Sharon C. Braunagel

Determination of the protein composition of the occlusion-derived virus of Autographa californica nucleopolyhedrovirus

The occlusion derived form of baculovirus is specially adapted for primary infection of the host midgut epithelium. As such, the virion must contain the proteins essential for host range determination and initiation of infection. Because knowledge of virion composition is a prerequisite for functional investigation, this study used a combination of techniques to identify the proteins present within or associated with the occlusion-derived virus (ODV) virion. Thirty-one proteins, including proteins known to be essential for viral DNA replication, were identified with confidence. An additional 13 proteins were identified by using one of the three techniques. A comparison of gene conservation among the ODV proteins encoded in the 16 sequenced baculoviridae genomes is presented. With knowledge of the composition of ODV, it is now possible to target proteins and study their role(s) during primary infection.

Molecular biology of the baculovirus occlusion-derived virus envelope.

Study of the biology of the occlusion-derived virus (ODV) of the baculovirus Autographa californica nucleopolyhedrovirus provides opportunities to reveal new discoveries into the mechanism of several cellular pathways. The synchronous pulse of multiple ODV envelope proteins that integrate into the endoplasmic reticulum (ER) and traffic to the nuclear membranes on their way to the ODV envelope provide a unique tool to study the mechanisms of integral membrane protein trafficking from the ER to the outer and inner nuclear membrane. Studies of the formation of virus-induced, intranuclear membrane microvesicles provide insight on mechanisms that alter fluidity and regulate budding of the inner nuclear membrane. Since ODV is specially adapted for primary infection of the insect gut, studies of the structure and function of ODV envelope proteins reveals insights on the mechanism of viral invasion of the gut and this knowledge is fundamental for the development of new strategies for insect control. This review focuses on recent advances in understanding the source of the ODV envelope and the molecular events that sort and traffic integral membrane proteins from the ER to the ODV envelope. The composition of ODV is reviewed, however it is worth noting that the function of many ODV proteins are currently unknown.

Importin-α-16 is a translocon-associated protein involved in sorting membrane proteins to the nuclear envelope

A viral inner nuclear membrane–sorting motif sequence (INM-SM) was used to identify proteins that recognize integral membrane proteins destined for the INM. Herein we describe importin-α-16, a membrane-associated isoform of Spodoptera frugiperda importin-α that contains the C-terminal amino acid residues comprising armadillo helical-repeat domains 7–10. In the endoplasmic reticulum (ER) membrane, importin-α-16 is adjacent to the translocon protein Sec61α. Importin-α-16 cross-links to the INM-SM sequence as it emerges from the ribosomal tunnel and remains adjacent to the INM-SM after INM-SM integration into the ER membrane and release from the translocon. Cross-linking results suggest that importin-α-16 discriminates between INM- and non–INM-directed proteins. Thus, it seems that during and after cotranslational membrane integration, importin-α-16 is involved in the trafficking of integral membrane proteins to the INM.

The metalloprotease gene ofSerratia marcescens strain SM6

SummaryUtilizing the DNA sequence of the metalloprotease fromSerratia strain E-15, we isolated and sequenced the homologous gene fromSerratia strain SM6. These two genes are similar at both the DNA and protein sequence level. Expression of the protease gene inEscherichia coli was achieved by use of thelac promoter. This resulted in the production and excretion of an immunologically detectable but inactive protein of slightly higher molecular weight than that fromSerratia. We introduced the cloned gene into previously described protease mutants. The observed pattern of protease expression suggested that these mutations fall into three classes.

Identification of BV/ODV-C42, an Autographa californica Nucleopolyhedrovirus orf101-Encoded Structural Protein Detected in Infected-Cell Complexes with ODV-EC27 and p78/83

ABSTRACT orf101 is a late gene of Autographa californica nucleopolyhedrovirus (AcMNPV). It encodes a protein of 42 kDa which is a component of the nucleocapsid of budded virus (BV) and occlusion-derived virus (ODV). To reflect this viral localization, the product of orf101 was named BV/ODV-C42 (C42). C42 is predominantly detected within the infected-cell nucleus: at 24 h postinfection (p.i.), it is coincident with the virogenic stroma, but by 72 h p.i., the stroma is minimally labeled while C42 is more uniformly located throughout the nucleus. Yeast two-hybrid screens indicate that C42 is capable of directly interacting with the viral proteins p78/83 (1629K) and ODV-EC27 (orf144). These interactions were confirmed using blue native gels and Western blot analyses. At 28 h p.i., C42 and p78/83 are detected in two complexes: one at approximately 180 kDa and a high-molecular-mass complex (500 to 600 kDa) which also contains EC27.

Early sorting of inner nuclear membrane proteins is conserved

Spodoptera frugiperda (Sf9) importin-α-16 is a translocon-associated protein that participates in the early sorting pathway of baculovirus integral membrane proteins destined for the inner nuclear membrane (INM). To discern whether sorting intermediate protein complexes like those observed in insect cells are also formed with mammalian INM proteins, cross-linked complexes of importin-α-16 with human lamin B receptor (LBR) and nurim were examined. Both LBR and nurim cross-link with Sf9 importin-α-16 during cotranslational membrane integration and remain proximal with importin-α-16 after integration into the endoplasmic reticulum membrane and release from the translocon. Human cells encode several isoforms of importin-α; to determine whether any of these isoforms may recognize INM-directed proteins, they were tested for their ability to cross-link with the viral-derived INM sorting motif sequence. One cross-linked adduct was detected with a 16-kDa isoform encoded from KPNA4 (KPNA-4–16). KPNA-4–16 was easily detected in microsomal membranes prepared from KPNA4–16 recombinant virus-infected cells and was also detected in microsomes prepared from HeLa cells. Together these observations suggest that elements of the early sorting pathway of INM-directed proteins mediated by importin-α-16 are highly conserved, and mammalian KPNA-4–16 is a candidate partner in sorting integral membrane proteins to the INM.

Effects of Deletion and Overexpression of the Autographa californica Nuclear Polyhedrosis Virus FP25K Gene on Synthesis of Two Occlusion-Derived Virus Envelope Proteins and Their Transport into Virus-Induced Intranuclear Membranes

ABSTRACT Partial deletions within Autographa californica open reading frame 61 (FP25K) alter the expression and accumulation profile of several viral proteins and the transport of occlusion-derived virus (ODV)-E66 to intranuclear membranes during infection (S. C. Braunagel et al., J. Virol. 73:8559–8570, 1999). Here we show the effects of a full deletion and overexpression of FP25K on the transport and expression of two ODV envelope proteins, ODV-E66 (E66) and ODV-E25 (E25). Deletion and overexpression of FP25K substantially altered the levels of expression of E66 during infection. Compared with cells infected with wild-type (wt) virus, the levels of E66 were reduced fivefold in cells infected with a viral mutant lacking FP25K (ΔFP25K) and were slightly increased in cells infected with a viral mutant overexpressing FP25K (FP25Kpolh). In contrast, no significant changes were observed in the levels of E25 among wt-, ΔFP25K-, and FP25Kpolh-infected cells. The changes observed in the levels of E66 among the different viral mutants were not accompanied by changes in either the time of synthesis, membrane association, protein turnover, or steady-state transcript abundance. Deletion of FP25K also substantially altered the transport and localization of E66 during infection. In cells infected with the ΔFP25K mutant virus, E66 accumulated in localized regions at the nuclear periphery and the outer nuclear membrane and did not traffic to intranuclear membranes. In contrast, in cells infected with the FP25Kpolh mutant virus E66 trafficked to intranuclear membranes. For comparison, E25 was normally transported to intranuclear membranes in both ΔFP25K- and FP25Kpolh-infected cells. Altogether these studies suggest that FP25K affects the synthesis of E66 at a posttranscriptional level, probably by altering the translation of E66; additionally, the block in transport of E66 at the nuclear envelope in ΔFP25K-infected cells suggests that the pathway of E66 trafficking to the inner nuclear membrane and intranuclear microvesicles is specifically regulated and must be influenced by factors that do not control the traffic of E25.

Baculovirus Data Suggest a Common but Multifaceted Pathway for Sorting Proteins to the Inner Nuclear Membrane

ABSTRACT Multiple unique protein markers sorted to the inner nuclear membrane (INM) from the Autographa californica nucleopolyhedrovirus occlusion-derived virus (ODV) envelope were used to decipher common elements of the sorting pathway of integral membrane proteins from their site of insertion into the membrane of the endoplasmic reticulum (ER) through their transit to the INM. The data show that during viral infection, the viral protein FP25K is a partner for all known ODV envelope proteins and that BV/ODV-E26 (designated E26) is a partner for some, but not all, such proteins. The association with the ER membrane of FP25K, E26, and the cellular INM-sorting protein importin-α-16 is not static; rather, these sorting proteins are actively recruited to the ER membrane based upon requirements of the proteins in transit to the INM. Colocalization analysis using an ODV envelope protein and importin-α-16 shows that during viral infection, importin-α-16 translocates across the pore membrane to the INM and then is incorporated into the virus-induced intranuclear membranes. Thus, the association of importin-α-16 and INM-directed proteins appears to remain at least through protein translocation across the pore membrane to the INM. Overall, the data suggest that multiple levels of regulation facilitate INM-directed protein trafficking, and that proteins participating in this sorting pathway have a dynamic relationship with each other and the membrane of the ER.

Truncated Isoforms of Kap60 Facilitate Trafficking of Heh2 to the Nuclear Envelope

Isoforms of importin‐α have been identified in insect and human cells, and cross‐linking experiments suggest that at least one isoform in each species participates in the targeting of integral membrane proteins to the inner nuclear membrane (INM). To directly test this hypothesis, an assay was developed using Saccharomyces cerevisiae. The data show that internal promoters are present within KAP60, and the nested transcripts are translated into three isoforms: Kap60‐44, Kap60‐30 and Kap60‐10. In the absence of the isoforms, the INM protein Heh2‐green fluorescent protein (GFP) localized to cytoplasmic membranes, whereas its wild‐type localization at the nuclear periphery was restored when the Kap60‐44 isoform was reintroduced. An INM‐sorting sequence has been identified that cross‐links with the isoform of importin‐α that directs trafficking toward the nuclear envelope (NE). When this sequence in HEH2 was mutated, Heh2 was again localized to cytoplasmic membranes. Thus, this report provides the first evidence that isoforms of Kap60 exist in yeast, and these isoforms participate in the molecular sorting and enrichment of INM proteins to the NE. Herein, we provide additional support for the hypothesis that trafficking of INM proteins to the NE is a regulated process.

Sequence, genomic organization of the EcoRI-A fragment of Autographa californica nuclear polyhedrosis virus, and identification of a viral-encoded protein resembling the outer capsid protein VP8 of rotavirus

We present the sequence and genomic organization of the EcoRI-A fragment of the Autographa californica multicapsid nuclear polyhedrosis virus, which represents 11% of the AcMNPV genome. Fifteen putative open reading frames and their respective amino acid sequences are described. One open reading frame is similar to the VP8 protein of rotavirus.