Analysis of the interaction of the adenovirus L1 52/55-kilodalton and IVa2 proteins with the packaging sequence in vivo and in vitro

Abstract

We previously showed that the adenovirus IVa2 and L1 52/55-kDa proteins interact in infected cells and the IVa2 protein is part of two virus-specific complexes (x and y) formed in vitro with repeated elements of the packaging sequence called the A1-A2 repeats. Here we demonstrate that both the IVa2 and L1 52/55-kDa proteins bind in vivo to the packaging sequence and that each protein-DNA interaction is independent of the other. There is a strong and direct interaction of the IVa2 protein with DNA in vitro. This interaction is observed when probes containing the A1-A2 or A4-A5 repeats are used, but it is not found by using an A5-A6 probe. Furthermore, we show that complex x is likely a heterodimer of IVa2 and an unknown viral protein, while complex y is a monomer or multimer of IVa2. No in vitro interaction of purified L1 52/55-kDa protein with the packaging sequence was found, suggesting that the L1 52/55-kDa protein-DNA interaction may be mediated by an intermediate protein. Results support roles for both the L1 52/55-kDa and IVa2 proteins in DNA encapsidation.

FIG. 1.

The L1 52/55-kDa and IVa2 proteins interact in vivo with the packaging sequence (PS). Chromatin immunoprecipitation assays were performed on 293 cells infected with wild-type Ad5 at 1, 5, and 10 PFU. (A) Cross-linked nuclear lysates were used in immunoprecipitations with antibodies to L1 or IVa2 or with immunoglobulin G (IgG). After immunoprecipitation, the DNA (ChIP DNA) was isolated and used as template for PCR using primers to amplify the packaging sequence. No template (no temp) was used as a negative control for the PCR. (B) Input chromatin DNA was used as a control to detect the presence of the packaging sequence by PCR. (C) ChIP DNA and input chromatin DNA were amplified with primers specific for a 315-bp fragment of the L1 52/55-kDa protein ORF (52/55-kDa primers). (D) ChIP DNA and input chromatin DNA from mock-infected 293 cells were used to amplify the packaging sequence.

FIG. 2.

The L1 52/55-kDa and IVa2 proteins are independent in their interaction with the packaging sequence (PS). Cross-linked nuclear lysates from 293 cells infected with wild-type Ad5 (Ad5 wt), pm8001 (8001), or pm8002 (8002) or mock-infected cells (mock) were used for ChIP assays with antibodies to L1 52/55-kDa and IVa2 proteins. (A) ChIP DNA was used as a template to amplify the packaging sequence by PCR. (B) Input chromatin DNA was used as a control to detect the packaging sequence. 3T3 cells were used as a negative control for the packaging sequence and no template (no temp) as a negative control for the PCR. (C) ChIP DNA was used to amplify the major late promoter (MLP) as a negative control. (D) Presence of the major late promoter sequence was tested by PCR amplification of the input chromatin DNA. (E) Input proteins were tested by Western blotting to detect expression of L1 52/55-kDa and IVa2 proteins. As a positive control, cell lysate from 293-L1 cells was used in the L1 blot, and 293-IVa2 cell lysate was used in the IVa2 blot. The MagicMark protein ladder (Invitrogen) (M) is shown as the protein standard.

FIG. 3.

Purified IV2 protein binds directly to A1-A2 and A4-A5 repeats in the packaging sequence. Electrophoretic mobility shift assays were performed using purified hexahistidine-tagged IVa2 and GST-L1 52/55-kDa proteins. (A) Titration of the proteins at the indicated concentrations using a ³²P labeled A1-A2 probe. Combinations of L1 52/55-kDa and IVa2 proteins were performed by maintaining one of the proteins at a constant concentration of 20 nM and increasing the concentration of the second protein from 0 to 20 nM. (B) Supershift assays were performed with purified IVa2 protein alone (IVa2) or in combination with L1 52/55-kDa protein (IVa2 + L1) using antibodies (Ab) to IVa2 (rabbit), L1, or TAg proteins, and a ³²P-labeled A1-A2 probe. (C) Electrophoretic mobility shift assays were performed using ³²P-labeled probes containing A1-A2, A4-A5, or A5-A6 repeats (as indicated at the bottom) and either nuclear extract (293 NE) prepared from mock-infected (mock) or Ad5-infected (Ad5 wt) 293 cells or purified IVa2 or L1 52/55-kDa proteins. Complexes x and y are labeled with arrows. (D) EMSAs were performed with nuclear extract from mock-infected 293 cells or Ad5-infected 293 cells and ³²P-labeled A1-A2 or A5-A6 probes. Supershifts were performed using antibodies to IVa2 (antipeptide), L1 52/55-kDa, CDP, and hexon proteins, as indicated. The bracketed portion indicates cellular complexes with the A5-A6 probes. w, virus-specific complex with A5-A6 probe.

FIG. 4.

IVa2 and L1 binding to the packaging sequence using nuclear extracts. (A) Electrophoretic mobility shift assays were performed with nuclear extracts prepared from mock-infected 293 (293 mock) cells, wild-type Ad5 (Ad5 wt)-infected 293 cells, pm8001-infected cells, pm8002-infected cells, mock-infected 293-IVa2 cells, or mock-infected 293-L1 cells and a ³²P-labeled A1-A2 probe. Supershifts were performed using antibodies (Ab) to IVa2 (antipeptide, lanes labeled with asterisks), L1 52/55 kDa, or CDP proteins. Complexes x, y, x2, and y2 are labeled with arrows. Cellular DNA-protein complexes are labeled as C-1 and C-2. (B) Western blots using nuclear extracts from mock-infected 293 (293 mock), 293-IVa2, and 293-L1 cells and 293 cells infected with wild-type Ad5 (Ad5 wt), pm8001, or pm8002 and probed with rabbit polyclonal antibodies to IVa2 and L1 52/55-kDa proteins.

FIG. 5.

Binding of the 40-kDa form of IVa2 protein to the packaging sequence. Electrophoretic mobility shift assays were performed with nuclear extracts prepared from mock-infected 293 cells (293 mock), wild-type Ad5-infected 293 cells (Ad5 wt), or pm8002-infected cells and the ³²P-labeled A1-A2 probe. Supershifts were performed using goat polyclonal anti-IVa2, anti-L1 52/55-kDa protein, or anti-CDP antibodies (Ab). Complexes x, y, x2, and y2 are labeled with arrows.