Analysis of the Autographa californica Multiple Nucleopolyhedrovirus Overlapping Gene Pair lef3 and ac68 Reveals that AC68 Is a Per Os Infectivity Factor and that LEF3 Is Critical, but Not Essential, for Virus Replication

Abstract

bAutographa californica multiple nucleopolyhedrovirus ac68 is a core gene that overlaps lef3 which encodes the single-stranded DNA binding protein. A knockout (KO) virus lacking both lef3 and ac68 was generated (lef3-ac68 2؋KO) to enable the functional study of ac68. To produce an ac68KO virus that did not impact lef3 expression, the lef3-ac68 2؋KO virus was repaired with a DNA fragment containing lef3 and ac68, in which ac68 contained point mutations so that only LEF3 was expressed. Repair of lef3-ac68 2؋KO with just ac68 generated an lef3KO virus. Analysis of the ac68KO virus showed that viral DNA replication and budded virus (BV) levels were unaffected compared to levels in the double-repair or wild-type (WT) control virus. Bioassay analyses of Trichoplusia ni larvae injected with BV directly into the hemolymph, bypassing the gut, showed no difference in mortality rates between the ac68KO and the WT viruses. However, in oral bioassays the ac68KO occlusion bodies failed to kill larvae. These results show that the core gene ac68 encodes a per os infectivity factor (pif6). The lef3KO virus was also analyzed, and virus replication was drastically reduced compared to WT virus, but very low levels of lef3KO virus DNA replication and BV production could be detected. In addition, in transfected cells P143 was transported to the nucleus in the absence of LEF3. This study therefore shows for the first time that even though the loss of LEF3 severely impairs virus replication, it is not absolutely essential for P143 nuclear import or viral replication. Baculoviruses are large double-stranded DNA viruses, which specifically infect insects mainly from the orders Lepidoptera, Hymenoptera, and Diptera. Baculoviridae consists of alpha-, beta-, gamma-, and deltabaculoviruses (20,35). Of the baculoviruses completely sequenced to date, 32 genes have been shown to be present in all genomes, and these have been categorized as core genes (17,31,46). Many of these core genes have been analyzed in Autographa californica multiple nucleopolyhedrovirus (AcM-NPV), the Alphabaculovirus type species, and most appear to be required for an essential viral function such as replication or transmission (25,39,40). One exception, however, is the core gene ac68 for which no clear function has been identified, and the published results have been inconsistent (24, 25).The AcMNPV ac68 open reading frame (ORF) encodes a predicted protein of 192 amino acids (aa) that contains a C-terminal transmembrane domain (1, 24, 25). Using reverse transcription-PCR (RT-PCR) and 5= rapid amplification of cDNA ends (RACE), ac68 has been reported to be an early gene; however, Western blot analysis demonstrated that it is a late gene and that the observed size of AC68 is smaller than predicted (24,25). Functional analysis of ac68 by the construction of a knockout (KO) virus (designated ac68KO) did not reveal any impact on viral replication kinetics in tissue culture. In vivo bioassays indicated no difference in 50% lethal dose (LD 50 ) values, but the mean time to de...

Comparative Proteomics Reveal Fundamental Structural and Functional Differences between the Two Progeny Phenotypes of a Baculovirus

“…The homologue of HA68, another BV-specific membrane protein, was found to play a role in BV production (61). Among 11 ODV-specific envelope proteins, 7 proteins are essential for oral infectivity (P74, PIF1, PIF2, PIF3, PIF4, PIF5, and PIF6), and 1 protein plays an important role in infection (ODV-E66) (21,22,(30)(31)(32)(33)(34). In addition to the proteins discussed above, VP91 was also found to be a component of the PIF complex (62).…”

Autographa californicaNucleopolyhedrovirus Ac76: a Dimeric Type II Integral Membrane Protein That Contains an Inner Nuclear Membrane-Sorting Motif

“…11A). Considering the localization of these proteins (43)(44)(45)(46)(47)(48)(49), along with the identification of the INM-SM-like sequence of residues 15 to 48 of Ac76, we predicted that these proteins could also contain atypical INM-SM-like sequences. Thus, the INM-SM of baculovirus should be subdivided into two types, (i) the classical type, in which the TM domain is located at the N terminus of a protein with associated positively charged amino acids close to the C-terminal end of the TM domain, such as ODV-E66, PIF1, PIF2, PIF3, PIF4, Ac83, Ac91, and Ac150, except for ODV-E25, which lacks the positively charged residues, and (ii) the atypical type, in which the TM domain can be located in the middle or nearly at the C terminus of the protein, with associated positively charged amino acids close to the Nand/or C-terminal end of the TM domain, such as Ac76, ODV-E18, PIF5, PIF6, Ac108, Ac78, F-protein, and P74.…”

Bombyx mori nucleopolyhedrovirus BmP95 plays an essential role in budded virus production and nucleocapsid assembly

“…The initiation of baculovirus infection in the midgut is mediated by PIFs. So far, seven PIF proteins have been identified, P74 (Faulkner et al, 1997), PIF1 (Kikhno et al, 2002), PIF2 (Pijlman et al, 2003), PIF3 (Ohkawa et al, 2005), PIF4 (Fang et al, 2009), PIF5 (ODV-E56) (Sparks et al, 2011;Xiang et al, 2011a) and PIF6 (Ac68) (Nie et al, 2012). After ODVs are released from the polyhedra, the peritrophic matrix (PM) is the first barrier for ODV infection of the midgut epithelial cells.…”