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Self‐Assembly of Measles Virus Nucleocapsid‐like Particles: Kinetics and RNA Sequence Dependence

Abstract Measles virus RNA genomes are packaged into helical nucleocapsids (NCs), comprising thousands of nucleo‐proteins (N) that bind the entire genome. N‐RNA provides the template for replication and transcription by the viral polymerase and is a promising target for viral inhibition. Elucidation of mechanisms regulating this process has been severely hampered by the inability to controllably assemble NCs. Here, we demonstrate self‐organization of N into NC‐like particles in vitro upon addition of RNA, providing a simple and versatile tool for investigating assembly. Real‐time NMR and fluorescence spectroscopy reveals biphasic assembly kinetics. Remarkably, assembly depends strongly on the RNA‐sequence, with the genomic 5′ end and poly‐Adenine sequences assembling efficiently, while sequences such as poly‐Uracil are incompetent for NC formation. This observation has important consequences for understanding the assembly process.

eluted from the beads in 20 mM Tris pH 8, 150 mM NaCl, 400 mM imidazol. TEV cleavage was set up over night and at the same time the protein was dialyzed against 20 mM Tris pH 8, 150 mM NaCl, 5 mM beta-mercaptoethanol (BME). The protein was then purified by gel filtration (Superdex 200 column, GE Healthcare™) equilibrated with 50 mM phosphate pH 7, 150 mM NaCl, 5 mM BME.

Electron microscopy
The dependence of the assembly of NCLPs on RNA sequence was investigated by mixing P 50 N 405 with RNA of different sequence composition: ACCAGA corresponding to six nucleotides from the 5' end of the measles virus genome (5'-RNA 6 ), UUUUUU (polyU-RNA 6 ), AAAAAA (polyA-RNA 6 ) as well as a 60 nucleotide RNA from the 5' end of the genome (5'-RNA 60 ) with sequence: ACCAGACAAAGCUGGGAAUAGAAACUUCGUAUUUUCAAAGUUUUCUUUAAUAUAUUG CAA All RNAs were purchased from Integrated DNA Technologies, Leuven, Belgium and all were terminated with OH at both 3' and 5'. The protein was mixed with the RNAs reaching final concentrations of 31 µM of P 50 N 405 and 50 µM of RNA and incubated at room temperature for 24 hours. Four microliters of sample at approximately 0.1 mg/ml were adsorbed onto the clean face of a carbon film on a mica sheet (carbon/mica interface) and negatively stained with 2% (w/v) sodium silicotungstate (pH 7.5). Images were acquired with a FEI Tecnai12 LaB6 electron microscope working at 120 kV and with nominal magnifications of 35000 and 49000X, using a Gatan OriusTM SC1000 CCD camera.
The assembly of NC particles was studied in real time by electron microscopy by mixing P 50 N 405 with 5'-RNA 6 reaching final concentrations of 22 µM of P 50 N 405 and 50 µM of 5'-RNA 6 . The sample was visualized at the following time points after mixing: 4 min, 9 min, 25 min and 67 min. In addition the P 50 N 405 sample was visualized without RNA as a control showing no formation of NCLPs (data not shown).
The influence of the presence of N TAIL on the assembly of NCLPs was investigated by mixing P 50 N 525 with either 5'-RNA 6 or 5'-RNA 60 reaching final concentrations of 20 µM of P 50 N 525 and 50 µM of RNA.

NMR spectroscopy
The spectral assignment of P 50 N 405 , P 50 N 525 and P 50 N 405 in the presence of 5'-RNA 60 was obtained using 13 C, 15 N-labeled samples employing a set of BEST-TROSY triple resonance experiments [2] correlating Cα, Cβ and C' resonances. The NMR experiments were acquired at 25°C in a buffer consisting of 50 mM phosphate, 150 mM NaCl, 5 mM BME at pH 7.0 on a Bruker spectrometer equipped with a cryo-probe operating at a 1 H frequency of 700 MHz.
The assembly of NCLPs was followed by real-time NMR using a series of SOFAST 1 H-15 N HMQC experiments using a sample of 15 N-labeled N 0 P complex (P 50 N 405 or P 50 N 525 ). The formation of NCLPs was initiated by addition of 5'-RNA 6, 5'-RNA 60 , polyA-RNA 6 , polyU-RNA 6 or 5'-DNA 6 . A series of SOFAST HMQC [3] experiments were recorded with 100 complex points in the indirect dimension, a 200 ms recycling delay and four transients providing a time resolution of approximately four minutes.
The spectra were recorded on a Bruker spectrometer operating at a 1 H frequency of 950 MHz at 25°C in a buffer consisting of 50 mM phosphate, 150 mM NaCl, 5 mM BME at pH 7.0. In addition, two additional SOFAST HMQC experiments were recorded in the absence of RNA and at the end of the time course (after 24 hours) with 256 complex points in the indirect dimension and 16 transients (Fig.   3b, S5).
The experiments allow us to follow the evolution of the NMR spectra as P 50 is released from the N 0 P complex and the NCLPs are formed. A time trace of the formation of the NCLPs were obtained by extracting the NMR signal intensities from each of the SOFAST HMQC spectra and plotted as function of time after addition of the RNA.
NC assembly by fluorescence anisotropy P 50 N 405 or P 50 N 525 was diluted to the desired concentration into 50 mM Na-phosphate pH 7, 150 mM NaCl, 5 mM βME directly into the fluorescence cuvette. RNA 10 -FAM (10 nuleotide RNA derived from the 5' end of the MeV genome, with a fluorescein derivative attached to its 3' end; ACCAGACAAA-FAM) was added to N 0 P immediately prior to kinetics acquisition, mixed quickly through pipetting and fluorescence kinetics were recorded at an emission wavelength of 520 nm upon excitation with 470 nm light. Parallel and perpendicular polarization directions were recorded alternatingly and used pairwise to calculate the fluorescence anisotropy r with the G-factor correcting for detection differences between parallel ( ∥ ) and perpendicular ( ! ) polarized fluorescence light. G was determined on a daily basis according to standard protocols, and remained stable between days. Fluorescence spectra were recorded at the end of the assembly kinetics with an excitation wavelength of 460 nm and an emission range of 470-650 nm.
Assembly kinetics were analysed following exponential kinetics derived from a simple binding model assuming pseudo first order conditions: n was set to 2 depending on the requirements of the respective sample. A global fit was employed enforcing linearity between k i and N 0 P concentration. The global fit for varying P 50 N 405 concentrations was achieved by considering the time between RNA addition and the start of the measurement, fixing r t=0 to 0.05 and considering two independent rates (n=1). The apparent rate k i in the fitting equation was replaced by k i *·c i , with k i * the ith assembly rate constant and c i the concentration of N 0 P. c i was fixed for the individual curves and the amplitudes A i were fitted independently for the different N 0 P concentrations. Due to the more complex assembly kinetics, the fit of P 50 N 525 NC assembly was simplified by keeping r t=0 as a fitting parameter per kinetic time trace. Only the two smallest protein concentrations (7.5 µM and 3.8 µM) required a third (fast) kinetic component in this case. Figure S1. The three constructs of N and their nomenclature.    Figure) and that N contains two extra residues ( 406 VE 407 ) before the non-cleavable his-tag used for purification. The main differences between the two spectra are, as expected, located at residues 44-56 of P and residues 26-29 of N.  Figure S5.
Comparison of 1 H-15 N correlation spectra of P 50 N 405 with and without RNA. Spectra are shown of P 50 N 405 (red), P 50 (green) and P 50 N 405 after incubation with 5'-RNA 60 for 24 hours at 25°C (blue). The apparent rates for the assembly process in this case were 6.4×10 -4 s -1 and 7.0×10 -5 s -1 .

Figure S12
Comparison of the 1 H-15 N HSQC spectra of P 50 N 405 in the absence and presence of polyU-RNA 6 .
Spectra are shown of the isolated P 50 N 405 (red) and the P 50 N 405 in the presence of a 2-fold molar excess of polyU-RNA 6 recorded after incubation at 25°C for 24 hours (blue), showing that no assembly was observed by NMR for polyU-RNA 6 .

Figure S13
Comparison of the 1 H-15 N correlation spectra of P 50 N 405 in the absence (red) and presence (blue) of 5'-DNA 6 (DNA sequence corresponding to 5' RNA 6 ), showing that no assembly was observed upon addition of DNA. Spectra were recorded before addition and 20 hours after addition of the DNA.