The ϕ6 cystovirus protein P7 becomes accessible to antibodies in the transcribing nucleocapsid: a probe for viral structural elements

Abstract

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 ϕ6 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 five-fold 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 ϕ6 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 Vκ 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 ϕ6 P7 surface. It is further demonstrated that within ϕ6 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.

Fig 1. SDS-PAGE gel demonstrating effectiveness of P7 purification procedure.

Lanes are: Marker (wide range protein ladder p7708); Control—undisrupted cells after IPTG induction; SN—supernatant after the pelleting of lysed cell’s debris; Pellet—resuspended cell debris pellet; Flow-Through—proteins unbound to Ni-NTA beads; 1^st and 2^nd Wash—proteins eluted from the Ni-NTA agarose column after 1^st and 2^nd rinses with washing buffer; Eluate—P7 protein eluted from the column.

Fig 2. SDS-PAGE gel of purified Mabs.

MW of the heavy chains are 52 kDa for 6C3; and 49 kDa for 2D11, 1F11 and 1C10. MW of the light chains are 28 kDa for 6C3; and 23 kDa for 2D11, 1F11 and 1C10.

Fig 3. Affinity calibration curve for the P7 Mabs. Mabs concentrations were 1 μg/ml.

The background absorption for each Mab was determined from the optical density of ELISA plate wells lacking the P7 antigen, and the background optical density was subtracted from the absorption data.

Fig 4. Reactivity of Mabs.

A) Comparison of the reactivity of the Mabs 1C10, 6C3, 2D11 and 1F11 to the native and denatured forms of P7. P7 was coated at a concentration of 31.2 ng/ml (1.6 ng per ELISA plate well). Optical density is measured at 405 nm. B) Western Blot of P7 protein.

Fig 5. Competition between Mabs determined by ELISA.

A) Single Mab (no competition); B) 6C3; C) 1C10; D) 2D11; and E) 1F11; in competition with the other three Mabs at concentrations of 1000 ng/ml. Optical Density is measured at 405 nm.

Fig 6. Sandwich ELISA showing that NC reveals P7 in transcription buffer with or without rNTP.

Absorption is measured at 405 nm.

Fig 7. Cryo TEM images of transcribing ϕ6 NCs.

A) Control sample (no rNTP in transcription reaction mixture); B) NC in 4 mM of rNTP in transcription reaction mixture. Arrows in (B) indicate some of the transcribing NCs (hexagonal configuration with a lower electron density).