Characterization of P91, a Protein Associated with Virions of anOrgyia pseudotsugataBaculovirus

Abstract

Polyclonal antiserum produced against preoccluded virions from the Orgyia pseudotsugata multinucleocapsid nuclear polyhedrosis virus (OpMNPV) was used to screen an OpMNPV lambda gt11 expression library. The insert from one of the immunoreactive phage isolates hybridized to OpMNPV orf86 (p91), a 2460-bp (819 amino acids) open reading frame that encodes a predicted protein of 91 kDa. Antibodies generated against a maltose binding protein-P91 fusion detected a band of approximately 91 kDa on Western blots of extracts of OpMNPV-infected Lymantria dispar cells. This band was first observed at 18 hr p.i. and was present at all later time points. Similar results using this antiserum were seen with a time course of Autographa californica-infected Spodoptera frugiperda cells. Localization of P91 by confocal immunofluorescence microscopy showed that the protein was concentrated near the nuclear membrane and at late times p.i. was most concentrated near polyhedra. Immunoelectron microscopy indicated that P91 was present in both the capsid and envelope surrounding the capsid of occlusion-derived virions. Fractionation studies employing NP-40 and Western blot analysis indicated that P91 was associated with the capsid structure.

Analysis of baculovirus IE1 in living cells: dynamics and spatial relationships to viral structural proteins

“…As seen in Fig. 4(g-i), local accumulation of GFP-ODV-E25 and P91-DsRed exhibited some degree of overlap, suggesting that these two proteins were, as reported previously, co-ordinately assembled in the ODV envelopment sites to generate ODVs (Hong et al, 1997;Russell & Rohrmann, 1997).…”

“…4a-c) and subsequently polyhedra formed in the region accumulating P91-GFP (data not shown). This distribution of P91-GFP was similar to that of untagged P91 (Russell & Rohrmann, 1997), indicating that the GFP tag did not interfere in the intracellular localization of the P91 molecules and that P91-GFP could be employed as a marker for live-cell imaging. The distribution pattern of GFP-ODV-E25 was likewise similar to that of untagged ODV-E25 (Braunagel et al, 1999;RosasAcosta et al, 2001), i.e.…”

“…In previous studies, a number of ODV-associated proteins, including P91 and ODV-E25, were shown to be localized around the periphery of the nucleus (Russell & Rohrmann, 1997;Braunagel et al, 1999;Rosas-Acosta et al, 2001;Slack et al, 2001). These distributions are in sharp contrast to the location of the IE1-associated structure that often occupies the central region of the nucleus at 24 h p.i.…”

“…In studies examining the intracellular localization of viral proteins by fluorescent microscopy, a number of ODVassociated proteins (e.g. ODV-E25, ODV-E66, P91 and P74) were shown to localize to the periphery of the nucleus (Russell & Rohrmann, 1997;Braunagel et al, 1999;RosasAcosta et al, 2001;Slack et al, 2001). More detailed observations with immunoelectron microscopy, however, have revealed that these proteins are mainly localized to the peristromal region in association with ODVs and intranuclear microvesicles (Russell & Rohrmann, 1993, 1997Braunagel et al, 1996a, b;Hong et al, 1997).…”

Identification of a Lymantria dispar Nucleopolyhedrovirus Isolate That Does Not Accumulate Few-Polyhedra Mutants during Extended Serial Passage in Cell Culture

“…Initially, the treatment of ODV was performed with the nonionic detergent NP-40 to remove virion envelopes (24). After the treatment of ODV with NP-40, a small amount of E1 was found in the supernatant fraction (Fig.…”