Chromosome-protein interactions in polyomavirus virions

Abstract

In this work, we sought to determine whether the components of the murine polyomavirus capsid establish specific interactions with the minichromosome encapsidated into the mature viral particles by using the cis-diamminedichloroplatinum(II) cross-linking reagent. Our data indicated that VP1, but not minor capsid proteins, interacts with the viral genome in vivo. In addition, semiquantitative PCR assays performed on cross-linked DNA complexes revealed that VP1 binds to all regions of the viral genome but significantly more to the regulatory region. The implications of such an interaction for viral infectivity are discussed.

FIG. 1.

cis-DDP cross-links encapsidated minichromosomes. (A) cis-DDP penetrates viral particles. Naked viral DNA and viral particles were incubated with or without cis-DDP at different concentrations, and treated DNA was analyzed by Southern blotting with the whole Py genome used as a probe. The level of hybridization was quantified by phosphorimager analysis. One hundred percent hybridization corresponds to the level of hybridization detected in cis-DDP-untreated samples. (B) Effect of cis-DDP concentration on the recovery of DNA-protein complexes. Viral particles were incubated with different concentrations of cis-DDP. After virus dissociation, samples were ultracentrifuged in denaturing CsCl gradients. The distribution of viral DNA in the fractions of the gradient was analyzed by slot blotting with the whole Py genome used as a probe.

FIG. 2.

VP1 binds viral genome in mature particles. Qualitative (A) and quantitative (B) densitometry analysis of the distribution of viral DNA and capsid proteins VP1 and VP2/3 in the fractions of the gradient. Virions treated with (+) or without (−) the cis-DDP reagent are indicated at the top of each lane in panel A. Aliquots of each fraction were analyzed by slot blotting. Viral DNA was detected by hybridization as described in the legend to Fig. 1. Viral proteins were detected by using monoclonal antibodies directed against VP1 or VP2/3 (13) followed by incubation with horseradish peroxidase-conjugated secondary antibodies and an enhanced chemiluminescence reaction. Solid and broken lines represent cis-DDP-treated and cis-DDP-untreated virions, respectively, in panel B. (C) VP1 is associated with the viral genome. Complexes containing fractions of cis-DDP-treated (35 μM) or cis-DDP-untreated (0 μM) virions were immunoprecipitated with (+) or without (−) anti-VP1 antibodies. After reversal of cross-links, the DNA purified from the immunoprecipitated (IP) and immunodepleted (ID) samples was used as a template in PCRs (with primer pair 9) to amplify the Py origin of replication region.

FIG. 3.

Experimental design of semiquantitative PCR analysis of VP1 cross-linked DNA fragments. (A) The relative amplification value (R.A.), measured as the ratio of amplification of Py to amplification of reference sequences, is a function of the initial amount of Py molecules present in the samples. (B) Representative PCR experiments performed in the presence of [α-P³²]ATP with primer pairs 4 and 8 amplifying the VP1-encoding and enhancer regions, respectively.

FIG. 4.

Semiquantitative PCR analysis of VP1 cross-linked DNA fragments. (A) Description of the primer pairs used to amplify target Py sequences. For a statistical analysis, each PCR was done in triplicateand repeated at least six times. The relative amplification value (A.V.) was calculated for each primer pair as the ratio of (Py/ref)_IP to [(Py/ref)_IP + (Py/ref)_ID]. The fold increase (F.I.) value was calculated as the ratio of the amplified value of each primer pair to the amplified value of primer pair 4 (lowest amplified value). ORI, origin of replication; LT, large T antigen; mT, middle T antigen; sT, small T antigen. (B) Graphical representation of the semiquantitative PCR analysis. The numbering of the Py genome starts at the EcoRI site (nt 1562). nct, nucleotide.