The ϕ6 Cystovirus Protein P7 Becomes Accessible to Antibodies in the Transcribing Nucleocapsid: A Probe for Viral Structural Elements

March The 6 Cystovirus Protein P7 Becomes Accessible to Antibodies in the Transcribing Nucleocapsid: A Probe for Viral Structural Elements Alexandra Alimova 0 1 2 3 Hui Wei 0 1 2 3 Al Katz 0 1 2 3 Linda Spatz 0 1 2 3 Paul Gottlieb 0 1 2 3 0 Funding: Funding provided by National Institute of General Medical Science Grant SC1-GM092781, www.nigms.nih.gov (PG); National Institutes of Health Research Centers in Minority Institutions (NIH/NCRR/ RCMI) CCNY/Grant G12-RR03060, www.nih.gov (PG). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript 1 Data Availability Statement: All data are contained within the paper 2 Academic Editor: Nicholas J Mantis, New York State Dept. Health , UNITED STATES 3 1 Sophie Davis School of Biomedical Education, City College of New York , New York, NY 10031 , United States of America, 2 Department of Physics, City College of New York , New York, NY 10031 , United States of America Protein P7 is a component of the cystovirus viral polymerase complex. In the unpackaged procapsid, the protein is situated in close proximity to the viral directed RNA polymerase, P2. Cryo-electron microscopy difference maps from the species 6 procapsid have demonstrated that P7 and P2 likely interact prior to viral RNA packaging. The location of P7 in the post-packaged nucleocapsid (NC) remains unknown. P7 may translocate closer to the fivefold axis of a filled procapsid but this has not been directly visualized. We propose that monoclonal antibodies (Mabs) can be selected that serve as probe- reagents for viral assembly and structure. A set of Mabs have been isolated that recognize and bind to the 6 P7. The antibody set contains five unique Mabs, four of which recognize a linear epitope and one which recognizes a conformational epitope. The four unique Mabs that recognize a linear epitope display restricted utilization of V and VH genes. The restricted genetic range among 4 of the 5 antibodies implies that the antibody repertoire is limited. The limitation could be the consequence of a paucity of exposed antigenic sites on the 6 P7 surface. It is further demonstrated that within 6 nucleocapsids that are primed for early-phase transcription, P7 is partially accessible to the Mabs, indicating that the nucleocapsid shell (protein P8) has undergone partial disassembly exposing the protein's antigenic sites. - The cystoviridae family of viruses, of which 6 was the first discovered species, contain three segments of double stranded RNA. Bacteriophage 6 and its relatives are model systems for virus assembly, genome packaging and dsRNA polymerization. The RNA packaging, replication, transcription mechanism, and overall structure resembles that of reoviruses making the species an excellent model system to study these important pathogens. The initial step in cystoviridae replication is the assembly of a closed and unexpanded, dodecahedral-shaped procapsid Competing Interests: The authors have declared that no competing interests exist. (PC). The RNA packaging proceeds in a specific order with the small (2948 bp) viral RNA segment packaged first, followed by the middle (4063 bp) and large (6374 bp) segments [13]. Step-wise expansion of the PC accompanies the RNA packaging [4]. Ultimately all three dsRNA segments are enclosed into a nucleocapsid (NC) surrounded by a lipoprotein envelope to constitute the mature viral particle. The outer layer of the NC is a shell composed of a matrix assembled of protein P8 [57] that upon cell penetration facilitates an endocytic plasma membrane penetration and is thought to disassemble during viral entry [8]. The P8 shell is composed of 200 trimers arranged as a T = 13 lattice that partially covers the filled PC [5,9,10]. During genome packaging the PC undergoes significant conformational morphogenesis with the sequential expansion revealing unique binding sites for each of the three viral RNA segments [11,12]. The PC is composed of four proteins, P1, P2, P4, and P7, which are responsible for RNA packaging, transcription, and genome replication [11,13]. Three of the four proteins (P1, P2 and P4) are known to have specific functions in regard to the packaging and replication of viral RNA. The entire PC framework is composed of P1 which has RNA binding activity. The atomic structure of P1 for both 6 and 8 has recently been determined and shown to be a flattened trapezoid in shape that adapts to two conformations, P1A and P1B, that undergo conformational changes when maturing from the unexpanded PC to the RNA packaged NC [1416]. A hexamer of the nucleotide triphosphorylase, P4, forms the packaging portal responsible for RNA transport into the expanding PC. The viral RNA-directed RNA polymerase (RdRP), P2, is required for the replication of the single stranded RNA to the doublestranded RNA (dsRNA) genome [5]. P7 is the least characterized of the PC proteins and its precise function still remains undetermined. It is required for efficient PC assembly and transcription [17], and RNA packaging [18,19]. In 6, P7 has a molecular mass of 17168 Da. The 6 virion can potentially contain 60 copies of P7 (three copies at each of the 20 three-fold symmetry axes); but there is a controversy regarding occupancy in recombinant PC particles: SunBamford and Poranen [20] noted that the same amount of P7 is in recombinant PC particles as in the complete virion. Our previous publication described approximately 20 copies of P7 protein per PC particle [21]; while NemecekQiaoMindichSteven and Heymann [16] observed even less occupancy for P7, at only 12 copies in a complete PC. The occupancy of P7 in mature viruses has not been determined and may differ from recombinant PC particles. There is evidence that P7 forms an elongated dimer in solution [17], but in both the PC and NC, P7 is seen to exist as a monomer. PoranenButcherSimonovLaurinmaki and Bamford [22] observed that an excess concentration of P7 accelerated assembly of P1 in vitro, indicating that P7 may stabilize P1. Study of self-assembly of PC particles shows potential competition between P2 and P7 proteins [16]. In the presence of an excess of P7, the number of P2 copies decreases from 12 to 6 copies, whereas the number of P7 copies increases from 36 to 60 copies per particle [20]. In prior work, we proposed that P7 is located inside the unexpanded PC core near the three-fold axes and that P7 stabilizes P2 in position close to the three-fold axis [21]. The structure of P7 from the related cystovirus 12 has been determined by X-ray crystallography [23]. Most interestingly, the protein is seen to be composed of two distinct domains in which the N-terminal core region (1129) of P7 forms a homodimeric /-fold, having structural similarities with breast cancer gene 1 (BRCA1) C-terminal domains implicated in multiple proteinprotein interactions in DNA repair proteins. The C-terminal tail consisting of approximately 30 to 40 amino acids is (...truncated)