Baculovirus lef-12 Is Not Required for Viral Replication

Abstract

In the baculovirus system, most of the viral proteins involved in expression of late genes have been identified by transientexpression assays (25). In Spodoptera frugiperda cells, 19 viral proteins, called late expression factors (LEFs), are required for expression of a reporter gene cloned under the control of a late Autographa californica nuclear polyhedrovirus (AcNPV) promoter. Baculovirus late gene expression is absolutely dependent on viral DNA replication, and therefore these 19 genes include early transcription factors and DNA replication proteins as well as proteins directly involved in late transcription (21).The identification of 19 LEFs is broadly applicable to all baculoviruses, although a few virus-specific and cell line-specific differences have been noted. For example, the apoptosis suppressor p35 can be replaced by inhibitor of apoptosis genes from other baculoviruses, and it is not required for expression in Trichoplusia ni cells because of the inherent resistance of this cell line to apoptosis (1,10,21,29). In addition, three AcNPV genes (ie-2, lef-7, and lef-12) are essential or strongly stimulatory for transient expression of late genes in Spodoptera frugiperda cells but not in Trichoplusia ni cells (20,22). Instead, transient late expression in T. ni requires HCF-1, which is nonessential for replication in S. frugiperda cells.IE-2 transactivates early gene expression, stimulates DNA replication, and inhibits the host cell cycle in S. frugiperda cells (5,6,21,24). IE-2 is expressed only transiently during the early phase of replication. Therefore, it seems likely that it is not directly involved in late gene expression but rather helps to establish viral replication factories. Little is known concerning the functions of LEF-7 and HCF-1, but they are also required for efficient transient DNA replication and therefore may also play an indirect role in late gene expression (9,22 MATERIALS AND METHODS Construction of lef-12 (orf41) mutant viruses.To construct mutant viruses that do not express functional LEF-12, a fragment of DNA encoding the ␤-galactosidase gene was cloned into the lef-12 open reading frame. To do this, a 6-kb PstI-KpnI fragment, which represents the left half of the PstI-F fragment of the AcNPV genome and contains lef-12, was cloned into the PstI and SmaI sites of pUC8. The resulting clone, named PstF-PK, was digested with ApaI and then treated with T4 DNA polymerase in the presence of deoxynucleoside triphosphates to make blunt ends. The repaired DNA was then ligated with BglII linkers (GAAGATCTTC). This DNA was subsequently digested with BglII and then ligated to a 3.1-kb fragment encoding the ␤-galactosidase gene which had previously been purified from plasmid DP502 after digestion with BamHI. Colonies expressing the LEF-12-␤-galactosidase fusion under the control of the lef-12 promoter were selected as blue colonies when grown in the presence of 5-bromo-4-chloro-3-indolyl-␤-D-galactopyranoside (X-Gal). DNA sequence analysis was performed to confirm that the construction wa...

Autographa californica Nucleopolyhedrovirus orf69 Encodes an RNA Cap (Nucleoside-2′- O )-Methyltransferase

“…But, overall, the protein expression and RNA mapping data (38) suggest that orf69/mtase1 stimulated late gene expression threefold. Similar results have been found for several of the LEFs, which are essential for transient late expression but are not required for viral replication (7,20,46,48). It is possible that these differences between transient expression and viral infection could reflect the fact that proteins like 2ЈOMTase offer a limited advantage to expression and that this is more significant in transient expression, which is less efficient than viral infection.…”

“…These genes are referred to as LEFs (48). The LEFs include immediate early transcription factors, DNA replication proteins, the four RNA polymerase subunits, and additional transcription factors (18,20,22,39). Levels of transient gene expression can be further stimulated by addition of other viral genes, such as the AcNPV orf69 gene.…”

An ac34 Deletion Mutant of Autographa californica Nucleopolyhedrovirus Exhibits Delayed Late Gene Expression and a Lack of Virulence In Vivo

“…However, the transient-expression system does not perfectly mimic the course of viral infection, and it is possible that the functional importance of factors that are regulated by other viral components during virus infection could be overlooked. Studies of gene deletion mutants have provided new insights into the roles of some genes, particularly regarding their requirements for DNA replication and/or late transcription in the context of the infection cycle (13,21,30,33,37,42,51,63). Our data demonstrate that Ac34 is required for efficient viral late transcription during the infection cycle.…”

“…In this respect, Ac34 is similar to some late expression factors, such as LEF6, LEF12, and PP31 (21,32,65). These genes do not appear to play essential roles in AcMNPV infection in Sf9 cells, but their presence accelerates the infection cycle and increases viral yields (21,32,65).…”

The effect of BM67 gene deletion on Bombyx mori nuclear polyhedrosis virus replication

“…This effectively inserts the foreign gene into the position of the target gene in the host genome, efficiently achieving gene knockout (Costantino et al, 2003). Red recombination technology has been widely applied to the functional analysis of the genes of AcMNPV and BmNPV (Guarino et al, 2002;Dickison et al, 2012). To investigate the function of Bm67, we generated a Bm67-deficient virus and studied the impact of this deletion on infectious virus production and viral genome replication and transcription, using wild type (encodes a complete BmNPV) and Bm67-repair viruses as controls.…”