Functional characterization of Autographa californica multiple nucleopolyhedrovirus gp16 ( ac130 )

Abstract

To investigate the function of Autographa californica multiple nucleopolyhedrovirus (AcMNPV) gp16, multiple gp16-knockout and repair mutants were constructed and characterized. No obvious difference in productivity of budded virus, DNA synthesis, late gene expression and morphogenesis was observed between gp16-knockout and repair viruses, but gp16 deletion resulted in six hours of lengthening in ST50 to the third instar Spodoptera exigua larvae in bioassays. GP16 was fractionated mainly in the light membrane fraction, by subcellular fractionation. A GP16-EGFP fusion protein was predominantly localized close around the nuclear membrane in infected cells, being coincident with formation of the vesicles associated with the nuclear membrane, which hosted nucleocapsids released from the nucleus. These data suggest that gp16 is not required for viral replication, but may be involved in membrane trafficking associated with the envelopment/de-envelopment of budded viruses when they cross over the nuclear membrane and pass through cytoplasm.

Autographa californica multiple nucleopolyhedrovirus ac75 is required for egress of nucleocapsids from the nucleus and formation of de novo intranuclear membrane microvesicles

“…The effect of the deletion of the ac75 on replication of the virus was first examined by fluorescence microscopy of the Sf9 cells transfected with either vAc ac75ko-gfp , vAc ac75ko-rep-gfp , or vAc gfp , a bacmid constructed by inserting an egfp under control of gp16 promoter [ 14 ]. The cells from each culture were viewed at various time points post transfection.…”

“…In these cases, the absence of the de novo intranuclear membrane microvesicles resulting from deletion of the individual viral genes coincided with nucleocapsids that were not enveloped and were not released from nuclei [ 19 – 23 ]. In the cells infected/transfected by wt AcMNPV or specific gene-knockout repair mutants, nucleocapsids bud through the nuclear membrane and are enveloped [ 9 , 14 , 24 ]. The de novo intranuclear membrane microvesicles are considered to be derived from the inner nuclear membranes that have been modified with virally encoded ODV-specific envelope proteins [ 25 ].…”

“…The fluorescent ring along the nuclear membrane observed at 12 hpi might comprise the nucleocapsids labeled by AC75-EGFP budding through the nuclear membrane since AC75 is associated the nucleocapsids. The bright fluorescent fragments and punctate spots present in the cytoplasm might be the progeny nucleocapsids released from nuclei, some of which are contained in large vesicles [ 9 , 14 , 24 ].…”

“…The resultant bacmid was named vAc ac75ko . pFB-PH, a plasmid containing a copy of AcMNPV polh with native promoter and pFB-P gp16 -egfp containing a copy of the enhanced green fluorescence protein gene ( egfp ) under control of AcMNPV gp16 promoter [ 14 ] were transformed into E . coli DH10B containing vAc ac75ko and pMON7124 to generate vAc ac75ko-PH and vAc ac75ko-gfp , respectively.…”

“…A fragment containing ac75 with its native promoter (nt 63126–63711) was PCR-amplified using primers P ac75 PF1 and ac75PR1 and inserted between the Bst1107 I and Xho I sites of pFD- polh , a plasmid constructed by inserting a copy of AcMNPV polh with native promoter between the Bst1107 I and Pst I, to obtain pFB-ac75-polh. Another PCR-amplified (with primers P ac75 PF1 and ac75PR2) fragment containing ac75 with its native promoter was inserted between the EcoR I and Pst I sites of pFD-P gp16 -gfp [ 14 ] to make pFD-P gp16 -gfp-ac75. pFB-ac75-polh and pFD-P gp16 -gfp-ac75 were separately transformed into E .…”