A baculovirus alkaline nuclease knockout construct produces fragmented DNA and aberrant capsids

Abstract

DNA replication of bacmid-derived constructs of the Autographa californica multiple nucleocapsid nucleopolyhedrovirus (AcMNPV) was analyzed by field inversion gel electrophoresis (FIGE) in combination with digestion at a unique Eco81I restriction enzyme site. Three constructs were characterized: a parental bacmid, a bacmid deleted for the alkaline nuclease gene, and a bacmid from which the gp64 gene had been deleted. The latter was employed as a control for comparison with the alkaline nuclease knockout because neither yields infectious virus and their replication is limited to the initially transfected cells. The major difference between DNA replicated by the different constructs was the presence in the alkaline nuclease knockout of high concentrations of relatively small, subgenome length DNA in preparations not treated with Eco81I. Furthermore, upon Eco81I digestion, the alkaline nuclease knockout bacmid also yielded substantially more subgenome size DNA than the other constructs. Electron microscopic examination of cells transfected with the alkaline nuclease knockout indicated that, in addition to a limited number of normal-appearing electron-dense nucleocapsids, numerous aberrant capsid-like structures were observed indicating a defect in nucleocapsid maturation or in a DNA processing step that is necessary for encapsidation. Because of the documented role of the baculovirus alkaline nuclease and its homologs from other viruses in homologous recombination, these data suggest that DNA recombination may play a major role in the production of baculovirus genomes.

Fig 1

AcMNPV bacmid constructs used in this report and the strategy employed to introduce marker genes and a unique Eco81I restriction site. (A) The transfer plasmid pfbie-GUS containing the GUS reporter gene and the Eco81I restriction site was transposed into a non-modified bacmid (parental), an alkaline nuclease (an), or a gp64 knockout bacmid to generate the constructs parental-GUS^(Eco81I), an-KO-GUS^(Eco81I), and gp64-KO-GUS^(Eco81I), respectively. (B) The transfer plasmid pfbie-GFP was transposed into an an knockout bacmid to generate an-KO-GFP. Transposition occurred at the polyhedrin locus (polh) and was performed in accordance with the bac-to-bac protocol (Invitrogen). The GUS and GFP marker genes are under control of the AcMNPV immediate early 1 (ie-1) promoter. Construction of the an and gp64 null bacmids containing the chloramphenicol resistance gene have been described previously (Okano et al., 2004).

Fig 2

FIGE analysis of viral DNA replication intermediates from Sf-9 cells infected with the parental-GUS^(Eco81I) recombinant virus. Uncut or Eco81I digested total DNA extracts were analyzed after electrophoresis (program 4) by either ethidium bromide staining (A, C, and E) or by Southern blot hybridization with labeled genomic viral DNA as the probe (B, D, and F). Hours post-infection (hpi) are indicated above the panels. M indicates mock infected cells, BV indicated DNA isolated from budded virus, and L (lambda) indicates MidRange PFG DNA size marker I (New England Biolabs). The arrows indicate single unit-size viral DNA and the asterisks indicate viral DNA that migrated larger-than-unit length. Size ranges in kilobases are indicated on the left of the panels.

Fig 3

FIGE analysis of viral DNA replication intermediates from gp64 or an knockout transfected Sf-9 cells. DNA was analyzed after electrophoresis (program 4) by either ethidium bromide staining (A, C, E and G) or by Southern blot hybridization with labeled genomic viral DNA as the probe (B, D, F and H). The bacmid constructs used for transfection and the time cells were harvested post-transfection (h.p.t.) are indicated above the panels. L indicates MidRange PFG DNA size marker I (New England Biolabs). The DNA in panels A, B, E, and F were uncut and the DNA in panels C, D, G, and H were digested with Eco81I. The arrows indicate single unit-size viral DNA and the asterisk indicates viral DNA that migrated larger-than-unit length. Size ranges in kilobases are indicated on the left of the panels.

Fig. 4

Real-time PCR analysis of viral DNA. At the designated time-point, total DNA was isolated from Sf-9 cells transfected with the indicated bacmid construct, digested with the restriction enzyme DpnI to eliminate input bacmid DNA, and analyzed by real-time PCR using SYBR green I. Values are displayed as the average from transfections performed in triplicate with error bars indicating standard deviations.

Fig. 5

Immunogold staining of thin sections generated from gp64-KO or an-KO-GFP transfected cells at 72 h.p.t. (A) Image of a gp64-KO transfected cell showing normal looking viral nucleocapsids containing an electron-dense core. (B) Image of an an-KO-GFP transfected cell also showing normal looking electron-dense nucleocapsids. (c–d) Images of an-KO-GFP transfected cells showing aberrant rod-shaped capsid structures lacking an electron dense core. For all samples, sections were stained with the primary monoclonal antibody to the AcMNPV p39 major capsid protein as undiluted tissue culture supernatant. The secondary 10 nm gold-conjugated antibody was used at 1:50 dilution. The bar equals 0.25 μm.