The pk-1 Gene of Autographa Californica Multinucleocapsid Nuclear Polyhedrosis Virus Encodes a Protein Kinase

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...

Section: Construction Of Vlef12-␤galsupporting

confidence: 65%

Baculovirus lef-12 Is Not Required for Viral Replication

Guarino

Mistretta²,

Dong

2002

“…The AcMNPV genome contains two genes, pk1 and pk2, with homology to eukaryotic protein kinases. Enzymatic activity is associated with the gene product of pk1 (16). pk2 encodes a truncated protein kinase homologue and is involved in the reduced phosphorylation of eukaryotic translation initiation factor 2␣ (6).…”

Section: Discussionmentioning

confidence: 99%

Actin Rearrangement-Inducing Factor of Baculoviruses Is Tyrosine Phosphorylated and Colocalizes to F-Actin at the Plasma Membrane

Dreschers

Roncarati

Knebel-Mörsdorf

2001

In previous studies we have identified actin rearrangement-inducing factor 1 as an early gene product of Autographa californica multicapsid nuclear polyhedrosis virus that is involved in the remodeling of the actin cytoskeleton. We have constructed viral recombinants with a mutated Arif-1 open reading frame that confirm the causal link of Arif-1 expression and the actin rearrangement observed as accumulation of F-actin at the plasma membrane at 3 to 7 h postinfection. Infection with Arif mutant viruses leads to the loss of actin accumulation at the plasma membrane in TN-368 cells, although in the course of infection, early actin cables and nuclear F-actin are observed as in wild-type-infected cells. By immunofluorescence studies, we have demonstrated the localization of Arif-1 at the plasma membrane, and confocal imaging reveals the colocalization to F-actin. Accordingly, the ϳ47-kDa Arif-1 protein is observed exclusively in membrane fractions prepared at 4 to 48 h postinfection, with a decrease at 24 h postinfection. Phosphatase treatment suggests that Arif-1 is modified by phosphorylation. Antibodies against phosphotyrosine precipitate Arif-1 from membrane fractions, indicating that Arif-1 becomes tyrosine phosphorylated during the early and late phases of infection. In summary, our results indicate that functional Arif-1 is tyrosine phosphorylated and is located at the plasma membrane as a component of the actin rearrangement-inducing complex.

“…Sequences of PK1 of baculoviruses Spodoptera litura nucleopolyhedrovirus (SpltNPV) (148), Choristoneura fumiferana granulovirus (ChfuGV) (73), Lymantria dispar nuclear polyhedrosis virus (LdNPV) (22), Helicoverpa armigera single nucleopolyhedrovirus (HaSNVP) (237), and Autographa californica nucleopolyhedrovirus (AcNPV) (188) were analyzed and were found to contain typical serine/threonine kinase domains. Recombinant PK1 of AcNPV, SpltNPV, and LdNPV phosphorylates exogenous substrates, such as histone H1 and myelin basic protein (MBP) (22,148,188). Based on genome sequence analyses, the existence of a viral protein kinase in Anagrapha falcifera multinuclear polyhedrosis virus (AfMNPV) and Neodiprion lecontei nucleopolyhedrovirus (NeleNPV) was also predicted (64,113).…”

Section: Baculovirusesmentioning

confidence: 99%

Viral Serine/Threonine Protein Kinases

Jacob

Broeke

Favoreel

2011

108

Phosphorylation represents one the most abundant and important posttranslational modifications of proteins, including viral proteins. Virus-encoded serine/threonine protein kinases appear to be a feature that is unique to large DNA viruses. Although the importance of these kinases for virus replication in cell culture is variable, they invariably play important roles in virus virulence. The current review provides an overview of the different viral serine/threonine protein kinases of several large DNA viruses and discusses their function, importance, and potential as antiviral drug targets.