Polyomavirus major capsid protein VP1 is capable of packaging cellular DNA when expressed in the baculovirus system

Abstract

Using the p2Bac dual multiple cloning site transfer vector, the polyomavirus major capsid protein gene VP1 was cloned for expression in the baculovirus-insect cell expression system. The 5-day-infected cellular lysate from this recombinant preparation was purified by cesium chloride density gradient centrifugation. Capsidlike particles were observed in the resulting preparation. The purified particle preparation was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and was shown to have accurately expressed the polyomavirus VP1 protein as cloned. It was found that the preparation revealed the presence of host histones in the stained gels, which is indicative of DNA packaging. To determine if cellular DNA was being packaged in the particles, Sf9 insect cells were prelabeled with [ 3 H]thymidine. The label was removed, and the cells were subsequently infected with a recombinant Autographa californica multiple nuclear polyhedrosis virus (AcMNPV) carrying the polyomavirus VP1 gene. Upon purification through three cesium chloride gradients and DNase I treatment, capsid-like particles, containing [ 3 H]thymidine-labeled DNA, were isolated which were found to coincide with hemagglutination activity. Studies have indicated that the AcMNPV appears to have the ability to fragment Sf9 cellular DNA. When infected with the recombinant AcMNPV carrying the VP1 gene of polyomavirus, these host DNA fragments are being packaged by the VP1 major capsid protein; further, these DNA fragments have been shown to be approximately 5 kb in size, which corresponds to the size of the native polyomavirus genome. These studies demonstrate that the recombinant polyomavirus VP1 protein has the ability to package DNA in the absence of the minor structural proteins VP2 and VP3 and independently of the polyomavirus T antigens.

Characterization of Self-Assembled Virus-Like Particles of Merkel Cell Polyomavirus

“…In addition to the major peak, a minor density peak was found at approximately 15 nm distance from the center of the VLP. Considering our evidence that cell-derived DNAs were contained in the MCPyV-LPs ( Fig 2B ) as well as structural information for other polyomaviruses [ 23 ], this density peak might express DNA population inside the particles.…”

Baculovirus as a highly efficient expression vector in insect and mammalian cells

“…Extended applications of VLP as gene delivery vehicles have also been reported [17,18] . To date, numerous VLP of different viruses, including HIV [19] , herpes simplex virus [20] , human papillomavirus [21] , polyomavirus [22] , parvovirus [23] , infectious bursal disease virus [24] , hepatitis C virus (HCV) [16] , and enterovirus 71 [25] , have been expressed using the baculovirus/insect cell expression system. Very recently, the synthesis of severe acute respiratory syndrome coronavirus (SARS-CoV) VLP using the baculovirus/insect cell system has also been reported [26,27] .…”

Murine pneumotropic virus VP1 virus-like particles (VLPs) bind to several cell types independent of sialic acid residues and do not serologically cross react with murine polyomavirus VP1 VLPs

“…1a, lane 2) were selected for further analysis and production of MPtV VP1. Baculovirus-infected Sf9 cells were grown for 72 h and not longer in order to minimize the number of capsids that had internalized cellular DNA (Gillock et al, 1997). MPtV VP1 was extracted from Sf9 cells in the form of self-assembled MPtV-VLPs, which were further purified using sucrose gradient followed by CsCl gradient centrifugation (Fig.…”