, lacking filaments, were labeled efficiently, as were noninfected cells Supporting Figure 9. Only the spherical-like particles were labeled with DiO, precluding its use in interrogating filamentous virion entry. Using cells plated on a fibronectin-patterned 35 mm coverslip-bottomed dish, we watched HEp-2 cells inoculated with hRSV for eight hours, delivering MTRIP probes, labeled with a spectrally distinct fluorophore from those labeling the virion, every four hours to monitor new gRNA production Figure 6 A.
The combination of cell patterning and MTRIP-labeling allowed the same cell to be relocated and imaged over multiple time points. By delivering MTRIPs with a spectrally distinct fluorophore at varying time points, we were able to quantify changes in the volume, intensity, and number of nascent gRNA puncta on a per-cell basis Figure 6 B,C,D, respectively.
It is clear that while the number of gRNA puncta did not change significantly, the sizes of the puncta increased dramatically, accompanied by a small, but significant increase in the normalized intensity. This finding suggested that once hRSV gRNA entered the cell, it was confined to certain sites of replication and did not diffuse readily throughout the cytoplasm. Our approach provides a method to analyze the distribution of RNA and multiple viral proteins within virions simultaneously, using both conventional and localization microscopy.
While the imaging analysis of virus on glass has been attempted before using either membrane labeling 3 or fluorescence in situ hybridization FISH , 30 these methods detected the viral envelope membrane labeling or an internal RNA FISH , not both at the same time. FISH requires fixation, membrane permeabilization, and the use of formamide, which can inhibit antibody binding for identification of proteins. However, the correlation between incorporation of multiple copies of RNA viral genomes and viral replication efficiency has yet to be established.
Future work could include using localization microscopy to investigate the distribution of other viral proteins such as the matrix M protein and cellular proteins such as F-actin within the filament. This approach might help better define principal mechanistic determinants of filament formation, which are still unclear.
We also analyzed the dynamics of a filamentous virion undergoing fusion for the first time. The only other filamentous virion to have been labeled and imaged in a live cell is Ebola, 10, 16 and that analysis was limited to the co-localization of endocytic markers with GFP-viral fusion proteins as Ebola traffics through the macropinosome. The passive motion of filaments during fusion merits further investigation. The velocity in the active states was not as high as those reported for virus surfing along filopodia, 1 and these states constituted a minority of the observed motion.
Rather, the largely passive motion we observed might be due to virus attachment to cell surface lectin-like molecules by the highly glycosylated hRSV G surface glycoprotein followed by diffusion along the membrane and binding of the particles to a bona fide cell surface receptor, such as nucleolin, 38 which triggers fusion. An alternative hypothesis might be that the virus diffuses after first being recruited to a lipid raft.
The ability to watch gRNA replication in a single cell allowed several important biological observations with implications for both hRSV and other viruses. For hRSV, we observed that, in general, the overall number of granules increased slowly over time, but that the volume increased at a higher rate, suggesting that these granules were replication complexes. These complexes have not been defined previously, only assumed.
The data also showed the inherent heterogeneity of cell features during hRSV infections, which previous imaging studies have revealed, but had yet to be quantified. For viral infections, this approach has the means to ask and answer fundamental questions regarding the sources of heterogeneity of viral infections, because the initial conditions of each cellular infection can be defined, and the infection characterized within the same cells.
This method clearly will assist in the quantification and development of mechanistic models of cell-to-cell variation during viral infections. Since each MTRIP is labeled with 8—12 fluorophores, fewer probes are required to produce a single molecule sensitive fluorescence intensity than other methods such as single fluorophore FISH, 41 increasing sensitivity. Organic fluorophores are also more resistant to photobleaching than fluorescent proteins. While quantum dots have also been used for virus labeling, 42 their size and toxic effects limit their ability to observe the viral gRNA over long periods of time.
For high frame rate imaging of viral dynamics, the blinking of the fluorescence intensity of quantum dots 43 might introduce artifacts in single-particle tracking and deconvolution algorithms.
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MTRIPs do not blink under normal buffer conditions and wide-field illumination, avoiding this problem. By designing different biotinylated oligonucleotides to bind to different virion sequences, they can be easily adapted to target to different regions on the gRNA, allowing for the possibility of increasing fluorescence intensity in particle tracking experiments and for increased localization accuracy in super-resolution microscopy.
Most importantly, the MTRIP labeling method does not affect the first cycle of virus replication, the titer during amplification, or the virus morphology. The approach could also be combined with conventional membrane-labeling techniques and fluorescent fusion protein technology to yield multicolored virions suitable for live-cell observation of virion infection over the entire replication cycle. MTRIP probe design is described in more detail elsewhere. To assemble probes, labeled oligonucleotides were mixed with neutravidin Pierce in a molar ratio and allowed to react at room temperature for 1 h.
Unbound oligonucleotides were removed by centrifugation in a 30 kDa filter Millipore. Cells were plated on No. Inoculant was allowed to incubate for 1 h before cells were covered with 1 mL per well of 1. A peroxidase substrate was added after washing TrueBlue, KPL and allowed to develop for 10 min before washing again.
This assay was done for three aliquots per batch of virus grown, and the average titer of the three aliquots was used to normalize the number of input virus between unlabeled and labeled virus for all subsequent experiments with that virus batch. Cells were infected with unlabeled or labeled virus at an MOI of 0.
Cycle threshold detection was performed using the Applied Biosystems software. Infections and extractions were repeated three times. Protein was transferred to 0.
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Blots were stained using a Snap i. Vacuum was immediately turned on for 20 s to remove solution.