Mutational analysis of the N-linked glycans on Autographa californica nucleopolyhedrovirus gp64.

gp64 is the major envelope glycoprotein in the budded form of Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV). gp64 is essential for AcMNPV infection, as it mediates penetration of budded virus into host cells via the endocytic pathway. In this study, we used site-directed mutagenesis to map the positions of the N-linked glycans on AcMNPV gp64, characterize their structures, and evaluate their influence on gp64 function. We found that four of the five consensus N-glycosylation sites in gp64 are used, and we mapped the positions of those sites to amino acids 198, 355, 385, and 426 in the polypeptide chain. Endoglycosidase H sensitivity assays showed that N-linked glycans located at different positions are processed to various degrees. Lectin blotting analyses showed that each N-linked glycan on gp64 contains alpha-linked mannose, all but one contains alpha-linked fucose, and none contains detectable beta-linked galactose or alpha2,6-linked sialic acid. The amounts of infectious progeny produced by AcMNPV mutants lacking one, two, or three N-linked glycans on gp64 were about 10- to 100-fold lower than wild-type levels. This reduction did not correlate with reductions in the expression, transport, or inherent fusogenic activity of the mutant gp64s or in the gp64 content of mutant budded virus particles. However, all of the mutant viruses bound more slowly than the wild type. Therefore, elimination of one or more N-glycosylation sites in AcMNPV gp64 impairs binding of budded virus to the cell, which explains why viruses containing these mutant forms of gp64 produce less infectious progeny.

Isolation and characterization of a class II alpha-mannosidase cDNA from lepidopteran insect cells.

Lepidopteran insect cells are used routinely as hosts for foreign glycoprotein expression by recombinant baculoviruses, but the precise nature of their N-glycosylation pathway remains poorly defined. These cells clearly have processing glucosidases and mannosidases that can convert precursors to Man3GlcNAc2 structures and fucosyltransferases that can add fucose to the oligosaccharide core. However, their ability to extend these structures to produce complex side chains like those found in mammalian cells remains to be determined. To begin to examine this pathway at the molecular genetic level, we isolated and characterized a class II alpha-mannosidase (alpha-mannosidase II) cDNA from Sf9, a lepidopteran insect cell line. In mammalian cells, this enzyme catalyzes the committed step in the pathway converting N-linked carbohydrates to complex forms. Degenerate primers against conserved regions in known class II alpha-mannosidase protein sequences were used to generate an alpha-mannosidase II-specific PCR product from Sf9 cell DNA. Sequence information from this product was used to isolate a partial cDNA clone, the 5' end was isolated by ligation-anchored PCR, and the full length alpha-mannosidase II cDNA was assembled. This cDNA contained a long open reading frame predicted to encode an 1130 amino acid protein with 37% identity to human Golgi alpha-mannosidase II and with a type II membrane topology, a feature of all known Golgi processing enzymes. Southern blotting indicated that alpha-mannosidase II is a single copy gene in Sf9 cells. Other Lepidoptera had related alpha-mannosidase II genes, but there was variation among different genera, and the Sf9 alpha-mannosidase II cDNA did not cross-hybridize with DNA from animals outside Lepidoptera. Steady-state levels of alpha-mannosidase II RNA were low in uninfected Sf9 cells and even lower after baculovirus infection. The in vitro-translated Sf9 alpha-mannosidase II protein had the expected size and was translocated and N-glycosylated by microsomal membranes. Expression of the Sf9 alpha-mannosidase II cDNA in the baculovirus system produced large amounts of a protein with the expected size and swainsonine-sensitive alpha-mannosidase II activity towards an aryl-alpha-mannoside substrate. These results demonstrate that Sf9 cells encode and express an alpha-mannosidase II with properties similar to those of the mammalian enzyme.

Influence of different signal peptides and prosequences on expression and secretion of human tissue plasminogen activator in the baculovirus system.

Foreign secretory pathway proteins are often produced in surprisingly low amounts in the baculovirus/insect cell expression system. One possible reason for this is that heterologous signal peptides might be inefficiently recognized by the insect cell protein translocation machinery. This idea was supported by a recent study showing that secretion of a plant protein in the baculovirus system was enhanced when its signal peptide was replaced with an insect-derived signal peptide (Tessier, D. C., Thomas, D. Y., Khouri, H. E., Laliberte, F., and Vernet, T. (1991) Gene (Amst.) 98, 177-183). We have extended these observations by measuring the effects of different signal peptide and signal peptide-prosequence combinations on baculovirus-mediated expression and secretion of human tissue plasminogen activator (t-PA). Replacement of the native prepropeptide with signal peptides from a lepidopteran insect secretory protein (cecropin B), a major baculovirus structural glycoprotein (64K), or an abundant, highly conserved lumenal protein of the rough endoplasmic reticulum (GRP78/BiP, a 78-kDa glucose-regulated protein/immunoglobulin heavy chain-binding protein), had no significant effect on t-PA expression or secretion. The same results were obtained with the signal peptide from honeybee prepromellitin, which was able to enhance secretion of plant propapain (Tessier et al., 1991 (above)). Similar results were obtained when heterologous signal peptides were combined with the native prosequence or when the intact cecropin B preprosequence was used. Translational initiation at an upstream, in-frame ATT, which could functionally inactivate any signal peptide, did not explain the low efficiency of t-PA secretion. Finally, deletion of the native signal peptide, prosequence, or both, failed to increase t-PA production. These results showed that insect-derived signal peptides and/or prosequences cannot always enhance the expression and/or secretion of foreign secretory pathway proteins in the baculovirus system. They also suggested that the inability of insect cells to recognize the processing signals in human t-PA efficiently is probably not the major factor preventing its high level production in this system.

Enhancement of polyhedrin nuclear localization during baculovirus infection.

Polyhedrin is the major component of the nuclear viral occlusions produced during replication of the baculovirus Autographa californica multicapsid nuclear polyhedrosis virus (AcMNPV). Since viral occlusions are responsible for the horizontal transmission of AcMNPV in nature, the biosynthesis, localization, and assembly of polyhedrin are important events in the viral replication cycle. We recently defined the sequence requirements for nuclear localization and assembly of polyhedrin. In this study, we examined the localization of polyhedrin at different times of infection. The results showed that nuclear localization of polyhedrin becomes more efficient as the occlusion phase of infection progresses. Several different factors were identified that might contribute to this overall effect, including a higher rate of polyhedrin nuclear localization and a higher rate of polyhedrin biosynthesis. We also examined the biosynthesis and processing of polyhedrin in cells infected with an AcMNPV few polyhedra (FP) mutant, which produces smaller numbers of viral occlusions that contain few or no virions. Compared with wild type, the FP mutant produced polyhedrin more slowly and localized it to the nucleus less efficiently at the beginning of the occlusion phase of infection (24 h postinfection). This supported the idea that the efficiency of polyhedrin nuclear localization is tightly coupled to its rate of biosynthesis. It also revealed that expression of the viral 25K gene, which is inactivated in the FP mutant, is directly or indirectly associated with an enhancement of polyhedrin biosynthesis and nuclear localization at the beginning of the occlusion phase of infection. This enhancement effect appears to be necessary to ensure the normal assembly of viral occlusions.

Requirements for nuclear localization and supramolecular assembly of a baculovirus polyhedrin protein.

This study defines the requirements for the nuclear localization, stable nuclear association, and supramolecular assembly of a baculovirus polyhedrin protein in lepidopteran insect cells. Fragments of the polyhedrin protein were genetically fused to two different nonnuclear reporter proteins and the intracellular distribution of the fusion proteins was analyzed in infected insect cells. Analysis by indirect immunofluorescence showed that the domain between amino acids 30 and 57 could mediate nuclear localization of polyhedrin. However, biochemical fractionation experiments showed that this domain was not sufficient for a detergent-stable association of polyhedrin with the nucleus. This required a slightly larger domain, between amino acids 30 and 110. Differential interference-contrast microscopy showed that the supramolecular assembly of polyhedrin into nuclear occlusion-like particles required the domain between amino acids 19 and 110. The most likely candidate for a minimal nuclear localization signal was the sequence KRKK, located between amino acids 32 and 35. Therefore, oligonucleotide-directed mutagenesis was used to change this sequence to NGNN and the intracellular distribution of the mutant protein was analyzed. The results showed that the mutant protein was predominantly localized in the cytoplasm of infected cells, where it assembled into large, cubic, occlusion-like particles. Thus, the KRKK sequence is necessary for the nuclear localization of polyhedrin, but nuclear localization is not required for its supramolecular assembly into occlusion-like particles.