The N terminus of rotavirus VP2 is necessary for encapsidation of VP1 and VP3

Abstract

The innermost core of rotavirus is composed of VP2, which forms a protein layer that surrounds the two minor proteins VP1 and VP3, and the genome of 11 segments of double-stranded RNA. This inner core layer surrounded by VP6, the major capsid protein, constitutes double-layered particles that are transcriptionally active. Each gene encoding a structural protein of double-layered particles has been cloned into baculovirus recombinants and expressed in insect cells. Previously, we showed that coexpression of different combinations of the structural proteins of rotavirus double-layered particles results in the formation of virus-like particles (VLPs), and each VLP containing VP1, the presumed RNA-dependent RNA polymerase, possesses replicase activity as assayed in an in vitro template-dependent assay system (C. Q.-Y. Zeng, M. J. Wentz, J. Cohen, M. E. Estes, and R. F. Ramig, J. Virol. 70:2736-2742, 1996). This work reports construction and characterization of VLPs containing a truncated VP2 (VPdelta2, containing amino acids [aa] Met-93 to 880). Expression of VPdelta2 alone resulted in the formation of single-layered delta2-VLPs. Coexpression of VPdelta2 with VP6 produced double-layered delta2/6-VLPs. VLPs formed by coexpression of VPdelta2 and VP1 or VP3, or both VP1 and VP3, resulted in the formation of VLPs lacking both VP1 and VP3. The presence of VP6 with VPdelta2 did not result in encapsidation of VP1 and VP3. To determine the domain of VP2 required for binding VP1, far-Western blot analyses using a series of truncated VP2 constructs were performed to test their ability to bind VP1. These analyses showed that (i) full-length VP2 (aa 1 to 880) binds to VP1, (ii) any N-terminal truncation lacking aa 1 to 25 fails to bind VP1, and (iii) a C-terminal 296-aa truncated VP2 construct (aa 1 to 583) maintains the ability to bind VP1. These analyses indicate that the N terminus of rotavirus VP2 is necessary for the encapsidation of VP1 and VP3.

FIG. 1

Electron micrographs of the VPΔ2-containing VLPs and their VP2-containing counterparts. Δ2-VLPs were expressed from the recombinant baculovirus BVP2C24 by infection of Sf9 cells and purified on a 5 to 20% sucrose gradient. Δ2/6-VLPs were coexpressed from the recombinant baculoviruses BVP2C24 and pAc461/SA11-6 by infection of Sf9 cells and purified by CsCl isopycnic centrifugation. Shown are the negative-stained structures of the VLPs containing the indicated protein species. (A to C) VPΔ2-containing VLPs; (D) native rotavirus double-layered particles (DLP); (E and F) VP2-containing VLPs as the counterparts of panels A to C. All micrographs are at the same magnification. Bar, 100 nm.

FIG. 2

Protein content of single- and double-layered VPΔ2-containing VLPs and their VP2-containing counterparts. Shown is a silver-stained SDS–10% polyacrylamide gel with the proteins of purified VLPs resulting from the expression of the indicated genes in the absence or presence of protease inhibitors. Protease inhibitors aprotinin and leupeptin, each at 1 μg/ml, were added daily.

FIG. 3

Protein content of the coexpressed 1/Δ2-, 1/2-, 1/Δ2/6-, and 1/2/6-VLPs. Shown is a silver-stained SDS–10% polyacrylamide gel with proteins of the purified VLPs resulting from the coexpression of indicated genes in the absence or presence of protease inhibitors. Protease inhibitors aprotinin and leupeptin, each at 1 μg/ml, were added daily. VP1/Δ2 and VP1/2 were coexpressed from the baculovirus recombinant pVL941/RF-1 with recombinant BVP2C24 or BacRF2A, respectively, and purified by centrifugation on a 5 to 20% sucrose gradient. VP1/Δ2/6 and VP1/2/6 were coexpressed from the baculovirus recombinants of pVL941/RF-1, pAc461/SA11-6 with recombinants BVP2C24 and BacRF2A, respectively, and purified by CsCl isopycnic centrifugation. To compare VP1 encapsidation, the protein amount loaded onto each lane was adjusted so that the amount of VPΔ2 was similar to or higher than that of VP2 band A, the VP1 binding protein (Fig. 6C). To create a cleaved VP2 band C for a marker of VPΔ2, 1/2/6-VLPs in lane 1 (also in lanes 1 of Fig. 4 and 5) were produced in the absence of protease inhibitors.

FIG. 4

Protein content of the coexpressed Δ2/3-, 2/3-, Δ2/3/6-, and 2/3/6-VLPs. (A) Silver-stained SDS–10% polyacrylamide gel with proteins of the purified VLPs resulting from the coexpression of the indicated genes in the absence or presence of protease inhibitors. Protease inhibitors aprotinin and leupeptin, each at 1 μg/ml, were added daily. (B) Autoradiogram of [α-³²P]GTP-bound VP3 to visualize the presence of VP3 in the VLPs shown in panel A. VPΔ2/3 and VP2/3 were coexpressed from the baculovirus recombinant pVL1393/SA11-3 with recombinants BVP2C24 and BacRF2A, respectively, and purified on a 5 to 20% sucrose gradient. VPΔ2/3/6 and VP2/3/6 were coexpressed from the baculovirus recombinants pVL1393/SA11-3 and pAc461/SA11-6 with recombinant BVP2C24 or BacRF2A, respectively, and purified by CsCl isopycnic centrifugation. To compare VP3 encapsidation, the protein amount loaded onto each lane was adjusted so that the amount of VPΔ2 was similar to or higher than that of the VP2 band A, the VP1 binding protein (Fig. 6C).

FIG. 5

Protein content of the coexpressed 1/Δ2/3-, 1/2/3-, 1/Δ2/3/6-, and 1/2/3/6-VLPs. (A) Silver-stained SDS–10% polyacrylamide gel with proteins of purified VLPs resulting from the expression of indicated genes in the absence or presence of protease inhibitors. Protease inhibitors aprotinin and leupeptin, each at 1 μg/ml, were added daily. (B) Autoradiogram of [α-³²P]GTP-bound VP3 to visualize the presence of VP3 in the VLPs shown in panel A. VP1/Δ2/3 and VP1/2/3 were coexpressed from the baculovirus recombinants pVL941/RF-1 and pVL1393/SA11-3 with recombinant BVP2C24 or BacRF2A, respectively, and purified by centrifugation on a 5 to 20% sucrose gradient. VPΔ2/3/6 and VP2/3/6 were coexpressed from the baculovirus recombinants pVL941/RF-1, pVL1393/SA11-3, and pAc461/SA11-6 with recombinants BVP2C24 and BacRF2A, respectively, and purified by CsCl isopycnic centrifugation. To compare VP1 and VP3 encapsidation, the protein amount loaded onto each lane was adjusted so that the amount of VPΔ2 was similar to or higher than that of VP2 band A, the VP1 binding protein (Fig. 6C).

FIG. 6

Interactions between VP1 and other VLP proteins tested by far-Western blotting. (A) Silver-stained SDS–10% polyacrylamide gel of purified VP1 used for far-Western blotting to verify the VP1 binding proteins. VP1 was purified by FPLC followed by passage through an immunoaffinity column of rabbit IgG against wild-type baculovirus proteins. (B) Silver-stained SDS–10% polyacrylamide gel of the proteins in the indicated VLPs. Each purified VLP was (i) mixed with Laemmli buffer containing βME and boiled for 5 min or (ii) mixed with sample buffer without βME, kept at 4°C, and then resolved on an SDS–10% polyacrylamide gel at 4°C. (C) VP1 binding proteins detected on far-Western blots. A duplicate of the gel in panel B was run, and the proteins were transfered to a PVDF membrane for far-Western VP1 binding assay. After renaturation of the proteins blotted to the PVDF membrane and blocking of free binding sites, 10 μg of VP1 per ml was added to probe the proteins capable of binding VP1. The bound VP1 was detected by guinea pig anti-VP1 antibody followed by goat anti-guinea pig IgG-alkaline phosphatase conjugate and enzyme substrates.

FIG. 7

Mapping the domain of VP2 required for binding VP1 by far-Western blotting. (A) Western blot of three truncated forms of VP2. Lysates of infected cells expressing three N- and C-terminus truncations of VP2 (aa 1 to 583, aa Met-26 to 583, and aa Met-45 to 583) were boiled with Laemmli sample buffer containing βME, resolved on an SDS–10% polyacrylamide gel, and blotted to nitrocellulose. They were probed by MAb 164AE22, which directs a VP2_Rf epitope located between aa 45 and 92. (B) VP1 binding of truncated forms of VP2. Three VP2 truncation mutants were mixed with Laemmli buffer containing βME, boiled for 5 min, resolved on an SDS–10% polyacrylamide gel, and blotted to a PVDF membrane. After renaturation of the blots, 10 μg of VP1 per ml was added to probe the peptide fragments capable of binding VP1 as described for Fig. 6C.