Pseudotyping Autographa californica Multicapsid Nucleopolyhedrovirus (Ac M NPV): F Proteins from Group II NPVs Are Functionally Analogous to Ac M NPV GP64

Abstract

GP64, the major envelope glycoprotein of budded virions of the baculovirus Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV), is involved in viral attachment, mediates membrane fusion during virus entry, and is required for efficient virion budding. Thus, GP64 is essential for viral propagation in cell culture and in animals. Recent genome sequences from a number of baculoviruses show that only a subset of closely related baculoviruses have gp64 genes, while other baculoviruses have a recently discovered unrelated envelope protein named F. F proteins from Lymantria dispar MNPV (LdMNPV) and Spodoptera exigua MNPV (SeMNPV) mediate membrane fusion and are therefore thought to serve roles similar to that of GP64. To determine whether F proteins are functionally analogous to GP64 proteins, we deleted the gp64 gene from an AcMNPV bacmid and inserted F protein genes from three different baculoviruses. In addition, we also inserted envelope protein genes from vesicular stomatitis virus (VSV) and Thogoto virus. Transfection of the gp64-null bacmid DNA into Sf9 cells does not generate infectious particles, but this defect was rescued by introducing either the F protein gene from LdMNPV or SeMNPV or the G protein gene from VSV. These results demonstrate that baculovirus F proteins are functionally analogous to GP64. Because baculovirus F proteins appear to be more widespread within the family and are much more divergent than GP64 proteins, gp64 may represent the acquisition of an envelope protein gene by an ancestral baculovirus. The AcMNPV pseudotyping system provides an efficient and powerful method for examining the functions and compatibilities of analogous or orthologous viral envelope proteins, and it could have important biotechnological applications.

GP64 of group I nucleopolyhedroviruses cannot readily rescue infectivity of group II f-null nucleopolyhedroviruses

“…The PCR fragment was cloned into the SmaI and BamHI sites of pFBSePolh Dual, thereby removing the AcMNPV p10 and polyhedrin promoters and generating pFBSepolh-(pSeF). The SeMNPV f gene and the AcMNPV gp64 gene were cloned as BamHI/NotI and EcoRI/EcoRI fragments from pDFBgusSe8 and pDFBgusGP64 (Lung et al, 2002) into the BamHI and NotI sites of pFBSePolh-(pSeF), generating pFBSePolh-SeF(pSeF) and pFBSePolh-GP64(pSeF), respectively. For the generation of control bacmids, a vector was generated containing only the SeMNPV polyhedrin gene behind its own promoter.…”

“…The SeMNPV polyhedrin gene with its own promoter was cloned as an SmaI/HindIII fragment (Pijlman et al, 2002) in the SmaI and KpnI sites of pFastBac Dual (Invitrogen), generating pFBSePolh Dual. The SeMNPV f gene and the AcMNPV gp64 gene, both under the control of the gp64 promoter, were cloned as XbaI/EcoRI and XbaI/SstI fragments from pDFBgusSe8 and pDFBgusGP64 (Lung et al, 2002) into the SmaI and SstI sites of pFBSePolh Dual. In this procedure, the AcMNPV p10 and polyhedrin promoters were removed, and plasmids pFBSepolh-SeF(pGP64) and pFBSepolh-GP64(pGP64) were obtained, respectively.…”

“…GP64 homologues are also found on Thogoto and Dhori viruses, which are tick-transmitted orthomyxoviruses that replicate in both ticks and mammals (Freedman-Faulstich & Fuller, 1990;Morse et al, 1992). It has therefore been suggested that the group I NPVs have acquired the gp64 gene later during evolution, either from the host or from another insect-infecting virus (Lung et al, 2002;Morse et al, 1992), thereby getting a selective advantage due to increased efficiency of either virus-receptor interaction and virus entry, or virus budding, obviating the need for a functional F protein. However, experimental evidence to support this view is lacking.…”

“…Recently, it was shown that the F proteins of the group II NPVs SeMNPV, LdMNPV and HearNPV are capable of substituting functionally for GP64 in AcMNPV (Long et al, 2006b;Lung et al, 2002). An AcMNPV bacmid lacking the gp64 gene was unable to produce BVs after transfection into insect cells (Monsma et al, 1996), whereas this defect could be rescued by insertion of the SeMNPV, LdMNPV or HearNPV f gene (Long et al, 2006b;Lung et al, 2002).…”

“…This protein is involved in attachment of BVs to the cell, is required for low-pH-triggered membrane fusion during virus entry and is necessary later in the process of infection for efficient budding of progeny nucleocapsids (NCs) into the haemolymph or cell-culture supernatant (Blissard & Wenz, 1992;Hefferon et al, 1999;Oomens & Blissard, 1999). envelope fusion proteins, the baculovirus F protein must be cleaved post-translationally by a proprotein convertase (furin) to become fusiogenic (Lung et al, 2002;Westenberg et al, 2002). Homologues of the F gene have been identified in other group II NPVs, in beta-and deltabaculoviruses and in members of the insect retrovirus family Errantiviridae, but also exist in group I NPVs (Herniou et al, 2003;Malik et al, 2000;Rohrmann & Karplus, 2001;Terzian et al, 2001).…”