Roles of Cellular NSF Protein in Entry and Nuclear Egress of Budded Virions of Autographa californica Multiple Nucleopolyhedrovirus

Abstract

In eukaryotic cells, the oluble-ethylmaleimide-ensitive actor (NSF)ttachment protein ceptor (SNARE) proteins comprise the minimal machinery that triggers fusion of transport vesicles with their target membranes. Comparative studies revealed that genes encoding the components of the SNARE system are highly conserved in yeast, insect, and human genomes. Upon infection of insect cells by the virus Autographa californica multiple nucleopolyhedrovirus (AcMNPV), the transcript levels of most SNARE genes initially were upregulated. We found that overexpression of dominant-negative (DN) forms of NSF or knockdown of the expression of NSF, the key regulator of the SNARE system, significantly affected infectious AcMNPV production. In cells expressing DN NSF, entering virions were trapped in the cytoplasm or transported to the nucleus with low efficiency. The presence of DN NSF also moderately reduced trafficking of the viral envelope glycoprotein GP64 to the plasma membrane but dramatically inhibited production of infectious budded virions (BV). Transmission electron microscopy analysis of infections in cells expressing DN NSF revealed that progeny nucleocapsids were retained in a perinuclear space surrounded by inner and outer nuclear membranes. Several baculovirus conserved (core) proteins (Ac76, Ac78, GP41, Ac93, and Ac103) that are important for infectious budded virion production were found to associate with NSF, and NSF was detected within the assembled BV. Together, these data indicate that the cellular SNARE system is involved in AcMNPV infection and that NSF is required for efficient entry and nuclear egress of budded virions of AcMNPV. Little is known regarding the complex interplay between cellular factors and baculoviruses during viral entry and egress. Here, we examined the cellular SNARE system, which mediates the fusion of vesicles in healthy cells, and its relation to baculovirus infection. Using a DN approach and RNA interference knockdown, we demonstrated that a general disruption of the SNARE machinery significantly inhibited the production of infectious BV of AcMNPV. The presence of a DN NSF protein resulted in low-efficiency entry of BV and the retention of progeny nucleocapsids in the perinuclear space during egress. Combined with these effects, we also found that several conserved (core) baculovirus proteins closely associate with NSF, and these results suggest their involvement in the egress of BV. Our findings are the first to demonstrate that the SNARE system is required for efficient entry of BV and nuclear egress of progeny nucleocapsids of baculoviruses.

Distinct Roles of Cellular ESCRT-I and ESCRT-III Proteins in Efficient Entry and Egress of Budded Virions of Autographa californica Multiple Nucleopolyhedrovirus

“…All expression plasmids are listed in Table 4. Initially, to generate the transient-expression vectors pIEnGFP and pIEcGFP, the ORF of enhanced green fluorescent protein (EGFP) and a fragment containing the ORF of EGFP and the poly(A) signal of the AcMNPV gp64 gene were amplified by PCR using Vps4-gfppBlue (23) as the template and separately inserted between the XbaI and BamHI or the EcoRI and HindIII sites of the plasmid pIE (81). For generating ESCRT-I components Tsg101-and Vps28-derived expression plasmids, the ORF and truncated forms of Tsg101 and Vps28 were isolated from X-pMD18-T with enzymes BamHI and EcoRI and then inserted into pIEnGFP vector.…”

“…The ORFs of ESCRT-III components were isolated from Y-pMD18-T using restriction enzymes XbaI and EcoRI (Vps2B, Vps20, Vps24, Snf7, Vps46) or XbaI and PstI (Vps60) and then cloned into the same enzyme sites of pIEcGFP or pIE-MCS-Myc (81) to produce the target genes fused with GFP or a c-Myc tag at the C terminus. The plasmids expressing HA-or c-Myc-tagged AcMNPV genes (Ac11, Ac76, Ac78, GP41, Ac93, p48, Ac142, Ac146, Lef3 genes) and the mCherry-based bimolecular fluorescent complementation (BiFC) system were constructed as described previously (81,82). The gene-specific BiFC plasmids Y-HA-NmpBlue, Z-HA-NmpBlue, U-HA-NmpBlue, Y-Myc-CmpBlue, Z-Myc-CmpBlue, U-Myc-CmpBlue (where Y, Z, U, Nm, and Cm represent ESCRT-III components, AcMNPV genes except Ac146, Vps4, and its mutants E231Q and K176Q, and the N and C termini of mCherry, respectively) were generated by insertion of the XbaI-EcoRI fragment isolated from Y-pMD18-T, pIE-Z-Myc, Vps4-gfppBlue, E231Q-gfppBlue, and K176Q-gfppBlue (23) into HA-NmpBlue or Myc-CmpBlue (81), respectively.…”

“…RNAi assay. The dsRNA-based RNAi assay was performed as described previously with modifications (81,85). A fragment (305 to 495 bp) of the coding sequence of the components of ESCRT-I (Tsg101, Vps28) or ESCRT-III (Vps2B, Vps20, Vps24, Snf7, Vps46, and Vps60), Vps4, or GFP was amplified by PCR.…”

The Autographa californica Multiple Nucleopolyhedrovirus ac51 Gene Is Required for Efficient Nuclear Egress of Nucleocapsids and Is Essential for In Vivo Virulence

“…As shown by TEM in our study and other studies (13,20), baculovirus nucleocapsids seem to pass through the NM to the Cyt by a budding process, but the mechanism by which viral structural proteins are involved in this process remains unknown. Viral proteins, including Ac11, Ac76, Ac78, GP41, Ac93, Ac103, Ac142, and Ac146, which are essential for nucleocapsid egress from the nucleus and ODV formation, have recently been shown to interact with the soluble cellular NSF (N-ethylmaleimide-sensitive factor) and ESCRT-III (endosomal sorting complex required for transport) proteins directly or indirectly (11,57). NSF functions by triggering the fusion of transport vesicles with their target membranes, and ESCRT-III is essential for membrane remodeling and is part of the scission machinery for sorting ubiquitinated membrane proteins into intraluminal vesicles.…”

“…NSF functions by triggering the fusion of transport vesicles with their target membranes, and ESCRT-III is essential for membrane remodeling and is part of the scission machinery for sorting ubiquitinated membrane proteins into intraluminal vesicles. These viral proteins have been speculated to form a complex to direct nucleocapsids to the Ac76-rich budding region on the NM and to recruit ESCRT-III proteins to promote the nuclear egress of nucleocapsids, and NSF may help cleave vesicles containing progeny nucleocapsids during nuclear egress (11,14,57).…”