Autographa californica Multiple Nucleopolyhedrovirus Core Gene ac96 Encodes a Per Os Infectivity Factor (pif-4)

Autographa californica multiple nucleopolyhedrovirus (AcMNPV) ac96 is a core gene, but its role in virus replication is still unknown. To determine its role in the baculovirus life cycle, we used the AcMNPV bacmid system to generate an ac96-null virus (vAc^96null). Our analyses showed that the absence of ac96 does not affect budded virus (BV) production or viral DNA replication in infected Sf9 cells. Western blotting and confocal immunofluorescence analysis showed that AC96 is expressed in both the cytoplasm and the nucleus throughout infection. In addition, AC96 was detected in the envelope fractions of both BV and occlusion-derived virus. Injection of vAc^96null BV into the hemocoel killed Trichoplusia ni larvae as efficiently as repaired and control viruses; however, vAc^96null was unable to infect the midgut tissue of Trichoplusia ni larvae when inoculated per os. Therefore, the results of this study show that ac96 encodes a new per os infectivity factor (PIF-4).The Baculoviridae comprise a large and diverse group of viruses that are pathogens of insects, mainly from the Lepidoptera, Hymenoptera, and Diptera. During the typical biphasic infection cycle, two structurally and functionally distinct enveloped virion phenotypes are produced: occlusion-derived virus (ODV) and budded virus (BV) (35). The primary infection cycle in animals begins in the midgut cell after occlusion bodies (OBs) are ingested. Upon ingestion, the OBs dissolve in the alkaline environment of the midgut, and the ODVs are released into the lumen of midgut (15, 16, 20). Virions pass through a disrupted peritrophic membrane, a process often facilitated by enhancins, a group of virus-encoded metalloproteases (38). Subsequently, ODVs bind to and fuse directly with the microvilli of midgut columnar epithelial cells. A protein receptor is proposed to mediate the process, since binding is proteinase sensitive and saturable (15, 16, 20). After the nucleocapsids are transported to the nuclei of the midgut cells, viral DNA is released, followed by gene expression, DNA replication, and assembly of progeny nucleocapsids. In the late phase of infection, newly formed nucleocapsids are transported to the cell membrane, bud from the cell, and acquire a new envelope from the basal membrane. The BVs spread via the hemolymph (16) and the tracheal system (8) into the other tissues of the insect, causing the secondary infection.Baculoviruses encode per os infectivity factors (PIFs) on the envelope surface of ODV to initiate the efficient primary infection in midgut. So far, four highly conserved core genes, p74 (pif-0), pif-1, pif-2, and pif-3, have been identified. The deletion of the Autographa californica multiple nucleopolyhedrovirus (AcMNPV) p74 gene results in the complete elimination of the per os infectivity of OBs, while virions purified from mutant OBs were infectious when injected into the hemocoels of Trichoplusia ni or Heliothis virescens larvae (13, 17, 22). P74 is proposed to function as an ODV attachment protein that binds to a specific 30-kDa receptor protein on the primary target cells within the midgut (17, 39). PIF-1 was originally identified in Spodoptera littoralis NPV, where the deletion of pif-1 (spli7) resulted in viruses that were unable to infect S. littoralis larvae per os (21). PIF-2 was first identified in Spodoptera exigua MNPV, and the disruption of pif-2 resulted in the complete loss of per os infectivity for the host (11, 31). PIF-1 and PIF-2 have also been shown to participate in the binding of ODV to target cells in the midgut (28). PIF-3 (ac115) is also an essential factor for oral infection of AcMNPV. Although PIF-3 is not required for ODV attachment and fusion, it may mediate a critical downstream event, such as the translocation of ODV along microvilli during primary infection (28).AcMNPV, the archetype Alphabaculovirus of the Baculoviridae, has a double-stranded DNA genome of approximately 134 kbp that contains 154 predicted open reading frames (ORFs) (1). Comparative analysis of the 49 completely sequenced baculovirus genomes reveals 31 core genes that are conserved in all baculovirus genomes and are therefore likely to serve important roles in baculovirus life cycles (14, 26, 32, 37). Most core genes are related either to DNA replication, gene expression, packaging and assembly, or per os infection (37). Four core genes, ac68, p33 (ac92), ac96, and ac109, still have no known function or sequence similarities to proteins of known functions.In this study, an ac96-null mutant was constructed utilizing an AcMNPV bacmid, and the results showed that in tissue culture, ac96 was nonessential and was not required for viral DNA replication, ODV production, or BV production. However, in vivo assays demonstrated that the ac96-null virus was unable to infect midgut tissue when T. ni larvae were inoculated per os. The core gene ac96 therefore encodes a new per os infectivity factor, PIF-4.