JVI Accepts, published online ahead of print on 15 January 2014 J. Virol. doi:10.1128/JVI.03483-13 Copyright © 2014, American Society for Microbiology. All Rights Reserved.

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Common mechanism for RNA encapsidation by negative strand RNA viruses

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Todd J Green, Robert Cox, Jun Tsao, Michael Rowse, Shihong Qiu, Ming Luo#

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Department of Microbiology

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University of Alabama at Birmingham

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Birmingham, Alabama, 35294, USA

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(Running title: NSV RNA encapsidation) #

send correspondence to:

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1720 2nd avenue south

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Birmingham, Alabama, 35294, USA

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Phone: 1-205-934-4259

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Fax: 1-205-934-0480

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Email: [email protected]

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Abstract

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The nucleocapsid of a negative strand RNA virus is assembled with a single nucleocapsid

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protein and the viral genomic RNA. The nucleocapsid protein polymerizes along the length of

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the single strand genomic RNA (vRNA) or its complementary RNA (cRNA). This process of

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encapsidation occurs concomitantly with genomic replication. Structural comparisons of several

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nucleocapsid-like particles show that the mechanism of RNA encapsidation in negative strand

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RNA viruses has many common features. Fundamentally, there is a unifying mechanism to keep

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the capsid protein protomer monomeric prior to encapsidation of viral RNA. In the nucleocapsid,

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there is a cavity between two globular domains of the nucleocapsid protein where the viral RNA

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is sequestered. The viral RNA must be transiently released from the nucleocapsid in order to

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reveal the template RNA sequence for transcription/replication. There are cross-molecular

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interactions among the protein subunits linearly along the nucleocapsid to stabilize its structure.

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Empty capsids can form in the absence of RNA. The common characteristics of the RNA

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encapsidation not only delineate the evolutionary relationship of negative strand RNA viruses,

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but also provide insights to their mechanism of replication.

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Importance

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What separates NSVs from the rest of the virosphere is that the nucleocapsid of NSVs serves

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as the template for viral RNA synthesis. Their viral RNA-dependent RNA polymerase (vRdRp)

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can induce local conformational changes in the nucleocapsid to temporarily release the RNA

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genome so that vRdRp can use it as the template for RNA synthesis during both transcription and

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replication. After RNA synthesis at the local region is completed, vRdRp processes downstream

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and the RNA genome is restored in the nucleocapsid. We found that the nucleocapsid assembly

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of all NSVs shares three essential elements: a monomeric capsid protein protomer, parallel

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orientation of subunits in the linear nucleocapsid, and a 5H+3H motif that forms a proper cavity

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for sequestration of the RNA. This observation also suggests that all NSVs are evolved from a

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common ancestor that has this unique nucleocapsid.

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Introduction

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All viruses contain a protein capsid that encapsidates the genomic polynucleotide. The capsid

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is assembled with multiple copies of one or a few types of protein subunits following certain

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symmetry. The most commonly studied symmetry is that of icosahedron which leads to spherical

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or prolate virus particles (1-3). By contrast, helical symmetry is used for assembly of filamentous

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capsids (4). The basic fold of the capsid protein subunit is found to be the same for a large

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number of virus families, even though they may not be related to each other on a genomic basis.

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The quintessential example of this is that of the β–barrel fold that was first found in small plant

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RNA viruses, yet now has been discovered in at least 15 different viral families (5). The

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architecture of the nucleocapsid is closely tied with the mechanism of replication in view of the

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fact that the assembly of the capsid packages the viral genome.

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For negative strand RNA viruses (NSVs), eight families have been recognized by the

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International Committee on Taxonomy of Viruses (ICTV). The unique feature that distinguishes

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NSV from the rest of the virosphere is that the nucleocapsid, instead of the naked genome, is

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used as the template for viral nucleotide synthesis. It is indubitable that the assembly of the NSV

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nucleocapsid is related to the unique mechanism of its viral RNA synthesis. In each of NSVs, the

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nucleocapsid is packaged inside a lipid envelope. The appearance of the nucleocapsid inside the

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envelope is different from virus to virus. In rhadboviruses, the nucleocapsid adopts a

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characteristic bullet shape (6). In paramyxoviruses, the nucleocapsid is filamentous or

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herringbone-like (7). In orthomyxoviruses, the nucleocapsid has a double helical structure (8, 9).

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When the nucleocapsid is released from the virion, they all have the appearance of a coil (10).

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The genomic RNA encapsidated in the nucleocapsid is protected from RNA nucleases to various

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degrees depending on the structure. This protective structure renders the RNA not readily 4

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accessible to the viral polymerase. Thus the viral polymerase must gain access to the

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encapsidated RNA in order to carry out viral RNA synthesis. Since this is a common mechanism

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of all NSVs, it is likely that the nucleocapsid of NSVs bears characteristic elements shared by all

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NSV families. Recognition of these elements helps in defining essential viral functions and

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revealing the underlining mechanism for NSV replication.

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By systematic analyses of the known structures of the NSV nucleocapsid, we discovered the

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common mechanism for genomic RNA encapsidation during replication. This unifying

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mechanism suggests a common origin of NSV families and presents a clear picture for the

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functions of the nucleocapsid protein.

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Materials and Methods

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The coordinates with PDB codes of 3PU4 (VSVN), 2WJ8 (RSVN), 4H5P (RVFVN) and

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4BHH (LACVN) were retrieved from PDB (11-14). The superposition was carried out with Fr-

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TM-align (15). The results were summarized in Table 1.

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The NA320-24-2P protein complex was produced in E. coli as reported previously (16). X-ray

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scattering data on VSV NA320-24-2P samples were collected on the SIBYLS beamline at the

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Advanced Light Source (Figure 1A). Scattering curves were processed with PRIMUS (17).

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Protein samples ranging from 0.75 to 4.3 mg/mL showed no sign of aggregation by Guinier plot

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analysis (Figure 1B). Rg, Dmax and Porod volumes were calculated with the ATSAS package

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(Table 2) (18). Bead models (ten in total) were generated from the scattering data with

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DAMMIN (Figure 2) (19) and an averaged bead model was calculated with DAMAVER (20).

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The average χ2 value for the bead models was 0.986. EOM (RANCH and GAJOE) was used to 5

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build a multi-domain model of the NA320-24-2P complex against the SAXS data (18, 21).

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Structures of the VSV P N-terminus (PNT, amino acids 6-34) (22), the dimeric P oligomerization-

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domain (POD) (23), PCTD/N protein complex with bound PNT (24) and an additional unbound PCTD

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were used as rigid bodies. The domain structures were derived from the structures with PDB

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codes: 2FQM, 3HHW, and 3PMK. No partial restraints were imposed on the individual rigid

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bodies. Fit of the bead and the multi-domain models to the experimental data are shown in

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Figure 1E. Inspection of the fitted curves showed a dip in the calculated curve at low q values

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suggesting that a larger organized species was in solution. Two copies of the EOM model were

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fit to the experimental curve with SASREF (25). This fit to the curve was a remarkable match

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yielding a χ value of 1.581 (single fit 12.865). The resulting models were superimposed onto the

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bead models from DAMMIN with SUPCOMB (19, 26). The fit is shown in Figure 2A, B.

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Results and Discussion

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Capsid protomer

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The atomic structure of nucleocapsid-like particles (NLPs) has been reported for three virus

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families of NSV, Rhabdoviridae, Paramyxoviridae (Genus Pneumovirus), and Bunyaviridae

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(Genera Phlebovirus and Orthobunyavirus) (11-14, 27-31). A comparison of representative

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structures from each genus was performed. Since the structures in the same genus are highly

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homologous, vesicular stomatitis virus (VSV) (11) is selected to represent rhabdoviruses;

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respiratory syncytial virus (RSV) (12), pneumoviruses; rift valley fever virus (RVFV) (13),

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phleboviruses; and La Crosse virus (LACV) (14), orthobunyaviruses. In each of these structures,

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it was found that the encapsidated RNA is sequestered in a protein cavity. The capsid protein (N, 6

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also known as nucleocapsid protein or nucleoprotein) is first synthesized as a monomeric protein

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(named N0, the capsid protein protomer) before being incorporated in the nucleocapsid. N0

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remains monomeric through a number of different ways. However, the fundamental requirement

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to support viral replication is to prevent N0 from oligomerization before encapsidation of viral

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RNA. The historic description that N0 is prevented from RNA binding is proven incorrect (16,

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32). N0 is not an RNA binding protein but rather a capsid protein that assembles a capsid to

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accommodate any RNA sequence. Any reported in vitro RNA binding measurements are mainly

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for nonspecific electrostatic interactions between the negative charges of RNA phosphate groups

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and the positively charged residues in N0. The nature of these nonspecific interactions has no

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difference from that between any other positively charged protein, for instance, the matrix

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protein of influenza virus, and RNA.

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For rhabdoviruses, the N0 form is kept monomeric by forming a complex with the

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phosphoprotein (P). To support viral replication continuously, it is required that the N and P

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proteins are expressed in a 1:1 molar ratio (33, 34). A complex of N0-P was isolated from insect

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cell expression of rabies virus (RABV) N and P proteins, which contains a N subunit and a dimer

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of P (35). The same N0-P complex was also isolated for a mutant of the VSV N protein when the

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mutant N protein was coexpressed with P in E. coli (16). The mutations in the VSV N protein

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changed a stretch of five residues to Ala (NA320-24) and prevented formation of NLP. Analytical

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unltracentrifugation experiments showed that the complex like the insect derived complex has a

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1:2 (N to P) stoichiometry. This complex was studied by small angle X-ray scattering (SAXS)

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techniques (Figure 1 and Table 2). The shape of the complex was determined by the ab initio

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method in DAMMIN and is shown in Figure 2 (19). Independently, previously determined

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crystal structures of N and domains of P were modeled against the scattering curves with EOM 7

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(18, 21). In this method, the linkers between P protein domains were also modeled by an ab

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initio approach yielding a complete model of the No-P2 protein complex. A complete model is

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presented in Figure 3. The model suggests that the dimeric P is associated with N0 through

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multiple sites of interactions. The observation that different parts from each monomer of the P

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dimer contribute to N binding helps to explain studies where functions of the N-terminus

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truncated P protein mutant can be restored when a C-terminus truncated P protein mutant is

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provided in trans (36).

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The P protein has a modular structure with a flexible N-terminal region, a structured

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oligomerization domain in the middle and a compact C-terminal domain (PCTD). Both the N- and

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C-terminal regions of P bind to the N protein. In one study, a complex was generated by binding

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the N-terminal 60 residues of VSV P with the ring structure of a N mutant in which the N-

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terminal 21 residues were deleted (22). The N-terminus of P interacts with the back of the C-

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terminal half (C-lobe) of N. An α-helix corresponding to residues 17 to 31 of P was shown to

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occupy the RNA cavity in the structure. Subsequent studies by Chen et al. (37) using a P3A

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mutant (triple mutations of Ser or Thr to Ala) showed that phosphorylation of P at positions

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Ser60, Thr62 and Ser64 is required to prevent N0 from encapsidating cellular RNA, which leads

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to a dead-end product of N and diminishes viral replication. In the SAXS model, residues Ser60,

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Thr62 and Ser64 were notably positioned to form charge interactions with Lys398, Arg399,

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Lys414 and Lys417 of N (named here as the “PO binding site”). The crystal structure of the

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VSV NLP in complex with PCTD showed P binds to the C-lobe of N, primarily the C-terminal

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extended loop and α-helix 13 (24). Combining the results of SAXS and crystallography suggests

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that P forms a stable complex with the monomeric N subunit involving interactions with the C-

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terminal loop, a positively charged patch at the PO binding site, the RNA cavity, and the back of 8

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the C-lobe of N. The oligomerization of N is prevented by binding of P whereby the binding site

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for the N-terminus of P overlaps with that for the N-terminus of N in the nucleocapsid. The

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region of P bound at the PO binding site can block the side-by-side contact between N subunits

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found in the nucleocapsid. Mutation studies showed that loss of any of these interaction sites

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could diminish N oligomerization, thus RNA encapsidation (16). Upon assembly of the

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nucleocapsid, the N0 bound P is removed. N begins to oligomerize and encapsidate viral RNA.

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The P protein is then recycled to bind another N0 subunit.

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For RSV, there is no structural report of an N-P complex. However, it was shown that the N-

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terminal 119 residues precede the oligomerization domain of P in primary sequence (38). The N-

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terminus of RSVP is required for P binding to N0 (39). The N-terminal region of RSVP was

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predicted to be intrinsically disordered, but residues 15-25 are predicted to be an α-helix. A

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homologous structure was recently reported for a complex between the N-terminus of P and N of

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Nipah virus (40). The N-terminus of P binds the C-terminal domain of N where it may exclude

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binding of the C-terminus of a neighboring N subunit to this surface to prevent N

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oligomerization. RSVP could bind to a similar site in RSVN, or another site of N which is

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required for binding the N-terminus of a neighboring N subunit. The exact binding site for the N-

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terminus of RSVP remains to be determined for RSVN.

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For RVFV, the structure of the N protein has been solved in two forms, as an apo-structure

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without RNA and an NLP. The first form of RVFVN has its N-terminal helix associated with its

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own RNA encapsidation cavity (41). Residues 20 to 26 form a helix (α2). Within this helix, the

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hydrophobic sidechain of Trp24 fits in the pocket where the bases of the RNA are sequestered in

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the nucleocapsid, as observed in the RVFV NLP. In the oligomeric structure of the RVFV NLP,

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the N-terminus of N is moved out of the RNA cavity to interact with the neighboring N subunit 9

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(13). These observations suggest that the RVFVN protein is kept RNA-free by sequestering its

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own N-terminus. During assembly, the N-terminus of RVFVN rearranges to support capsid

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formation and RNA encapsidation. In another study, the structures of RVFV empty capsids have

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also been solved (42). The empty capsids maintain the architecture of capsids with encapsidated

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RNA. The reason for formation of empty capsids in this case is not understood. It is possible that

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the N protein is not stable in the over-expressed bacterial milieu or an unknown protein, either

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viral or cellular is necessary to help keep N from self-assembling. Lastly, this could be an artifact

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resulted from producing the protein with an N-terminal thioredoxin-fusion.

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For LACVN, several structures have been produced. The N-terminal 17 residues of N are

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highly flexible and could be in a fold-back conformation in the N monomer, based on structures

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of the N protein complex after RNA was removed (14). The fold-back conformation is similar to

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sequestering the N-terminus in N of phleboviruses, but to a lesser extent. In LACV NLP, the N-

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terminus of N assumes a conformation that extends to its neighboring N subunit. The extended

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N-terminus has interactions with the bases and backbone of the encapsidated RNA, as well as

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amino acid interactions with its neighboring N subunit. The C-terminus (residues 218-235) is

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extended from the core of N and shows some conformational flexibility though not as much as

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the N-terminus. The elements required for keeping the orthobunyaviruses N in a monomeric

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form could be similar to those for N of phleboviruses.

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The essential requirement for N to be competent in encapsidating viral RNA is to remain

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monomeric. Different strategies may be used to achieve this, including occupying the sites

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required for oligomerization by P binding, such as in the case of rhabdoviruses and

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pneumoviruses, or sequestering the N-terminus by N itself, such as in the case of bunyaviruses.

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In rhabdoviruses, the proximity of P binding to the RNA cavity is purely coincidental and only 10

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for the formation of a stable N0-P complex. This complex is essential to prevent premature N

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oligomerization rather than prevent RNA binding. This concept is supported by the results in

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Chen et al. who showed that a phosphorylation deficient triple-mutant of P (P3A) that is mutated

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outside of the cavity binding region could not prevent encapsidation of cellular RNA by VSVN

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(37).

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Architecture of the nucleocapsid

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The N protein oligomerizes to encapsidate the single strand viral RNA. The N subunits are

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associated with each other in a parallel orientation. A linear nucleocapsid is assembled with the

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single strand viral RNA sequestered in the center. In rhabdoviruses, the nucleocapsid is a random

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coil when it is isolated from the virion (43). The nucleocapsid is packaged into a superhelical

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structure in the bullet shaped virion (6). The super symmetry is imposed on the nucleocapsid by

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the matrix protein that has a 1:1 interaction with the N protein in the virion. The matrix protein

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subunits have direct contacts among themselves to form the 2D helical mesh under the viral

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membrane envelope. The same helical symmetry is adopted by the nucleocapsid when packaged

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in the virion. The fact that the single strand viral RNA is completely encapsidated prior to being

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packaged into the virion defines that the true symmetry of the nucleocapsid is linear. In most

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NSVs, the nucleocapsid appears to be a random coil (10). The nucleocapsid in members of the

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Paramyxovirinae contains a number of helical segments, but the helical symmetry is not strict in

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terms of pitch and rotation of the N subunits (44). The Sendai virus nucleocapsid exists in at least

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four different helical states (45). Since the repeating unit in the nucleocapsid is linear along the

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encapsidated RNA and the helical symmetry is not required for RNA encapsidation, the exact

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symmetry of the nucleocapsid of members in Paramyxovirinae strictly speaking must be

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considered linear. In influenza virus (IFV), the linear nucleocapsid is twisted into a rough double 11

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helix, with interactions between the two associated strings (8, 9). The helical superstructure is a

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way to condense the nucleocapsid for packaging in the virion, but not essential for viral RNA

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encapsidation.

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The forces that stabilize the nucleocapsid involve extensive cross-molecular interactions

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among the N subunits. There are side-by-side interactions between N subunits aligned in parallel.

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These interactions are critical to capsid formation. The N protein can no longer assemble the

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nucleocapsid if these interactions are disrupted by mutation (16). The extent of the side-by-side

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interactions is different from virus to virus ranging from below 200 Å2 for LACVN up to ~2200

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Å2 for VSV. There is also a disparity to the amount of buried surface between adjacent C-

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terminal domains versus N-terminal domains of each viral N (11). The contact areas between

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neighboring domains in different NLPs are listed in Table 3. The contact areas must have

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plasticity because these calculated contact areas are derived from an artificial ring structure. In

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the authentic linear nucleocapsid, these contact areas are likely to be different. The difference

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between the two halves of the N protein may have some functional implications because the N

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protein needs to undergo a conformational change to reveal the sequestered template RNA

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during viral RNA synthesis. It is likely that the domain that has a lesser contact area with the

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neighboring domains should be opened to reveal the RNA. As shown in Table 3, the N-terminal

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domain of VSVN has a lesser contact area and is likely to be open during viral RNA synthesis,

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while the C-terminal domain remains associated to maintain the integrity of the nucleocapsid.

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Rearrangement of the N-terminal domain to open the cavity is further supported in lieu of the

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greater association between the C-terminal domains due to additional interactions, as noted

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below.

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In addition to the side-by-side contacts, the most obvious interactions between the N subunits

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are from the structural elements extended from the core of the N subunit. The core of N and the

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nucleotides associated with the core act as one structural unit. The number of nucleotides

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accommodated in the core is listed in Table 3. The registration of each N subunit may be defined

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by choosing one subunit as the origin (position 0). If the access to the RNA cavity faces away

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from the reader, -1 refers to the subunit on the left of the origin subunit (5’ end of the

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encapsidated RNA), and +1, on the right (3’end of the encapsidated RNA). In rhabdoviruses, the

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N-terminal arm (21 residues) extends away from the core to interact with the -1 N-terminal

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domain (N-lobe), whereas an extended loop in the C-lobe interacts with the +1 C-lobe. The N-

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terminal arm also interacts with the extended loop in the -2 C-lobe. In RSVN, the N-terminal arm

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(28 residues) interact with both N- and C-terminal domains of the -1 subunit, whereas the

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extended C-terminal arm, rather than a loop in this case, interacts with the C-terminal domain of

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the +1 N subunit. In RVFVN, only the N-terminal arm (32 residues) interacts with the -1 N-

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terminal domain. In LACVN, the N-terminal arm (17 residues) interacts with the -1 N-terminal

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domain involving only the first 8 residues. The remaining residues interact with the encapsidated

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RNA. The C-terminal arm of LACVN (18 residues) contains an α-helix that interacts with the C-

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terminal domain of subunit +1. Residues that are involved in capsid formation are illustrated in

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Figure 4.

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In IFVN, there is also an extended loop in the C-terminal region that interacts with the C-

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terminal domain in the +1 N subunit. It is not clear if the N-terminal region is involved in the

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interactions with the neighboring subunits in the nucleocapsid. An atomic model of the

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nucleocapsid or an NLP is needed to fully address this. It is also not clear how IFVN is

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maintained in a monomeric form prior to RNA encapsidation. There is no report of any viral 13

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protein that is involved in this function. In the reported structure of IFVN, the N-terminus of N

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could be in a sequestered conformation. If this is the case, IFVN may employ a method similar to

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that used by RVFVN and LACVN to remain monomeric. The extended loop in the C-terminal

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domain folds back on the surface of its own monomer as shown by the structure of an obligate

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monomeric mutant of IFVN (46). This folded conformation of the C-terminal extended loop

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should also contribute to maintaining a monomeric mode of IFVN prior to RNA encapsidation.

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The surface contact and domain-swap interactions between neighboring N subunits are

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essential for the assembly of NSV nucleocapsids. Similar interactions are also common in the

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nucleocapsid of other viruses, such as picornaviruses and adenovirus (47, 48). The only unique

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feature in the NSV nucleocapsid is that these interactions are linear along the encapsidated single

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strand viral RNA.

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Sequestered RNA in the cavity

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The nucleocapsid of NSVs needs to be capable of encapsidating all possible RNA sequences.

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The mechanism for specific encapsidation of viral RNA may be that viral RNA encapsidation is

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concomitant with viral replication, likely to be at the site of the viral RNA replication complex

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(24). At this point, the monomeric N protein assembles the nucleocapsid simultaneous with

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encapsidation of the genome. No consensus interactions with the bases of the encapsidated RNA

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were identified among all the reported NLP structures, including the cases in which

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homogeneous sequences were encapsidated in the NLP (49). Interactions of the backbone

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phosphate groups with positively charged sidechains were found for a fraction of the sequestered

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phosphate groups, but there is not a conserved pattern among NLPs of different viruses. This

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holds true for even the closely related NLP of VSV and RABV (32) and suggests that the

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positively charged residues in the RNA cavity are not the key factor responsible for RNA

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encapsidation.

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What is unique about the viral RNA in the NSV nucleocapsid is that the sequestered bases

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are stacked to form a motif similar to one-half of the A-form double helix of RNA (Figure 5A).

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In rhabdoviruses, the bases of nucleotides 1-4 are stacked and face the solvent side of the RNA

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cavity, whereas bases of nucleotides 5, 7 and 8 are stacked and face the interior of the N subunit.

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Similarly in RSV, bases of nucleotides 2-4 are stacked and face the solvent side of the RNA

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cavity, whereas bases of nucleotides 5-7 are stacked and face the interior of the N subunit. There

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is an additional “linker” nucleotide between the N subunits in rhabdoviruses and RSV. This

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nucleotide is still sequestered from solvent accessibility when N subunits oligomerize in the

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nucleocapsid, but it may allow some flexibility between the N subunits. The base of this linker

304

nucleotide can be stacked with the other bases, or an aromatic sidechain will take its place to

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stack with the other bases when the linker nucleotide is in a transitional conformation (11, 12). In

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the RVFV NLP, four bases are sequestered by the core domains each facing the interior of the N

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subunit. Among these four bases, the two in the center are stacked. The bases of the three

308

“linker” nucleotides are also in a stacked conformation and protected by the capsid structure. In

309

the LACV NLP, seven bases near the 5’ end (nucleotides -11 and 1-6) of the RNA strand are

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stacked and face the entrance to the RNA cavity. The aromatic sidechain of tyrosine-177 is

311

intercalated between the bases of nucleosides 7 and 8, and the base of nucleotide 8 is further

312

stacked with the base of nucleotide 9. The bases in this triple stacking face the RNA cavity.

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Lastly, the linker nucleotide 10 is sequestered in the RNA cavity, but its base is not stacked. The

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unique stacking arrangements observed in NSVs allow for maximized packaging of the RNA in

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the capsid. 15

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The base stacking is only stabilized when the viral RNA is in the cavity of the nucleocapsid.

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The N subunits can form a stable empty capsid without RNA (16).The encapsidated RNA is a

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resident in the nucleocapsid, perhaps also contributing to the overall stability of the

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nucleocapsid. The shape and the electrostatic environment of the cavity define how base stacking

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occur. The stability of the base-stacking motifs in the RNA cavity is dependent on the RNA

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sequence, with poly(rA) being the most stable and poly(rU) being the least stable (49). Specific

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sequences in the sequestered RNA genome regulate many viral functions, such as transcriptional

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initiation and termination. In terms of transcriptional termination, there is a highly conserved U7

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track at the end of each coding region in rhabdoviruses. The structure in this region is the least

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stable in the nucleocapsid. It is conceivable that such instability could promote dissociation of

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the viral transcription complex. Mutations in the N protein have been shown to alter the level of

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mRNA synthesis. In this case, genomic mutations have subsequently occurred resulting in an

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extension of the U track to U8 in rescued VSV (50). U rich sequences are also found in the

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intergenic regions in other NSV genomes, but not all.

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The RNA cavity is formed by secondary structural elements from the N- and C-domains in

331

the N protein. A highly conserved (5H+3H) motif consisting of 5 helices from the N-domain and

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3 helices from the C-domain has been identified to constitute the RNA cavity (51). When the

333

structures of VSVN and RSVN are superimposed using the Fr-TM-align method (15), the

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(5H+3H) motif from each N protein (α4-α8 in the N-domain, and α9-α11 in the C-domain of the

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VSVN protein (11); αN3, αN5-αN8, and αC1, η2, and αC3 of the RSVN protein (12)) could be

336

superimposed as a rigid body (Figure 5B, 5C and Table 1). η indicates a 310 helix. In addition,

337

two helices following the (5H+3H) motif in the C-domain could also be superimposed as part of

338

the core in these two N proteins (Figure 5B, 5C). In the case of RVFVN, only the first four 16

339

helices (α3, α5-α7 in RVFVN (13)) in the (5H+3H) motif may be aligned with those in VSVN or

340

RSVN (Figure 5B, 5C and Table 1). The 5th helix of the motif is present but moved away from

341

the RNA cavity, inducing reorientation of the following three helices in the (5H+3H) motif, as

342

well as helices following the motif. As a result, this RNA cavity is almost completely collapsed

343

compared to that in VSVN or RSVN. This small RNA cavity could only accommodate the bases

344

of four nucleotides, while excluding the ribose-phosphate backbone. LACVN (14) has the same

345

topology as RVFVN (Figure 5B, 5C and Table 1). The first four helices of LACVN in the

346

(5H+3H) motif (α2, η1, α3, and α4) correspond to those in RVFVN. However, there are two

347

noticeable differences between the two structures. One is that the linker between the first and

348

second helices is changed from a helix (α4) to a β–hairpin (β1-4). The other is that the third helix

349

in RVFVN (α4) is much shorter than that in LACVN (α3). The three helices in the C-terminal

350

domain of the (5H+3H) motif are the same in RVFVN and LACVN, as well as three additional

351

helices following the motif, in terms of topology. In addition, LACVN has an extra helix at the

352

C-terminus (α11). This helix is involved in interactions with the neighboring N subunit on the

353

right side (+1), as described above.

354

Summary

355

The nucleocapsid of NSVs is assembled by the universal assembly principle of all viral

356

nucleocapsids. Oligomerization of the protein subunits forms the capsid that encapsidates the

357

viral polynucleotide. Extensive interactions cross protein subunits, including domain-swaps.

358

These interactions are involved in the stabilization of the nucleocapsid. What is unique about

359

NSVs is that their nucleocapsid has a linear symmetry. The viral RNA is a string in the center of

360

the nucleocapsid ribbon. The nucleocapsid assembly of all NSVs shares three essential elements:

361

a monomeric capsid protein protomer, parallel orientation of subunits in the nucleocapsid, and 17

362

the 5H+3H motif that forms a proper cavity for sequestration of the RNA. During viral RNA

363

synthesis, the nucleocapsid does not disassemble to completely release the viral RNA template.

364

Instead, conformational changes must occur in protein domains to temporarily release the RNA

365

template on a local rather than global basis. The viral polymerase complex is a likely candidate

366

to induce this conformational change in order to gain access to the sequestered bases, some of

367

which face the interior of the N subunits. Once revealed, the RNA template is available for

368

transcription and genomic replication. After the viral polymerase complex passes, the RNA is

369

tidily repositioned in the encapsidation cavity and the integrity of the nucleocapsid is restored.

370

This new mechanism of RNA encapsidation allows the N protein to play a role in viral

371

replication and transcription. Mutations in the N protein may increase or decrease the level of

372

viral replication (52, 53). More interestingly, some of these mutations in the N protein did not

373

change the level of viral replication, but changed the level of transcription. This suggests that the

374

structure of the nuclecapsid itself has a role in viral transcription that is independent of

375

replication. For viral replication, the viral polymerase complex needs to load at the 3’ end of the

376

genome and process through the nucleocapsid. For viral transcription, on the other hand, the viral

377

polymerase complex needs to recognize the promoter and the transcription termination

378

sequences, in addition to loading and processivity. There may be a unique structural feature

379

associated with the regions where the promoter or the transcription termination sequence is

380

located in the nucleocapsid to allow such recognition.

381

Acknowledgement

382

We thank Dr. Mark Walter for assistance in collecting SAXS data. SAXS experiments were

383

conducted at the Advanced Light Source (ALS), a national user facility operated by Lawrence

18

384

Berkeley National Laboratory on behalf of the Department of Energy, Office of Basic Energy

385

Sciences, through the Integrated Diffraction Analysis Technologies (IDAT) program, supported

386

by DOE Office of Biological and Environmental Research. Additional support comes from the

387

National Institute of Health project MINOS (R01GM105404). The work is supported in part by a

388

NIH grant 1R01AI10630 to ML and 1R56AI01087 to TG. References

389 390 391 392 393 394 395 396 397 398 399 400 401 402 403 404 405 406 407 408 409 410 411 412 413 414 415 416 417 418 419 420 421 422

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Mavrakis M, Iseni F, Mazza C, Schoehn G, Ebel C, Gentzel M, Franz T, Ruigrok RW. 2003. Isolation and characterisation of the rabies virus N degrees-P complex produced in insect cells. Virology 305:406-414. Chen M, Ogino T, Banerjee AK. 2007. Interaction of vesicular stomatitis virus P and N proteins: identification of two overlapping domains at the N terminus of P that are involved in N0-P complex formation and encapsidation of viral genome RNA. J Virol 81:13478-13485. Chen L, Zhang S, Banerjee AK, Chen M. 2013. N-terminal phosphorylation of phosphoprotein of vesicular stomatitis virus is required for preventing nucleoprotein from binding to cellular RNAs and for functional template formation. J Virol 87:3177-3186. Llorente MT, Garcia-Barreno B, Calero M, Camafeita E, Lopez JA, Longhi S, Ferron F, Varela PF, Melero JA. 2006. Structural analysis of the human respiratory syncytial virus phosphoprotein: characterization of an alpha-helical domain involved in oligomerization. J Gen Virol 87:159-169. Mallipeddi SK, Lupiani B, Samal SK. 1996. Mapping the domains on the phosphoprotein of bovine respiratory syncytial virus required for N-P interaction using a two-hybrid system. J Gen Virol 77 ( Pt 5):1019-1023. Yabukarski F, Leyrat C, Tarbouriech N, Jensen MR, Blackledge M, Ruigrok R, Jamin M. 2013. Crystal structure of the N0-P complex of Nipah virus and of VSV provide new insights into the encapsidation mechanism. Proceedings of XV International Conference on Negative Strand RNA Viruses:84. Raymond DD, Piper ME, Gerrard SR, Smith JL. 2010. Structure of the Rift Valley fever virus nucleocapsid protein reveals another architecture for RNA encapsidation. Proc Natl Acad Sci U S A 107:11769-11774. Ferron F, Li Z, Danek EI, Luo D, Wong Y, Coutard B, Lantez V, Charrel R, Canard B, Walz T, Lescar J. 2011. The hexamer structure of Rift Valley fever virus nucleoprotein suggests a mechanism for its assembly into ribonucleoprotein complexes. PLoS Pathog 7:e1002030. Blocquel D, Bourhis J-M, Éléouët J-F, Gerlier D, Habchi J, Jamin M, Longhi S, Yabukarski F. 2012. Transcription et réplication des Mononegavirales : une machine moléculaire originale. Virologie 16:3. Bhella D, Ralph A, Yeo RP. 2004. Conformational flexibility in recombinant measles virus nucleocapsids visualised by cryo-negative stain electron microscopy and real-space helical reconstruction. J Mol Biol 340:319-331. Egelman EH, Wu SS, Amrein M, Portner A, Murti G. 1989. The Sendai virus nucleocapsid exists in at least four different helical states. J Virol 63:2233-2243. Chenavas S, Estrozi LF, Slama-Schwok A, Delmas B, Di Primo C, Baudin F, Li X, Crepin T, Ruigrok RW. 2013. Monomeric nucleoprotein of influenza A virus. PLoS Pathog 9:e1003275. Rossmann MG, Arnold E, Erickson JW, Frankenberger EA, Griffith JP, Hecht HJ, Johnson JE, Kamer G, Luo M, Mosser AG, et al. 1985. Structure of a human common cold virus and functional relationship to other picornaviruses. Nature 317:145-153. Liu H, Jin L, Koh SB, Atanasov I, Schein S, Wu L, Zhou ZH. 2010. Atomic structure of human adenovirus by cryo-EM reveals interactions among protein networks. Science 329:1038-1043. Green TJ, Rowse M, Tsao J, Kang J, Ge P, Zhou ZH, Luo M. 2011. Access to RNA encapsidated in the nucleocapsid of vesicular stomatitis virus. J Virol 85:2714-2722. Harouaka D, Wertz GW. 2012. Second-site mutations selected in transcriptional regulatory sequences compensate for engineered mutations in the vesicular stomatitis virus nucleocapsid protein. J Virol 86:11266-11275. Luo M, Green TJ, Zhang X, Tsao J, Qiu S. 2007. Structural comparisons of the nucleoprotein from three negative strand RNA virus families. Virol J 4:72-78.

21

517 518 519 520 521 522 523 524 525 526 527 528 529

52.

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54. 55. 56. 57.

Nayak D, Panda D, Das SC, Luo M, Pattnaik AK. 2009. Single-amino-acid alterations in a highly conserved central region of vesicular stomatitis virus N protein differentially affect the viral nucleocapsid template functions. J Virol 83:5525-5534. Harouaka D, Wertz GW. 2009. Mutations in the C-terminal loop of the nucleocapsid protein affect vesicular stomatitis virus RNA replication and transcription differentially. J Virol 83:1142911439. Svergun DI. 1992. Determination of the regularization parameter in indirect-transform methods using perceptual criteria. . J. Appl. Crystal. 25:495-503. Volkov VV, Svergun DI. 2003. Uniqueness of ab initio shape determination in small-angle scattering. Journal of Applied Crystallography 36:860-864. Kozin MB, Svergun DI. 2001. Automated matching of high- and low-resolution structural models. Journal of Applied Crystallography 34:33-41. PyMol. The PyMOL Molecular Graphics System, Version 1.3, Schrödinger, LLC.

530 531 532

22

533

Figure legends

534

Figure 1. Small-angle X-ray scattering (SAXS) analysis of the N0–P complex from VSV. A)

535

The experimental SAXS profile for three concentrations of the protein complex, 0.75 (black), 2.4

536

(blue) and 4.3 (red) mg/mL. Scattering curves were scaled with PRIMUS (17). The

537

concentrations and associated coloring schemes are used in (A) – (D). B) A Guinier plot of the

538

experimental SAXS profile with the fit shown. Plots are shown on a relative scale. C) A Kratky

539

plot. D) The pair-distribution function as calculated in GNOM (54).

540

Figure 2. Ab initio shape modeling of the VSV N0-P complex. A) DAMMIN (19) was used

541

to produce 10 bead models. These 10 models were used to produce an averaged model with

542

DAMAVER (55). The averaged model (white spheres) is shown with aligned “dimer” of the N0-

543

P complex (red and blue). The dimer was determined with SASREF (25) and superimposed with

544

SUPCOMB (56). 90° rotations relating each orientation are noted. B) A representative single

545

bead model from DAMMIN is shown in beads of cyan. The orientations are the same as in (A).

546

The protein model is not adjusted to fit the single DAMMIN bead model. C) The plot of a single

547

DAMMIN model against the experimental scattering curve. D) Fit of the N0-P model shown in

548

Figure 3 to the experimental SAXS curve. E) SASREF (25) was used to rigid body fit two copies

549

of the complex against the SAXS data. The fit of the SASREF derived dimer is shown.

550 551 552

Figure 3. Maintaining a monomeric N. A complete multi-domain model of theN0-P complex

553

from VSV as determined with EOM is presented. The N protein is shown in red, while the

554

monomers of the P protein dimer are colored, yellow and green. Previously determined domains 23

555

are shown in cartoon representation, while ab initio generated loops are shown as C-α ribbons.

556

The three orange spheres represent positions of Ser60, Thr62 and Ser64 (sites of

557

phosphorylation) of the P protein. These residues have a proximity to the negatively charged

558

patch formed by residues Lys398, Arg399, Lys414 and Lys417 of the N protein (colored dark

559

blue on the surface). This region is circled and denoted the “PO binding site”. All cartoon

560

drawings in this and following figures were prepared with PyMol (57).

561 562

Figure 4. Oligomerization of the N subunits in the nucleocapsid. Surface representations of

563

the nucleocapsid proteins of (A) VSV, (B) RSV, (C) RVFV and (D) LACV are shown for three

564

subunits. Each subunit has been radially spaced to expose the surfaces that contribute to subunit

565

interactions. The monomers are colored red, green and yellow while residues that contact the

566

adjacent protomers are shaded in black.

567 568

Figure 5. Comparisons of the RNA cavity. (A) Ribbon drawings that show the N subunit

569

with encapsidated RNA for VSV, RSV, RVFV and LACV. The rainbow color code of the

570

polypeptide is blue for the N-terminus to red for the C-terminus. The encapsidated RNA is

571

shown again below each N subunit as stick and ribbon models. The nucleotides are numbered

572

from 5’ to 3’. The number 1’ for RVFV means that this nucleotide is the equivalent of nucleotide

573

1 in the next N subunit in the NLP. (B) Superposition of the N proteins. Cα tracing of the aligned

574

coordinates was shown. In the first three panels, the Cα tracing for VSVN was shown in gray.

575

The superimposed Cα tracing was colored in rainbow from N-terminus (blue) to C-terminus

576

(red). In the fourth panel, the Cα tracing for LACVN was shown in skyblue; RVFVN, in 24

577

rainbow. Only the aligned portions are shown (see Table 1). (C) Topological cartoons

578

representing the helices in the N protein core. Each circle represents a helix that is labeled

579

according to their secondary structure assignment in the references (11-14).

580

25

Table 1. Superposition of the N proteins

RSVN(2-375) vs VSVN(2-422) RVFVN(49-126) vs VSVN(131-206) LACVN(35-124) vs VSVN(131-206) LACVN(35-124) vs RVFVN(49-126)

Residues aligned

RMSD (Å2)

TM-score

292(421) 53(76) 50(76) 55(78)

4.97 3.13 4.08 3.88

0.52 0.44 0.35 0.40

Residue number in parenthesis is the length of the reference structure. Table 2. Radius of gyration (Rg), maximum size (Dmax) and Porod volume (Vp) as calculated from the SAXS curves for the VSV N0-P complex. concentration (mg/mL) 0.75 2.4 4.3

Rg (Å) 60.94 65.07 66

Vp (Å3) 5.82 x 105 6.27 x 105 6.52 x 105

Dmax (Å) 209 227.5 229

Rg was calculated with AUTORG, Dmax with DATGNOM and Vp with DATPOROD. All of which are from the ATSAS package (18). Table 3. Area of contacts between the N proteins and number of nucleotides bound in the N protein Surface Virus Area/Protomer 23093.9 VSV 20134.4 RSV 13884.2 RVFV 13775.4 LACV

Buried Interface Complex (Å2) 5648.0 5117.6 3576.6 1982.7

Buried Interface Core Buried Interface N-lobe (Å2) Core C-lobe (Å2) 552.4 1572.4 997.1 248.0 344.6 165.0 63.9

581

26

Buried Interface Arms/Loops (Å2) 3523.2 3872.5 3232.0 1753.8

# of nucleotides in the core 8 6 4 10