Biosynthesis and Processing of the Autographa californica Nuclear Polyhedrosis Virus gp64 Protein

Jarvis, Donald L.; Garcia, A.

doi:10.1006/viro.1994.1646

Cited by 90 publications

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“…GP64 is temporally ‡ Corresponding author. E-mail address: joshz@helix.nih.gov.© 1999 by The American Society for Cell Biology 4191 expressed during both the early and late phases of viral infection (Blissard and Rohrmann, 1989;Jarvis and Garcia, 1994;Kogan et al, 1994;Oomens et al, 1995;Garrity et al, 1997) and oligomerizes to form two electrophoretically distinct trimers (Oomens et al, 1995;Markovic et al, 1998). This protein is both necessary and sufficient for mediating pH-dependent membrane fusion (Blissard and Wenz, 1992), is essential for cell-to-cell transmission of the enveloped virion (Monsma et al, 1996), and has host cell receptor-binding activity (Hefferon et al, 1999).…”

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confidence: 99%

A Discrete Stage of Baculovirus GP64-mediated Membrane Fusion

Kingsley

Behbahani

Rashtian

et al. 1999

MBoC

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Viral fusion protein trimers can play a critical role in limiting lipids in membrane fusion. Because the trimeric oligomer of many viral fusion proteins is often stabilized by hydrophobic 4-3 heptad repeats, higher-order oligomers might be stabilized by similar sequences. There is a hydrophobic 4-3 heptad repeat contiguous to a putative oligomerization domain of Autographa californica multicapsid nucleopolyhedrovirus envelope glycoprotein GP64. We performed mutagenesis and peptide inhibition studies to determine if this sequence might play a role in catalysis of membrane fusion. First, leucine-to-alanine mutants within and flanking the amino terminus of the hydrophobic 4-3 heptad repeat motif that oligomerize into trimers and traffic to insect Sf9 cell surfaces were identified. These mutants retained their wild-type conformation at neutral pH and changed conformation in acidic conditions, as judged by the reactivity of a conformationally sensitive mAb. These mutants, however, were defective for membrane fusion. Second, a peptide encoding the portion flanking the GP64 hydrophobic 4-3 heptad repeat was synthesized. Adding peptide led to inhibition of membrane fusion, which occurred only when the peptide was present during low pH application. The presence of peptide during low pH application did not prevent low pH-induced conformational changes, as determined by the loss of a conformationally sensitive epitope. In control experiments, a peptide of identical composition but different sequence, or a peptide encoding a portion of the Ebola GP heptad motif, had no effect on GP64-mediated fusion. Furthermore, when the hemagglutinin (X31 strain) fusion protein of influenza was functionally expressed in Sf9 cells, no effect on hemagglutinin-mediated fusion was observed, suggesting that the peptide does not exert nonspecific effects on other fusion proteins or cell membranes. Collectively, these studies suggest that the specific peptide sequences of GP64 that are adjacent to and include portions of the hydrophobic 4-3 heptad repeat play a dynamic role in membrane fusion at a stage that is downstream of the initiation of protein conformational changes but upstream of lipid mixing. INTRODUCTIONThe mechanism of membrane fusion is currently a field of intense investigation, both for intracellular trafficking and enveloped viral infection. For viral fusion protein function, we have previously suggested that fusion protein oligomers form transient and cooperative higher-order structures or rings . These transient ring structures are proposed to facilitate conversion of kinetic energy, released during protein conformational change, into work on the target and host membranes required for membrane fusion. Indeed, biochemical evidence for baculovirus GP64 aggregates during membrane fusion has recently been reported (Markovic et al. 1998). Whether and how viral fusion proteins might function in a coordinated and cooperative manner is a major question in the field of membrane fusion. Because hydrophobic 4-3 heptad repeats are observed...

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confidence: 99%

A Discrete Stage of Baculovirus GP64-mediated Membrane Fusion

Kingsley

Behbahani

Rashtian

et al. 1999

MBoC

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“…The two helices have quite different characters. The first, PLLILGS (3)(4)(5)(6)(7)(8)(9), is apolar, whereas the second, IQ-AIHDAQR (17)(18)(19)(20)(21)(22)(23)(24)(25), contains polar and charged amino acids. This will result in different interactions with cell membranes.…”

Section: Discussionmentioning

confidence: 99%

“…Interestingly, the ATG coding for the middle methionine of the signal peptide is contained in a perfect Kozak consensus sequence AAGATGG, ensuring proper translation initiation [36]. Thus, the recombinant poneratoxin seems to be synthesized through the initiation from the second ATG in the open reading frame, similar to insect protein gp64 [25], a source of signal peptide. However, the signal peptide was not cleaved from the poneratoxin resulting in an uncleaved intracellular form, with no extra-cellular poneratoxin detected.…”

Section: Discussionmentioning

confidence: 99%

“…However, the mass spectroscopy of the recombinant poneratoxin contained in the formic acid extract showed that it has molecular mass of 4861, compatible with a longer peptide starting with the methionine in the middle of the signal peptide (theoretical mass 4861). It is relevant that when gp64 (called also gp67) is expressed during AcMNPV infection, the second ATG in the open reading frame is used as the translation initiation codon and that downstream sequences encode a functional signal peptide [25]. In addition, the preparation showed the presence of a second species with mass of 4888, suggesting the postranslational modification by formylation of the initiator methionine (theoretical mass 4889), which explained the difficulties encountered in the N-terminal sequencing.…”

Section: Peptide Synthesis and Expression Of Recombinant Poneratoxinmentioning

confidence: 99%

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Poneratoxin, a neurotoxin from ant venom

Szołajska

Poznański

Ferber

et al. 2004

European Journal of Biochemistry

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Poneratoxin is a small neuropeptide found in the venom of the ant Paraponera clavata. It is stored in the venom reservoir as an inactive 25-residue peptide. Here we describe both chemically synthesized poneratoxin and poneratoxin obtained by expression in insect cells. When expressed in insect cells, poneratoxin was observed attached to cell membranes. Both synthetic and recombinant ponerotoxins were soluble below pH 4.5. The structure of synthetic poneratoxin was characterized by circular dichroism and solved by nuclear magnetic resonance. In an environment imitating a lipid bilayer, at pH within the range of insect hemolymph, synthetic poneratoxin has a V shape, with two a-helices connected by a b-turn. Insect larvae were paralyzed by injection of either of the purified toxins, with the recombinant one acting faster. The recombinant toxin-producing baculovirus reduced the average survival time of the insect host by 25 h compared with unmodified virus. Mass spectrometry analysis showed that the recombinant toxin has an N-terminal 21-residue extension, possibly improving its stability and/or stabilizing the membrane-bound state. The potential use of poneratoxin for the construction of biological insecticide is discussed.

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“…Dual early and late transcription initiation sites, on the other hand, have been revealed in several NPV genes expressed in cell culture. The gp64 envelope glycoprotein gene, for instance, is transcribed under such an arrangement from the genomes of both OpMNPV (Blissard & Rohrmann, 1989) and AcMNPV (Jarvis & Garcia, 1994), with lower levels of gp64 mRNA arising from an early start site being superseded by higher levels initiating from late sites. At the level of the whole insect, used here in the absence of a permissive cell line, cells are liable to be infected asynchronously so that no such early-to-late progression can be observed.…”

Section: Discussionmentioning

confidence: 99%

Sequence and in vivo transcription of Lacanobia oleracea granulovirus egt.

Smith

Goodale

1998

Journal of General Virology

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We have determined the nucleotide sequence and located the major in vivo transcript termini of the Lacanobia oleracea granulovirus (LoGV) egt gene. The open reading frame encodes a 460-amino acid polypeptide having extensive sequence similarity to ten nucleopolyhedrovirus (NPV) ecdysteroid UDPglucosyltransferase (EGT) proteins ; the degree of similarity is particularly high within several previously identified EGT ' domains ', and eight invariant amino acid residues are conserved. A phylogenetic tree, constructed by the neighbour joining method, showed LoGV EGT to be the most highly diverged of the eleven baculovirus sequences compared. Database searching revealed that part of a published DNA sequence from Cryptophlebia leucotreta granulovirus appears to encode the N-terminal

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Biosynthesis and Processing of the Autographa californica Nuclear Polyhedrosis Virus gp64 Protein

Cited by 90 publications

References 0 publications

A Discrete Stage of Baculovirus GP64-mediated Membrane Fusion

A Discrete Stage of Baculovirus GP64-mediated Membrane Fusion

Poneratoxin, a neurotoxin from ant venom

Sequence and in vivo transcription of Lacanobia oleracea granulovirus egt.

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