The structural protein ODV-EC27 of Autographa californica nucleopolyhedrovirus is a multifunctional viral cyclin

Abstract

Two major characteristics of baculovirus infection are arrest of the host cell at G2/M phase of the cell cycle with continuing viral DNA replication. We show that Autographa californica nucleopolyhedrovirus (AcMNPV) encodes for a multifunctional cyclin that may partially explain the molecular basis of these important characteristics of AcMNPV (baculovirus) infection. Amino acids 80-110 of the viral structural protein ODV-EC27 (-EC27) demonstrate 25-30% similarity with cellular cyclins within the cyclin box. Immunoprecipitation results using antibodies to -EC27 show that -EC27 can associate with either cdc2 or cdk6 resulting in active kinase complexes that can phosphorylate histone H1 and retinoblastoma protein in vitro. The cdk6-EC27 complex also associates with proliferating cell nuclear antigen (PCNA) and we demonstrate that PCNA is a structural protein of both the budded virus and the occlusion-derived virus. These results suggest that -EC27 can function as a multifunctional cyclin: when associated with cdc2, it exhibits cyclin B-like activity; when associated with cdk6, the complex possesses cyclin D-like activity and binds PCNA. The possible roles of such a multifunctional cyclin during the life cycle of baculovirus are discussed, along with potential implications relative to the expression of functionally authentic recombinant proteins by using baculovirus-infected cells.

Figure 1

Comparison of the cyclin box of −EC27 (amino acids 80–110) with the cyclin box of cyclins. Shaded areas represent conservative amino acids compared against the −EC27 sequence. Rules for assignment of conservative amino acids are: G=A=S=T; V=L=I=M=F=Y=W; n = Q=D=E; r = K=H .

Figure 2

−EC27-associated kinase activity. (A) Immunoprecipitation using antibody to −EC27 was performed from infected Sf9 cell lysates at indicated times p.i. The kinase activity of the precipitated complex was then assayed by using either histone H1 or pRb as the in vitro substrate. (B) Phosphorylated histone H1 or pRb bands were quantitated by using a PhosphorImager SF (Molecular Dynamics).

Figure 3

Identification of protein kinases interacting with −EC27. Sf9 cell lysates were prepared at 60 h p.i. and used for immunoprecipitation with antibodies to cdc2, cdk2, -4, -5, -6, -7, and Suc1. Immunoprecipitated complexes were separated by SDS/PAGE and Western blot performed by using antisera to −EC27.

Figure 4

−EC27 Association with Cdc2 and Cdk6 results in kinase activity. (A) At 60 h p. i. Sf9 cell lysates were immunodepleted using antibodies to indicated cdks. −EC27 immunoprecipitated complexes from these depleted lysates were obtained and then tested for kinase activity by using either histone H1 or pRb as in vitro substrate. Samples were separated by using SDS/PAGE, and phosphorylated bands of histone H1 and pRb are shown. (B) Phosphorylated histone H1 and pRb bands were quantitated by using a PhosphorImager SF (Molecular Dynamics). These data were normalized, assigning the levels of phosphorylation in undepleted lysates as 100%. (C) Lanes 1–2: Comparing the levels of cdc2 and cdk6 in Sf9 cell lysates before and after immunodepletion with cdc2 and cdk6 antibodies respectively. Lane 3: At 60 h p.i. cell lysate was immunoprecipitated by using −EC27 antibody and analyzed by Western blot by using antibody to either cdc2 or cdk6.

Figure 5

Association of PCNA with Cdk6–EC27 complex and AcMNPV nucleocapsid. (A) Twenty-five micrograms of total protein from infected Sf9 cell lysates (h p.i.) or −EC27 immunoprecipitated complex (ODV-EC27 IP) from 150 μg of whole-cell lysate (h p.i.) were separated by using SDS/PAGE and analyzed by using Western blot and antibodies to either −EC27 (α-ODV-EC27) or PCNA (α-PCNA). (B) Antibodies to Suc1, cdc2, and cdk6 were used to immunoprecipitate the protein complex from Sf9 cell lysate (60 h p.i.). The proteins were separated on SDS/PAGE gels and analyzed by Western blot by using PCNA antibodies. Proteins of purified BV and ODV (20 μg) were separated by using SDS/PAGE and analyzed by Western blot by using PCNA antibodies. BV was fractionated into envelope (BV-E) and nucleocapsid (BV-C) and preparations (10 μg/sample) were probed by using PCNA antibodies (α-PCNA). (C) ImmunoGold localization of PCNA to nucleocapsid of ODV (arrows).

Figure 6

−EC27 immunoreactive proteins of BV and ODV and model of interaction of −EC27 with Cdc2, Cdk6 and PCNA. (A) Purified BV and ODV were separated by using SDS/PAGE and analyzed by Western blot using −EC27 antibodies. The 27-kDa form is unique to ODV (this data is reproduced from ref. 13). (B) −EC27 can associate with either cdc2 (cyclin B-like activity) or cdk6 (cyclin D-like activity). The cdk6−EC27 complex is also capable of binding to PCNA. Protein “?” represents other potential, currently unknown partners.