Expression of PE38 and IE2, viral members of the C3HC4 finger family, during baculovirus infection: PE38 and IE2 localize to distinct nuclear regions

European Journal of Biochemistry

2002

Baculoviruses are well known for their large, circular, double-stranded DNA genomes. The type member, AcMNPV, is the best characterized and undergoes a succession of early, late and very late gene expression during its infection cycle. The viral genes involved in DNA replication have previously been identified and their products are required for the activation of late gene expression. In this study, we FLAG-and HA-tagged the replication late expression factors of AcMNPV, examined their expression and functional activities by CAT assay and Western blot analysis, and determined their subcellular localization in transfected cells by subcellular fractionation and immunofluorescent microscopy. We found that all replication LEFs with the exception of P143 and P35 resided in the nucleus of transfected cells. We further investigated the interactions among various replication LEFs using both yeast two-hybrid and coprecipitation strategies. A summary of the interactive properties of the replication LEFs is presented and a model for a putative AcMNPV replication complex is offered.

Section: R E S U L T S Expression and Characterization Of Haand Flag-supporting

confidence: 86%

Reconstructing the replication complex of AcMNPV

Hefferon

European Journal of Biochemistry

2002

“…Wild-type PE38 localized predominantly to the nucleus in a punctate pattern (Fig. 5, panels 1 and 2), similar to on August 5, 2020 by guest http://jvi.asm.org/ the pattern previously observed for wild-type PE38 expressed under the transcriptional control of its own promoter (21). The same punctate nuclear localization was observed for FLAG-PE38C109A, FLAG-PE38C138A, and FLAG-PE38L242A ( Fig.…”

Section: Below)supporting

confidence: 82%

“…The mechanism by which PE38 augments IE1-induced apoptosis remains unclear. Full-length PE38 is known to localize in a punctate pattern in the nucleus of transfected or infected cells (21). However, during the late phase of infection, levels of the 38-kDa polypeptide decline and a smaller polypeptide (20 kDa) which cross-reacts with PE38 antiserum is observed only in the cytoplasmic fraction (21).…”

Section: Discussionmentioning

confidence: 99%

“…The pe38 gene of AcMNPV was first characterized as a gene expressed immediately upon infection (20). PE38 is present during the early phase of infection as a nuclear 38-kDa protein, but during the late phase, it appears to be converted to or expressed as a cytoplasmic 20-kDa protein in a process which is controlled by viral factors (21). Upon transient expression, pe38 generates only a 38-kDa product which localizes to punctate regions of the nucleus.…”

mentioning

confidence: 99%

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The Baculovirus PE38 Protein Augments Apoptosis Induced by Transactivator IE1

Prikhod’ko

1999

J Virol

While studying apoptosis induced by baculovirus transactivator IE1 in SF-21 cells, we found that the levels of IE1-induced apoptosis were increased approximately twofold upon cotransfection with the baculovirus early pe38 gene. However, no apoptotic activity was observed in cells transfected with pe38 alone, even when placed under the control of a constitutive promoter. Thus,pe38 was able to augment IE1-induced apoptosis but was unable to induce apoptosis when expressed in SF-21 cells alone. PE38, the full-length product of pe38, is a nuclear protein with RING finger and leucine zipper motifs. Deletion of the amino-terminal region, which contains a putative nuclear localization motif, resulted in cytoplasmic localization of the PE38 mutants. These N-terminal deletion mutants were unable to enhance IE1-induced apoptosis. Mutation of a single conserved leucine (L242) of the leucine zipper motif also eliminated the ability of PE38 to augment apoptosis induced by IE1. In contrast, PE38 mutants with alanine substitutions for conserved cysteine residues (C109 or C138) of the RING finger motif were able to increase IE1-induced apoptosis to levels equivalent to those of wild-type PE38. We propose that PE38 is one of at least two viral factors which collectively evoke a cellular apoptotic response during baculovirus infection.

“…IE2 is a nuclear protein which contains a RING finger motif at its center (23,40). RING fingers, or C 3 HC 4 motifs, form a cross-braced zinc coordinating structure (11,12,24) and are found in a number of proteins of diverse function.…”

mentioning

confidence: 99%

Role of Baculovirus IE2 and Its RING Finger in Cell Cycle Arrest

Prikhod’ko

1998

J Virol

The ie2 gene of Autographa californicanuclear polyhedrosis virus (AcMNPV) is known to transactivate transient expression from viral promoters in a host cell-specific manner. We report that transfection ofSpodoptera frugiperda (SF-21) cells with ie2was sufficient to arrest the cell cycle, resulting in the accumulation of enlarged cells with abnormally high DNA contents. By 72 h posttransfection, more than 50% of ie2-transfected cells had DNA contents greater than 4N. There was no evidence of mitotic spindle formation in these cells, and expression ofie2 appeared to block cell cycle progression in S phase. Several ie2 mutants were analyzed to further define the region of IE2 responsible for arresting the cell cycle. Analysis of these mutants showed that deletion of the RING finger motif eliminated the ability of IE2 to arrest the cell cycle but did not affect its ability to transactivate the ie1 promoter. Moreover, mutation of a single conserved cysteine (C251) of the RING finger motif abolished the ability of IE2 to block cell cycle progression but had no apparent effect on its trans-regulatory activity. In contrast, a mutant of IE2 containing a deletion of residues 94 to 173 was able to block cell division but lacked trans-regulatory activity. Thus, the ability of IE2 to arrest the cell cycle depended on the integrity of the RING finger motif and was distinct from and independent of its ability to trans-activate theie1 promoter. IE2 also arrested the division of cells derived from other insect species, Trichoplusia ni (TN-368 and BTI-TN-5B1-4) and Helicoverpa zea (Hz-AM1).