Characterization of virus-like particles produced by a recombinant baculovirus containing the gag gene of the bovine immunodeficiency-like virus

VIROLOGY

178,435-451

(1990)

Characterization Containing LYNN RASMUSSEN, Laboratory

of Virus-like Particles Produced by a Recombinant the gag Gene of the Bovine Immunodeficiency-like

JANE K. BAfTLES,

of Cell and Molecular

Structure,

Program Received

WILLIS H. ENNIS, KUNIO NAGASHIMA, Resources, February

Inc., NC/-Frederick 13, 1990; accepted

Cancer May

Research

Baculovirus Virus’ MATHEW

AND Faci/ty,

Frederick,

A. GONDA’ Maryland

21701

2 7, 1990

The entire gag gene of the bovine immunodeficiency-like virus (BIV) was inserted behind the strong polyhedron promoter of Autographa californica nuclear polyhedrosis virus (AcNPV). The resultant recombinant baculovirus (AcNPVBIVBBg) was used to infect insect cells in order to overexpress and characterize BIV gag gene products. The infection resulted in the high-level expression of a protein similar in size to the predicted BIV gag precursor (Pr53gng). BIV Pr53g”9 was detected in AcNPV-BIVgW-infected insect cells and in culture supernatants. Electron microscopy of these cells revealed an abundance of virus-like particles (VLPs) in the cytoplasm, budding from the cell membrane, and free in the culture medium. The size and morphology of the VLPs were similar to those of the immature forms of BIV observed in infected mammalian cells. The VLPs sedimented at a density of 1 .16 g of sucrose per milliliter in linear gradients and were shown to contain the majority of the supernatant Pr53 gW. Antigenic determinants on Pr53gW from VLPs were recognized by BIV and HIV-1 antiserum, and serum from rats immunized with VLPs reacted with recombinant and viral BIV Pr53gag and processed products. The protease (PR) activity in BIV virions was capable of processing recombinant Pr53gag; this activity was blocked by pepstatin A, a potent aspartyl PR inhibitor. Baculovirus-expressed BIV Pr53gag appears to be an excellent source of gag precursor; it may prove useful for structural studies and enable the development of assays to detect retroviral PR inhibitors. The data further suggest that unprocessed BIV Pr538w plays a major role in the assembly of BIV particles. The expression of other BIV structural genes in insect cells may prove instructive in the study of molecular events involved in the assembly and processing of these BIV proteins. M 1990 Academic Press, Inc.

INTRODUCTION The bovine immunodeficiency-like virus (BIV) is an exogenous, nononcogenic, infectious retrovirus that is purported to cause lymphadenopathy, lymphocytosrs, central nervous system lesions, weakness, and emaciation in cattle (Van Der Maaten et a/., 1972). It is sporadically found in cattle herds in the U.S. BIV has the morphology of a lentivirus and a reverse transcriptase (RT) with a preference for Mg’+ cations. It shares immunologic cross-reactivity in the major capsid protein with the human immunodeficiencyvirus type 1 (HIV-l), simian immunodeficiency virus (SIV), and equine infectious anemia virus (EIAV) (Gonda et a/., 1987). Sequence determinations for the highly conserved RT domain of the pol gene demonstrate that BIV is a unique member of the lentivirus subfamily of retroviruses (Gonda et a/., 1987; Garvey et al., 1990; reviewed in Gonda et al., 1990). Two biologically active proviruses of BIV have been molecularly cloned (Braun era/., 1988) and sequenced

(Garvey et a/., 1990). The genomic organization of BIV is most similar to that of the primate lentiviruses; it has gag, PO/, and env genes and, in addition, five putative nonstructural/regulatory genes in or overlapping the central region between pal and env. The nonstructural/ regulatory gene products of lentiviruses are not known to be Incorporated into the virus particle, but are believed to play an important role in their pathogenesis (Haseltine, 1988). While a significant amount of knowledge has been amassed concerning the structure, assembly, and processing of the structural gene products of HIV-1 and its lentivirus relatives (Vigne et a/., 1982; Casey et a/., 1985; Robey et al., 1985; Henderson et al., 1987; Hussain et al., 1988; Mervis er al., 1988; Lillehoj et al., 1988; Veronese et al., 1988; Erickson-Witanen et al., 1989; Peng et a/., 1989), comparatively little is known about BIV, other than what has been deduced from the DNA sequence of the functional BIV clones (Garvey et al., 1990) or experimentally determined using polyvalent sera to BIV (Gonda et al., 1987; Battles et al., manuscript in preparation). The use of broadly reactive sera prepared to whole virus or obtained from virusinfected animals is useful In the initial serological characterization of putative viral proteins. However, be-

’ The U.S. Government’s right to retain a nonexcluslve royalty-free license In and to the copyright covenng this paper, for governmental purposes, IS acknowledged. ‘To whorrl correspondence and reprint requests should be addressed.

435

0042-6822/90

$3.00

Copyrght D 1990 by Academic Press. Inc 411 rtqhts pi reproduction I” any form resewd

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RASMUSSEN

cause of the complexities associated with the expression and processing of retroviral proteins, these multivalent sera often do not permit the precise identification of individual viral subunit or precursor proteins. This identification is made all the more difficult in lentivirus-infected cells by the presence of additional nonstructural/regulatory proteins, some with molecular masses similar to those of the structural gene products. Alternative approaches to their identification and study are to physically isolate, chemically synthesize, or express in heterologous systems individual viral proteins for experimental study. Dissection of lentivirus genomes into defined subgenomic fragments for incorporation into recombinant baculoviruses or vaccinia viruses in order to overexpress individual gene products is one method that has recently proved to be very instructive (Madisen et al., 1987; Delchambre et al., 1989; Gheysen et al., 1989; Karacostas et al., 1989; Over-ton et a/., 1989). To better characterize the individual gene products of BIV and to discern their role in the morphogenesis and assembly of the virus particle, we have utilized the strong polyhedron gene promoter, in a baculovirus-insect cell system, to express open reading frames (ORFs) predicted to be BIV structural genes. In this report, we describe the development of a recombinant baculovirus containing the entire gag ORF of BIV. When insect cells are infected with this baculovirus, insoluble particulates are synthesized and secreted by the cells; these particles resemble, in most respects, immature BIV virions and contain the putative BIV gag precursor (Pr53gag). The baculovirus-expressed BIV Pr53g”g behaves antigenically and structurally like native viral protein and has been further used in in vitro assays to characterize the proteolytic activity in purified BIV virions. MATERIALS Viruses

AND

METHODS

and cells

Spodoptera frugiperda (Sf-9) insect cells (Brown and Faulkner, 1977) were used for all baculovirus experiments. Sf-9 cells were maintained as suspension cultures in supplemented Grace’s insect medium (Gibco) containing 7.5% fetal bovine serum (FBS) (HyClone) and gentamicin sulfate (50 pug/ml) at 23-27”. Wild-type Autographa californica nuclear polyhedrosis virus (AcNPV) and recombinant AcNPVs were propagated in Sf-9 cells. Terminal dilution assays, to isolate recombinant baculoviruses, were performed in 96-well, flatbottom tissue-culture plates (Costar), according to previously published methods (Summers and Smith, 1986).

ET AL

BIV was grown in a bovine cell line (BLAC-20) established from the long-term culture of bovine leukocytes in this laboratory. The cultures were maintained in Dulbecco’s modified Eagle’s high glucose medium (Gibco) supplemented with 10% FBS, 2 mn/l L-glutamine, and antibiotics in a humidified atmosphere with 5% CO, at 37”. DNA manipulations All plasmid DNA manipulations were carried out essentially as described by Maniatis et al. (1982). DNAmodifying enzymes were used according to the manufacturer’s protocols. Wild-type AcNPV was obtained from low-speed-clarified culture fluids of AcNPV-infected Sf-9 cells, and AcNPV DNA for cotransfections was purified according to procedures previously described (Summers and Smith, 1986). Construction of AcNPV-transfer containing BIV gag gene

vector

The identification of the gag ORF in the sequence of BIV has previously been reported (Garvey et a/., 1990). Figure 1 diagrams the procedures used to isolate and clone the BIV gag gene into the AcNPV-transfer vector pVL941 (Luckow and Summers, 1989). The pBlVlO6Smaplasmid (Braun et al., 1988), containing a 9.6-kb insert in the Smal site of the vector pBluescript (Stratagene), which represents most of the proviral genome of BIV, was digested with Smal and Accl. The ends were made flush with Klenow fragment. The modified 1.5kb Smal-Accl fragment contains all of the gag gene starting at the 5’ end of the gag ORF and a very short segment [55 nucleotides (nt)] of the BIV protease (PR) gene, in thepo/ORF, which overlaps but is in a different reading frame than the gag ORF. Accl digestion cleaves the BIV-PR DNA immediately upstream of a region that codes for the amino acid sequence Asp-ThrGly, which aligns with the aspartyl PR active site of HIV1 (Seelmeier et al., 1988; Garvey et al,, 1990; Gonda et a/., 1990). The pVL941 transfer vector was prepared for subcloning by digesting it with BamHl and filling in the ends with Klenow fragment. The blunt-ended vector was dephosphorylated with bacterial alkaline phosphatase and ligated to the 1.5-kb BIV gag fragment with T4 ligase. The ligated mixture was used to transform the HB 10 1 strain of Escherichia co/i and the resultant colonies were lifted on nylon membranes. Recombinants containing BIV gag sequences were detected by hybridization with a BIV-specific 32P-labeled probe, according to the method of Southern (1975). A recombinant plasmid containing the gag gene in the proper ori-

BACULOVIRUS

EXPRESSION

entation for expression, relative to the AcNPV strong polyhedron promoter, was selected for cotransfection with wild-type AcNPV DNA.

OF

BIV gag

rescence microscopy and photographed Tri-X Pan film, ASA 400. Electron

Isolation of recombinant AcNPVs containing BIV gag inserts A recombinant AcNPV containing the BIV gag gene was derived by cotransfecting Sf-9 cells with purified AcNPV DNA and the pVL941 transfer vector containing the BIV gag gene insert, as previously described (Over-ton el al., 1987). Sf-9 cells, seeded in 96-well plates, were infected with terminal dilutions of virus and screened for recombinant DNA by dot-blot hybridization using a BIV-specific 3zP-labeled DNA probe (Summers and Smith, 1986). Antisera to BIV and baculovirus-expressed BIV Pr53gag Rabbit antiserum was made to detergent-disrupted, purified BIV virions as previously described (Gonda et a/., 1987). Bovine sera were obtained from a calf experimentally infected with BIV and from specific pathogenfree [BIV, bovine leukemia virus (BLV), bovine syncytial virus, and bovine viral diarrhea virus] control animals (kindly provided by M. J. Van Der Maaten). Antiserum to AcNPV-BIVg”g-expressed BIV Pr53gag was raised in rats. The inoculum consisted of detergent-disrupted, sucrose-gradient-purified, insoluble particulates containing BIV Pr53yag. The disrupted antrgen preparation was administered in complete Freund’s adjuvant for the first inoculation and incomplete Freund’s adjuvant for all subsequent inoculations. The antigen concentration was 50 pg/ml, and four 0.5-ml inoculations were delivered subcutaneously at 2-week intervals, at multiple sites. The animals were exsanguinated, and the blood was pooled and processed for serum 3 weeks following the final inoculation. immunofluorescence

Uninfected or AcNPV-BIVgag-infected Sf-9 cells were seeded onto glow-discharged glass slides and incubated at 27” for 4 hr to allow the cells to adhere. The cells on slides were dried at 65”, and then fixed and extracted in a 1 :l mixture of 100% acetone: 100% methanol for 6 min. The slides were incubated with a diluted rabbit anti-BIV serum for 0.5 hr at room temperature. The primary antibody was detected by incubating cells on slides for 0.5 hr at 23” with a diluted fluorestein-labeled affinity-purified goat anti-rabbit IgG (Kirkegaard and Perry). The prepared slides were viewed with a Leitz Orthoplan microscope equipped for fluo-

437

using Kodak

microscopy

Virus-infected cells were prepared for thin-section electron microscopy as previously described (Gonda et a/., 1985). Thin sections were stained with uranyl acetate and lead citrate; stained sectrons were viewed and photographed using a Hitachi H-7000 electron microscope operated at 75 kV. For immunoelectron mrcroscopy, uninfected Sf-9 and Sf-9 cells infected with wild-type AcNPV or AcNPV-BIVgay were fixed with phosphate-buffered saline (PBS), pH 7.2, containing 4% formaldehyde and 0.05% glutaraldehyde. Fixed cells were rinsed In a 50 mM ammonium chloride solution, dehydrated with two washes of 70% ethanol, and infiltrated in a 1: 1 mixture of 100% ethanol and LR White resin (Polysciences, Inc.) prior to final embedding tn pure resin. Thin sections (80 nm) were cut from blocks polymerrzed at 60” and mounted on 300.mesh nickel grids for immunostaining. Prior to immunostarnrng, thin sections on grids were incubated with PBS containing 1% bovine serum albumin (BSA) to block nonspecrfic adhesion of antibodies to the embedded tissue and resin. PBS containing 1% BSA and 0.5% Tween 20 was used as a wash buffer after each labeling step. BIV gag antigen was specifically localized with a polyclonal rabbit antrserum made to BIV (Gonda et al., 1987) and diluted 1 :lO to 1:50. Binding of the primary antibody to thin sections was detected with 10 nm colloidal gold-labeled, affinity-purified goat anti-rabbit IgG used according to the manufacturer’s directrons (Janssen Biotech). The virus structure in immunostarned sections was enhanced with uranyl acetate and lead citrate as it was in thin sections used in morphologic analyses (Gonda et a/., 1985). Radiolabeling

Indirect

GENE

of cells and viruses

Uninfected Sf-9 and Sf-9 cells Infected with wild-type AcNPV or AcNPV-BIVg”” were metabolically labeled (20-24 hr postinfection) with 100 &i/ml each of 35Slabeled cysteine and methionrne (Amersham). Labeled cells were lysed by adding 400 mM NaCI, 1 mM EDTA, 50 mM Tris (pH 8.0), containing 1% Triton X-100, 10 pug/ml ol-2-macroglobulin, and 10 @g/ml aprotinin (NET lysis buffer); lysates were clarified at 16,000 gfor 2 min in a mrcrofuge (Eppendorf). BIV-infected and uninfected BLAC-20 cells were metabolically labeled with 35S-labeled cysteine and methtonine when maximum cytopathic effects (syncytrum formation) were evident, as described elsewhere (Battles et al.. manuscript In

438

RASMUSSEN

preparation). 35S-labeled BLAC-20 lysates were prepared as for radiolabeled Sf-9 cells described above. The supernatants from metabolically labeled uninfected and BIV-infected BLAC-20 cells were saved for recovery of mock virus and BIV virions, respectively. BIV virions were concentrated by pelleting through a 20% (v/v) glycerol cushion for 1 hr at 100,000 g; the virions were lysed on ice in NET. In experiments to determine if BIV Pr53gag was myristoylated, AcNPV-BIVg’g-infected Sf-9 cells were radiolabeled with 3H-labeled myristic acid, according to previously published methods (Over-ton et al., 1989). Radioimmunoprecipitation

and gel electrophoresis

Radioimmunoprecipitations were performed, as described in detail by others (Jones, 1980; Smith and Pifat, 1982; Battles et al., manuscript in preparation). Briefly, 35S-labeled cell lysates were incubated with antisera. Rabbit anti-bovine IgG was added as a secondary antibody to reactions containing bovine antisera. Immune complexes were precipitated by the addition of protein A Sepharose beads (Sigma). Precipitated proteins were resolved by SDS-polyacrylamide gel electrophoresis (PAGE). Sucrose-gradient Pr53ga%ontaining

purification particles

and analysis

of

Insoluble pat-ticulates containing BIV Pr53gag were purified from the culture fluids of AcNPV-BIVgag-infected Sf-9 cells by adding polyethylene glycol 6000 and NaCl to final concentrations of 8 and 2.370, respectively, and stirring at 4” overnight. The protein precipitate containing Pr53g”g was collected by centrifugation at 9000 g, resuspended in 10 ml\/l Tris (pH 8.0) 100 mM NaCI, and 1 mlVI EDTA (TNE), and layered onto a 20-600/o linear sucrose gradient made in TNE (Benton eta/., 1978). Particulates were sedimented through the gradients at 100,000 g for 1.5 hr. Fractions (0.5 ml) were collected and analyzed for protein concentration and antigen content using ELISA and Western blot, as described below. Additional cultures of Sf-9 cells infected with AcNPV-BIVgag at a multiplicity of 10 were grown in the presence of 35S-labeled cysteine and methionine for use in experiments requiring radiolabeled Pr53gag. The 35S-labeled particles were harvested and purified in the same manner as described above for unlabeled particles. The band sedimenting at 1.16 g of sucrose per milliliter was collected and stored at -20” until use. Protein

quantitation

The protein content of purified particulates containing Pr53gag was determined with the BCA protein assay

ET AL.

reagent (Pierce Chemical Co.) according to protocols recommended by the manufacturer. Absorbance of the reaction was read at 550 nm on a spectrophotometer. The values obtained were compared to a standard curve created from known concentrations of BSA. BIV antigen

detection

by ELISA and Western

blot

BIV virions or particulates containing Pr53g”g were dissociated by adding 1o/oTriton X-l 00 and incubating at 23“ for 30 min with agitation. Dissociated antigen was diluted in coupling buffer (100 mM sodium bicarbonate, pH 9.6); dilutions (100 PI/well) were dispensed into a 96-well EIA plate (TitertekILinbro) and incubated at 4” overnight. The antigen-containing wells were washed in PBS with 0.02% Tween 20 (wash buffer) and blocked with 0.5% human serum albumin in PBS (350 PI/well) for 1 hr at 23”. Wells were cleared with wash buffer (lx), and a diluted serum (100 rJ/well) was added and incubated for 2 hr at 37”. Unreacted antibodies were removed with wash buffer (5X) and a diluted horseradish peroxidase-labeled goat anti-rabbit IgG (Kirkegaard and Perry) (100 PI/well) was added and incubated for 2 hr at 37”. The plates were cleared with wash buffer (5X), and the peroxidase substrate diammonium 2,2’-azino-bis[3-ethylbenzothiazoline-6-sulfonate] (ABTS) (Kirkegaard and Perry) was added. The optical density was determined on a vMAX plate reader (Molecular Devices Corp.) using a single wavelength at 405 nm. For Western blots, BlVvirion proteins or recombinant Pr53gag from sucrose-gradient-purified particles were resolved by SDS-PAGE and electrotransferred onto nitrocellulose membranes, according to methods previously described (Burnette, 1981). The antisera used to detect BIV proteins in Western blots were a rat antiBIV Pr53gag and rabbit anti-BIV. Antibodies bound to BIV proteins were detected with 35S-labeled protein A. Goat anti-rat IgG was added as an intermediate step to membranes incubated with rat serum to enhance the binding of protein A. Assay to detect proteolytic in purified BIV virions

activity

The proteolytic activity in purified preparations of BIV virions was measured using the recombinant BIV gag precursor as a substrate. Pr53gag-containing particles labeled with [35S]cysteine and -methionine were disrupted with 2% NP-40 to make the Pr53gag accessible to proteolytic cleavage; BIV virions were similarly disrupted to release their proteolytic activity (Yoshinaka et a/., 1985). Keeping the substrate concentration and temperature constant (23”) three parameters were

BACULOVIRUS

EXPRESSION

OF

BIV gag

GENE

439

evaluated: input of proteolytic activity, measured as the concentration of lysed BIV; incubation time; and the ability of an aspartyl PR inhibitor, pepstatin A (Seelmeier et a/., 1988), to interfere with the proteolytic activity in BIV virions. The ability of PRs in the lysed-virus preparation to process BIV Pr53gag was assessed by subjecting the reaction mixture to SDS-PAGE. PR activity was determined by visually inspecting autoradiographs of gels for a reduction in the 35S-labeled BIV precursor band and the appearance of putative processed-precursor products. A panel of heterologous retroviruses [BLV, EIAV, caprine arthritis-encephalitis virus (CAEV), visna virus, and HIV-l] were also tested for their ability to cleave Pr53g”g. RESULTS AcNPV-BIVgag

expression

in insect cells

The pVL941 transfer vector was constructed to enable the synthesis of nonfused foreign proteins by inactivating the single ATG start site downstream of the polyhedron promoter and prior to the BamHl cloning site (Luckow and Summers, 1989). Thus, it is necessary for the foreign gene inserted in pVL941 to supply its own ATG start site. It was not necessary to reconstruct this signal sequence for the BIV gag ORF, since a strong ATG start signal (Kozak, 1986) was situated 3 nt downstream of the 5’Smal site (Fig. 1) (Garvey et al., 1990). A recombinant baculovirus, AcNPV-BIVgag, was recovered from Sf-9 cells cotransfected with DNA from wild-type AcNPV and the pVL941 transfer vector containing the entire BIVgag ORF. This recombinant baculovirus was purified to homogeneity (Summers and Smith, 1986), and working stocks of AcNPV-BIVgag were prepared. To determine whether translation could be efficiently initiated from the ATG contributed by the BIV gag gene, AcNPV-BIV~“~ was used to infect Sf-9 cells. Antigen production in infected cells was grossly assessed using a polyvalent antiserum to BIV in indirect immunofluorescence experiments (Fig. 2). BIV antigen was readily detected in >90% of Sf-9 cells infected with AcNPV-BIVgag for more than 48 hr (Figs. 2a and 2b) but not in uninfected cells (Fig. 2~). The immunoreactivity was localized both in the cytoplasm and around the cell surface membrane. While the cytoplasmic fluorescence was primarily diffuse (Fig. 2a), fluorescence at the cell surface was particulate, suggesting an aggregation of the expressed viral protein (Fig. 2b). Electron microscopy insect cells

of AcNPV-BIVgag-infected

Recently, studies using recombinant baculoviruses have shown that the gag genes of HIV-1 and SIV are

FIG. 1. Diagram of the baculovirus gene. For details,

outlInIng the cloning strategy transfer vector that contains see Materials and Methods

for the constructbon the entire BIV gag

efficiently expressed in insect cells and that insoluble particulates containing the gag gene precursors, which resemble retrovirus particles by electron microscopy, are formed (Delchambre et al., 1989; Gheysen et a/., 1989). Electron microscopy was used to examine the particulate nature of the recombinant baculovirusexpressed BIV gag gene products in insect cells (Figs. 3 and 4). At low magnification, an abundance of VLPs resembling BIV were observed in the AcNPV-BIVgaginfected cells (Fig. 3). Baculovirus particles were also produced; they were found predominantly in the nucleus and less frequently in the extracellular spaces. BIV VLPs formed at the plasma membrane, and there was also a surprising accumulation of intracellular particles (ICPs) in the cytoplasm but not in the nucleus of the infected cells (Figs. 3 and 4e). The appearance of ICPs in the AcNPV-BIVgag-infected insect cells was specifically related to BIV gag expression driven by the strong polyhedron promoter, rather than to AcNPV

440

RASMUSSEN

ET AL

The assembly and morphogenesis of VLPs in AcNPV-BIVgag-infected insect cells are consistent with those of a retrovirus. Early buds with a crescent nucleoid formed at the plasma membrane (Figs. 4a and 4b). The nucleoid became more concentric in advanced stages of the budding process (Fig. 4c) until the VLPs were released extracellularly (Fig. 4d). Extracellular VLPs were 100-l 20 nm in diameter and resembled immature BIV produced in mammalian cells (Gonda et al., 1987, 1990; Braun et a/., 1988; Boothe and Vat Der Maaten, 1974). The nucleoid of budding and extra cellular VLPs had the characteristic lentivirus strut ture, being more electron dense in the inner region of the core and having a less electron-dense region be tween the inner core and the outer membrane. The envelopes of these particles were smooth. No VLPs with bar- or cone-shaped cores representative of mature extracellular BIVforms were observed (Braun eta/., 1988; Gonda et a/., 1987, 1989, 1990). The ICPs resembled immature BIV cores, were present in >50% of infected cells, and appeared to form on cytoplasmic membranes studded with ribosomes (Fig. 4e); these were associated with either polysomes or areas of rough endoplasmic reticulum. The ICPs were also 100-l 20 nm in diameter. Electron microscopy of BIV from infected mammalian cells is shown for comparison in Figs. 4f4i. With the noted absence of mature extracellular forms, the BIV VLPs appeared to be morphogenetically identical to BIV and other lentiviruses (Boothe and Van Der Maaten, 1974; Gonda et al., 1985, 1987, 1989; Gelderblom et al., 1989). lmmunoelectron microscopy of AcNPV-BIVg”g-infected insect cells was used to demonstrate the specific incorporation of BIV gag antigen into the VLPs. Using a rabbit serum with reactivity to BIV gag products in indirect immunogold staining of thin sections, BIV VLPs free in the extracellular spaces and budding from the plasma membrane and ICPs in the cytoplasm were labeled with colloidal gold (Figs. 5A-C). Baculoviruses in AcNPV- or AcNPV-BIVgag-infected Sf-9 cells showed no specific staining with this rabbit serum. Physical FIG. 2. Fluorescence microscopy of AcNPV-BIVgag-infected insect cells. Rabbit anti-BlV serum was used to detect BIV-specific antigen in recombinant baculovrrusinfected cells. (a and b) Sf-9 cells In fected with AcNPV-BIVga4 (c) Uninfected Sf-9 cells. Note the diffuse intracellular staining in most cells in (a) and the speckled cell-surface staining in (b). Uninfected Sf-9 cells (c) were not reactive with BIV antiserum.

structural gene expression, as ICPs were not seen in AcNPV-infected insect cells (data not shown). Many BIV VLPs were found free in the supernatant.

characteristics

and purification

of BIV VLPs

To better understand the physical nature and content of the VLPs, supernatants from AcNPV-BIVgag-infected Sf-9 cells containing VLPs were sedimented through 20-60% sucrose gradients. Figure 6 shows an analysis of a typical gradient. Three distinct bands (designated top, middle, and bottom) and a pellet were apparent (Fig. 6A). Gradient fractions were collected and examined for protein concentration and BIV antigen by ELISA (Fig. 6B) and Western blot (Fig. 6C) using a polyvalent rabbit anti-BIV serum.

BACULOVIRUS

EXPRESSION

OF

BIV gag

GENE

441

FIG. 3. Electron microscopy of AcNPV-BIVgag-Infected Sf-9 cells. The electron mlcrograph shows AcNPV-BIV gag-lnfected Sf-9 cells at low magniflcatlon. Many free extracellular virus-like particles (VLPs) (100--l 20 nm In diameter) can be seen on the perimeter of the cell and tn a vacuole (V). The nuclear region has begun to degenerate and contains some nonoccluded baculovlruses (AC). In addltlon to the extracellular VLPs, there are many Intracellular particles (ICP) of the same size.

The gradient fractions containing peak protein concentrations occurred in the top band; fractions representing this band also showed a small amount of reactivity with anti-BIV serum by ELISA. The greatest BIV antigen content appeared in the middle (opalescent) band, which had a sucrose concentration of 1.16 g/ml;

this is the same density at whrch retrovirus particles sediment. The protein content of this band was 50% less than that of the top band, which suggested that what BIV antigen was there was more concentrated. Very little BIV antigen was detected In the bottom band, and none was evident in the pellet. Electron mrcro-

FIG. 4. Electron microscopy of the morphogenesis of VLPs in AcNPV-BIV g%nfected Sf-9 cells and BIV-infected bovine cells. (a-e) VLPs and/ or ICPs In AcNPV-BIVg%nfected Sf-9 cells. (f-i) BIV-infected bovine cells. (a and f) Early stage of budding. (b, c, and g) Progressively later stages of budding, (d and h) Extracellular VLPs and immature extracellular BIV, respectively. (i) Mature extracellular BIV with characteristic bar- or coneshaped core. (e) Accumulation of ICPs in Sf-9 cells infected with AcNPV-BIVgag. Arrows in (e) indicate the position of ribosomes surrounding the ICPs. 442

BACULOVIRUS

EXPRESSION

OF

BIV gag

GENE

443

FIG. 5. lmmunoelectron mrcroscopy of AcNPV-BIV Q?nfected Sf-9 cells. Thin sections of cells were labeled wrth a rabbrt antr-BIV serum wrth reactrvrty to BIV gag proteins (see results of serum in Fig. 7) tn Indirect rmmunogold starning. (A) Portion of cell showing abundance of BIV VLPs at the cell surface that are heavrly decorated with colloidal gold. The cytoplasm (Cyt) of the cell IS rndrcated. (B) ICPs In the pennuclear space decorated with collordal gold (compare to images of ICPs In Fig. 4e). (C) Sectron of immunostarned cell that has baculovrruses tn the cytoplasm and BIV VLPS at the cell surface. The VLPs are labeled with collordal gold whereas the baculovrrus particles (AcNPV) are not.

RASMUSSEN

444

ET AL.

Processing and antigenic produced in insect cells

B 3.5 3.0

0 = Protein

Cont.

‘.O f

2.5 0.6

=E 2.0 P 6 d

0.6

1.5 1.0

g 5 = e 5 e 8

0.4 2 0.5

i? 0

0.0

1

3

5

7

9

11

13

15

17

19

21

23

5

7

9

11

13

15

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23

o.2

Fraction

3

Number

FIG. 6. Sucrose-gradient purification and physical characterization of VLPs from supernatants of AcNPV-BIVgag-infected insect cells. VLPs in the supernatants of Sf-9 cells infected by AcNPV-BIVgag were harvested and layered onto a 20-60% sucrose gradient, as described under Materials and Methods. The VLPs were centrifuged at 100,000 g for 90 min; 0.5.ml fractions were collected for analysrs. (A) Photograph of 20-600/o linear gradient after centrifugation at 100,000 g. (B) Plots of protein concentration (0) and ELISA results using a rabbit anti-BIV serum (0) to locate fractions containing BIV Pr53gag from VLPs in the gradient. (C) Western blot analysis of gradient fractions using rabbit anti-BIV serum. BIV virions were included in the Western blot as a positive control (c) for the rabbit antiserum. The lbcations of virion-derived BIV Pr53gag and CA (~26) on the control strip are indicated and serve as molecular weight markers for identifying the recombinant BIV Pr53gagcontained In the VLPs. Three major bands were resolved in the gradrent and were identified as the top (T), middle (M). and bottom (B), as indicated. The middle band, which contained the majority of the VLPs and BIV Pr530ag, had a density of 1.16 g/ml of sucrose.

scopic examination of material from the three bands conclusively demonstrated that the majority of VLPs migrated in the middle opalescent band (data not shown). Western blots (Fig. 6C) confirmed the ELlSA and electron microscopy data and further identified a protein identical in size to that of the unprocessed BIV gag precursor, BIV Pr53g”g (Garvey et a/., 1990; Battles et al., manuscript in preparation), as the major immunoreactive constituent of the VLPs.

of BIV Pr53gag

The gag precursor of lentiviruses is a polyprotein consisting of matrix (MA), capsid (CA), and nucleocapsid (NC) functional regions (standard nomenclature for retrovirus proteins proposed by Leis er al. (1988)). The gag precursor is processed into its mature internal structural subunits by the viral PR in the virus particle after budding. The MA, CA, and NC proteins (~17, 26, and 14, respectively) of BIV have been serologically recognized in BIV-infected cells and virions (Gonda et al., 1987; Battles eta/., manuscript in preparation). Several additional proteins of 45-47 kD (p45-47), intermediate in size to the precursor and mature processed gag proteins of BIV, have been detected by radioimmunoprecipitation in BIV-infected bovine cells but not in virions (Fig. 7). These intermediates are believed to be gag related (Battles et al., manuscript in preparation); however, they are also in the predicted size range of the transmembrane protein of the env gene (Garvey et al., 1990), which interferes in their identification by

,.1..11..,,111111,,.11.,,

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properties

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; Rabbit SWZmZ-

Anti-W

r

_

3

7 9

1

2

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FIG. 7. Characterization of baculovirus-expressed BIV Pr53gag and BIV proteins by radroimmunoprecipitation. Cells used in the labeling experiments were AcNPV-infected and AcNPV-BIVgag-infected Sf-9 cells, and uninfected and BIV-infected BLAC-20 cells. Cells and viruses were metabolically labeled with [?S]cysteine and -methionine. as described under Materials and Methods. (Lanes l-6) Proteins precipitated with rabbit anti-BIV serum; (lanes 8-13) bovine antr-BIV serum; and (lanes 15-l 7) rabbit anti-HIV-l serum. (Lanes 7 and 14) blank. (Lanes 1 and 8) Proteins precipitated from AcNPV-infected Sf9 cells; (lanes 2,9, and 15)AcNPVBIV @Qnfected Sf-9 cells; (lanes 3 and 10) uninfected BLAC-20 cells; (lanes 4, 1 1, and 16) BIV-infected BLAC-20 cells; (lanes 5 and 12) mock virions; (lanes 6, 13, and 17) BIV virions. BIV proteins [p174 (gag-pol polyprotein), gplO0 (SU), p53 (Pr53gag), CA (~26). MA (17) and NC (pl4)] precipitated by the various antisera are designated on the left of the figure. The dots emphasize the location of virus-specific single or double protein bands that are difficult to resolve in an average exposure, as shown. Molecular werght markers are indicated on the right side of the figure.

BACULOVIRUS

EXPRESSION

some polyvalent sera having BIV gag and env protein reactivity. Moreover, it has not been determined whether (1) the ~45-47 proteins represent authentic intermediate products in the cleavage of the gag precursor by the viral PR or (2) gag gene products that might have resulted from alternate translation initiation sites downstream of the first ATG in the BIVgag ORF (Garvey et a/., 1990) as proposed for HIV-1 intermediates (Mervis et a/., 1988). The synthesis of BIV Pr53gag by a recombinant baculovirus in insect cells in the absence of other viral genes has allowed us to investigate the identity of the ~45-47 proteins in more detail. Infection of insect cells with AcNPV-BIVgag results in the overexpression of the BIV gag precursor, the formation of ICPs, and the budding of VLPs that are released into the supernatant. AcNPV-expressed BIV Pr53ga” is readily recognized in SDS-PAGE of radioimmunoprecipitations using polyclonal antiserum made to lysed BIV virions or antiserum produced in response to BIV infection (Fig. 7). These antisera recognize BIV surface (SU) envelope (gpl 00), CA, NC, and/or MA proteins in addition to the gag precursor, putative gag intermediates, and the gag-pol polyprotein (~174) present in either BIV virions or BIV-infected bovine cells (Fig. 7) (Battles et al., manuscript in preparation). We intentronally excluded the BIV PR coding sequences from the recombinant baculovirus and did not, therefore, anticipate that in the absence of a PR, the synthesized Pr53g”g would be further processed to mature structural proteins in insect cells or the VLPs. An unprocessed BIV precursor, along with several minor bands that appear to be derived from baculovirus or Sf-9 cells, were resolved by SDS-PAGE analysis of VLPs from sucrose gradient-purified supernatants of AcNPV-BIVgag-infected Sf-9 cells; the minor bands were not immunoreactive with anti-BIV serum (data not shown). The initial Western blots of gradient-purified material (Fig. 6C) suggested that the baculovirus-expressed BIV precursor found in the supernatants was not processed into mature protein subunits, and no proteins in the size range of the MA, CA, NC, or putative gag intermediates (~45-47) were resolved in immunoprecipitates of AcNPV-BIVgag-infected insect cells (Fig. 7). From these data we conclude that the first ATG in the gag ORF is the primary start site for initiation of translation and that PRs present in AcNPV-BIVgag-infected cells are not effective in cleaving the BIV precursor rn insect cells. The BIV CA protein and gag precursor are immunologically recognized by polyclonal antisera to HIV-1 on Western blots; this immunologic relationship is reciprocal (Gonda eta/., 1987). To further determine if the baculovirus-expressed BIV precursor antigenically be-

OF

BIV gag

GENE

445

\\\

lo-

! *

051 00. 04

-?i?x\ y 0.8

I -i 16

32

64

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Serum

I, 256 Dilutions

L; d AJ 5121024204840968192

-

(x10')

FIG. 8. ELISA of rat antlsera made to BIV VLPs containing Pr53g”g. Serologic analyses were performed using BIV whole (0, 0) and VLPs (Pr53gag) (A, A) in seDarate ELlSAs

BIV virus

haves like viral proteins made in mammalian cells, and to provide additional evidence for the precursor origins of the gag intermediates, radiolabeled AcNPV-BIVyaginfected insect cells were lysed and immunoprecipitated with anti-HIV-l serum; the immunoprecipitates were resolved on SDS-PAGE (Fig. 7). The BIV CA protein, gag precursor and putative intermediates, and gag-pol polyprotein were all recognized by the HIV-1 antiserum in lysates of bovine cells infected with BIV, and the recombinant BIV Pr53g”Q was detected in lysates of AcNPV-BIVgag-infected insect cells. The data derived from this experiment show that the recombinant BIV Pr53g”g is recognized by antibodies to epitopes conserved on the HIV-l and BIV CA protein. Furthermore, these findings suggest that the putative p4547 proteins are intermediates in the cleavage of the BIV gag precursor, since they contain the BIV CA protein. A myristic acid residue is believed to be required for the transport of the gag precursor of several retroviruses, including HIV-l, to the cell membrane and thus may play an important role in the formation of the virus particle (Rein eta/., 1986; Mervis eta/., 1988; Veronese e2 al., 1988; Gottlinger et a/., 1989). Insect cells are known to carry out a number of co- or post-translational modifications, including fatty acid acylation (Luckow and Summers, 1988; Delchambre et al., 1989; Gheysen et al., 1989; Over-ton et al., 1989). To determine if BIV Pr53gaQ could be myristolyated, AcNPV-BIVg”“infected Sf-9 cells were radiolabeled with 3H-labeled myristic acid (Over-ton et al., 1989). We were unable to detect a myristoylated Pr53g”g protein in the AcNPV BIVgag-infected insect cells (data not shown). Antibodies to recombinant authentic BIV proteins

BIV Pr53gag recognize

Gradient-purified BIV VLPs were used to raise antisera in rats. We anticipated that srnce the BIV Pr53g’g

446

RASMUSSEN

precursor protein carries epitopes for CA, MA, and NC polypeptides, it would be a good immunogen for deriving a reagent to characterize the mature viral proteins. The BIV gag precursor titers of the rat anti-BIV serum were assessed in BIV whole virus and recombinant VLP (Pr53gag) ELlSAs; endpoint titers of 2.5 X 1 O3 and 4.1 X 1 O3 were obtained in the whole virus and recombinant VLP ELlSAs, respectively (Fig. 8). SDS-PAGE of radioimmunoprecipitations of uninfected, AcNPV-infected, and AcNPV-BIVgag-infected Sf-9 cells using the rat anti-Pr53gag serum indicated the presence of AcNPV proteins as well as Pr53gag in the inoculum, since the rats produced antibodies to AcNPV and Pr53gag (Fig. 9A). The AcNPV reactivity could easily be removed by adsorption with AcNPV-infected Sf-9 cells. Regardless, the AcNPV reactivity in unadsorbed rat anti-BIV p53 serum did not impede the recognition of the BIV gag-pol polyprotein, gag precursor and intermediates, mature gag cleavage products, or CA and MA proteins in BIV-infected BLAC-20 cells (Fig. 9A). Surprisingly, no band of the size predicted for the NC protein was observed in this experiment. Resolution of the BIV NC protein in radioimmunoprecipitation appears to be antiserum dependent since not all sera recognize BIV p14 (compare results of Fig. 7 and 9). Western blot analysis of BIV virions and BlVVLPs using adsorbed rat anti-BIV Pr53gagserum confirmed the radioimmunoprecipitation results (Fig. 9B). Recombinant

BIV Pr53gag is processed

by the BIV PR

Retrovirus precursors have been used to characterize the proteolytic activity in virions or of recombinant PRs in vitro (Yoshinaka et al., 1985; Erickson-Viitanen et al., 1989). Both techniques have yielded insight into the processing of retroviral gag proteins. We have developed a similar strategy (see Materials and Methods) for assessing the viral PR activity in purified preparations of NP-40-lysed BIV using 35S-labeled, gradientpurified BIV VLPs, which contain 35S-labeled BIV Pr53gag. First, the optimum concentration of BIV virions needed to significantly reduce the 35S-labeled Pr53gag in 40 pg of VLPs was determined; we found that 1 pg of BIV was effective in cleaving this amount of Pr53gag completely in 16 hr at 23” (Fig. 1OA). We next determined the optimum time needed for 1 pg of BIV to cause the complete cleavage of Pr53g”g in 40 pg of VLPs; we observed this to be 8 hr at 23” (Fig. 1OA). We next assessed pepstatin A’s ability, and the optimum concentration required, to completely inhibit the proteolytic activity in BIV. A l-pg sample of NP-40-lysed BIV was incubated with 40 pg of NP-40-disrupted VLPs

ET AL.

A. BIV Protein

1

23

4

5

Mkr

(kd)

- 200

p174-

p53

69

- 46

~26

-

B.

BIV Protein

1

2

3

4

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Mkr(kD)

p53-46

p26pl7FIG. 9. Characterization of rat antiserum to recombinant BIV Pr53gag by radioimmunoprecipitation and Western blotting. (A) Radioimmunoprecipiation. Uninfected and BIV-infected BLAC-20 ceils, and uninfected, AcNPV-infected, and AcNPV-BIVg8g-infected Sf-9 cells were metabolically labeled with [%]methionine and cysteine. lysed, and immunoprecipitated. (Lane 1) Proteins precipitated from BIV-infected BLAC-20 cells; (lane 2) uninfected BLAC-20 cells; (lane 3) uninfected Sf-9 cells; (lane 4) AcNPV-BIVQag-infected Sf-9 cells; and (lane 5) AcNPV-infected Sf-9 cells. BIV proteins [gag-pol polyprotein (~174) Pr53g”g (~53) gag intermediates (~45-47) CA (~26) and MA (pl7)] precipitated by the rat anti-BIV Pr53gag serum are indicated on the left of the figure. The recombinant Pr53g@J is the only BIV-specific protein immunoprecipitated in the AcNPV-BIVgaQ-infected Sf-9 cells (lane 4); all other proteins recognized bythe rat antrserum appear to be related to AcNPV (compare lanes 3, 4, and 5). (B) Western blotting. BIV virions and VLPs were lysed, subjected to SDS-PAGE, and transferred to nitrocellulose membranes. Nitrocellulose membranes were incubated with either rat anti-BIV Pr53g8Q serum (adsorbed to AcNPV-infected Sf-9 cells) or rabbit anti-BIV serum, as described under Materials and Methods. (Lanes 1 and 3) BIV virions; (lanes 2 and 4) BIV VLPs. (Lanes 1 and 2) Rat anti-BIV Pr53gJQ serum; (lanes 3 and 4) rabbit anti-BIV serum. BIV proteins [Pr53gaQ (~53) CA (~26) and MA (p17)] bound by the rat anti-BIV Pr53g8Q serum are indicated on the left side of the figure. Molecular weight markers are indicated on the right side of the figure.

and various concentrations (O-l mM) of pepstatin A at 23” for 8 hr (Fig. lOA). A 1 mM concentration of pepstatin A appeared to be effective in completely inhibit-

BACULOVIRUS

EXPRESSION

A. 2 2 =

1

2

3

4

5

6

7

6

9

10

11 12 13 IA

15

Q53...+

16 'I... Q53

A6 30-

B.

5 Y E r” I ,o r

OF

BIV gag

GENE

447

ing the proteolytic activity in BIV virions. The cleavage of Pr53g”g by the BIV PR resulted in the appearance of several new protein bands, ~45-47, ~30, ~26, ~17, and ~14; with the exception of the p30 band, these correlated well with the protein sizes predicted for the gag precursor intermediates, CA, MA, and NC of BIV. The p30 band is observed, albeit infrequently, in radioimmunoprecipitations of BIV proteins in BIV-infected bovine cells but not in virions (Fig. 7); thus, the p30 may also be an intermediate in the processing of the BIV gag precursor (Battles et a/., manuscript in preparation). Analysis of retroviral PR specificities using Pr53gagas substrate

1

23456

7

a

FIG. 10. In v&o processing of recombinant BlV Pr53gag by retrovrrus PRs. (A) Processrng of BIV Pr53gag by the BIV PR. BIV VLPs from AcNPV-BIVg?nfected Sf-9 cells were metabolically labeled wrth (35S]methronlne and cysteine and purified for use as PR substrate, as descrrbed under Materials and Methods. 35S-labeled VLPs and unlabeled BIV vrnons were drsrupted with 2% NP-40 prior to incubation. After incubation, the vinon-precursor mixture was subjected to SE&PAGE and autoradiographs were made. A reductron in the Intensity of the Pr53gag band In autoradiographs was indrcative of proteolyt~c activity In BIV vrnon preparatrons; this reduction coincided with the appearance of smaller protern bands, similar in srze to those observed for mature BIV virion proteins. (Lanes l-4) Incubation of 40 Fg of disrupted 35S-labeled BIV VLPs with increasing concentratrons of lysed BIV virions for 12 hr at 23”; concentrations of NP-QO-lysed BIV Included In lanes 1, 2. 3, and 4 were 0, 0.01, 0.1, and 1 .O pg, respectively. (Lane 5) Blank. (Lanes 661 1) Incubation of 1 rg of lysed BIVwith 40 fig of disrupted 35S~labeled VLPs over trme at 23”; incubatron times for lanes 6, 7, 8, 9, 10, and 1 1 were 0. 0.25, 1, 2, 4, and 8 hr. respectrvely. (Lane 12) Blank. (Lanes 13-l 6) incubation of 1 fig of lysed BIV and 40 pg of disrupted 3”S-labeled VLPs with increasing concentratrons of pepstatin A for 12 hrat 23”; concentrations of pepstatrn A In lanes 13, 14, 15. and 16 are 0, 0.05, 0.25, and 1 .O mM, respectrvely. The locatron of recombinant Pr53g”gis indicated by ~53; molecular werght markers are also indicated. (B) Processing of BIV Pr53Qag by heterologous retrovrrus PRs. BIV Pr53gag was used as the substrate and its reductron by homologous and heterologous retrovirus PRs was measured as described in (A) above. NP-40-lysed virions (5 pg) were Incubated with NP-40.disrupted 35S-labeled BIV VLPs (40 fig) for 12 hr. (Lanes l-6) lncubatron at 23”; (lane 8) 37”. (Lane 1) BLV; (lane 2) BIV, (lane 3) visna vrrus; (lane 4) EIAV; (lane 5) CAEV; (lane 6) HIV 1; (lane 7) blank; and (lane 8) EIAV. The dots rndrcate major cleavage products. BIV Pr53gag IS indicated by the arrow (+). The 14C molecular-weight markers are also shown.

Some retroviral PRs have the ability to cleave not only their own precursor but that of related retroviruses (Hafenrichter et a/., 1989). It was of interest to determine whether any other retrovirus PR could cleave the BIV precursor. On the basis of the phylogenetic relationships derived for the RT segment of the pal gene, the closest relatives to BIV are the members of the lentivirus subfamily of retroviruses (Gonda et a/., 1987, 1989; Garvey et al., 1990). A panel of lentiviruses, including visna virus, BIV, CAEV, HIV-I, and EIAV, and the oncovirus BLV, were used (Fig. 108). A 5-pg sample of each gradient-purified virus preparation was incubated with 40 pg of BIV VLPs containing 35S-labeied BIV Pr53gaY,for 12 hr at 23”. Under these conditions, only the PR activity in BIVvirions was effective in completely reducing Pr53gag.interestingly, the EIAV PR recognized cleavage sites in the BIV precursor. With the EIAV PR, however, some sites on the BIV Pr53gaQappeared more resistent to cleavage, and one gag intermediate, ~45, was incompletely cleaved. Moreover, there was no p30 band; this was true even at elevated temperatures (37”). DISCUSSION The genomes of replication-competent retroviruses invariably contain the structural genes, gag, pal, and env, encoding polyproteins that are incorporated into and are further processed in the virus particle. The gag and env gene products are utilized in the structural formation and the pal gene products in the enzymatic functions of the virus. The components that contribute to the formation of the virus initially aggregate at the cell-surface membrane, are assembled into well-structured particles with recognizable cores and envelopes, and are then released from the cell by budding. Maturation of immature extracellular particles is believed to

448

RASMUSSEN

occur after budding from the cell and is a result of autoprocessing of the gag and gag-pol precursors by the viral PR (Peng eta/., 1989). In the infectious virus particle, the core structure consists of both gag and pol proteins and viral RNA surrounded by a lipid membrane into which the env proteins have been inserted (Haseltine, 1988; Varmus, 1988; Gonda et al., 1989). Previous studies, using baculovirus vectors to overexpress the gag genes of the lentiviruses SIV and HIV1, have reported the synthesis of gag precursors and proteins in insect cells (Madisen eta/., 1987; Delchambre et a/., 1989; Gheysen et al., 1989; Over-ton et al., 1989). Several of these studies have demonstrated that the uncleaved gag precursor can be released from the recombinant baculovirus-infected cell as a structurally recognizable VLP (Delchambre et al., 1989; Gheysen et al., 1989). VLP assembly is accomplished in the absence of infectious RNA and env protein, as previously shown with defective avian and murine retroviruses (Kawai and Hanafusa, 1973; Levin et al., 1974; Shields et al., 1978; Goff, 1984). However, the recombinant HIV-1 and SIV gag precursors are posttranslationally modified at the N terminus of the MA protein with myristic acid in insect and mammalian cells; this modification appears to be important for gag precursor localization in the plasma membrane (Delchambre et a/., 1989; Gheysen et a/., 1989; Overton et al., 1989; Gottlinger et al., 1989). Our results with AcNPV-BIVgag-infected insect cells have shown that expression of the BIV gag precursor coincides with the appearance of VLPs that are ultrastructurally similar to the immature BIV observed in infected bovine cell cultures. The BIV Pr53gag, however, is not myristoylated in insect or bovine cells (Battles et al., manuscript in preparation). Visna virus and EIAV gag precursors are also not myristoylated (Schultz et al., 1988); in this respect, BIV resembles other ungulate lentiviruses and differs from the primate lentiviruses. Myristoylation is known to occur at the first Gly residue immediately after the N-terminal Met in the translation of the HIV-l and SIVgag ORFs (Delchambre et al., 1989; Gheysen et al., 1989; Overton eta/., 1989; Gottlinger et al., 1989); in the BIV gag ORF this residue is Lys, which may account for the lack of myristoylation in BIV Pr53gag (Garvey et al., 1990). Alternatively, the BIV gag precursor may be post-translationally modified by fatty acid groups other than myristic acid. In any case, the present studies suggest that expression of the unprocessed Pr53 gag appears to be all that is required for the efficient assembly and release of BIV VLPs in insect cells. This is supported by more recent studies with additional recombinant baculoviruses that incorporate both gag and pal genes of BIV; these con-

ET AL.

structs overproduce a gag-pol polyprotein with a functional PR that is capable of autoprocessing BIV gagpol polyprotein and gag precursor within the cell. Cells infected with the recombinant baculovirus containing the BIV gag-pof do not appear to efficiently produce VLPs, although RT activity is present in supernatants pelleted at 100,000 g (Rasmussen et al., unpublished data). Other than the differences noted, our present results are very similar to the previous findings with baculovirus expressed SIV and HIV-l gag precursors in insect cells (Delchambre et a/., 1989; Gheysen et al., 1989). We observed the abundant appearance of ICPs in AcNPV-BIVg”g-infected insect cells that overexpressed the Pr53gag (Figs. 3 and 4e). lmmunoelectron microscopy, using antisera to BIV gag products in the present report (Fig. 5) and to recombinant Pr53gag (Nagashima et al., unpublished data), has shown that the ICPs as well as VLPs in AcNPV-BIVg”g-infected insect cells contain gag determinants. The ICPs in insect cells resemble the intracytoplasmic particles found in bovine cells infected with BIV (Boothe and Van Der Maaten, 1974; Gonda and Nagashima, unpublished data) and equine fetal kidney cells infected with EIAV (Gonda et al., 1978). However, ICPs have been found only in cells infected with uncloned stocks of BIV, which contain many defective genomes (Braun et al., 1988), and not in those infected with functional BIV proviral molecular clones. This finding suggests that the appearance of ICPs in BIV-infected cells may be the result of an accumulation of unprocessed gag precursor; this accumulation may be related to the expression of gag precursor from truncated or PR-defective genomes. Infection of insect cells with AcNPV-BIVgag results in the expression of a single unprocessed protein, Pr53g”g. The 5’-most ATG in the gag ORF appears to be the authentic signal sequence for the initiation of gag precursor translation, since there was no other ATG sequence present between the polyhedron promoter and the beginning of the gag ORF. Although Pr53gag is the primary gag precursor product expressed in recombinant baculovirus-infected insect and BIV-infected mammalian cells, we have also resolved additional virus-specific proteins (~45-47) in radioimmunoprecipitations of BIV-infected cells using antibodies with reactivity to BIV and HIV-1 CA proteins; these observations suggest the synthesis of alternative gag precursors. The results of our present studies with the recombinant Pr53gag imply that these are intermediate cleavage products in the processing of Pr53g”g by the viral PR, and are not due to alternate start signals 3’ of the first ATG in the gag ORF (Garvey et a/., 1990).

BACULOVIRUS

EXPRESSION

The exact sequence of events that occur in the processing of the gag precursor by the BIV PR are not known. However, the hypothesis that natural cleavage sites are recognized by the BIV PR in the recombinant Pr53g”g is supported by the similarity in sizes of gag intermediates and mature proteins observed on SDSPAGE of radioimmunoprecipitations of BIV-infected cells and PR-processed gag precursor (Figs. 7 and 1 OA and 1OB). Moreover, the fact that cleavage sites in viral proteins used by viral PRs are very specific (Copeland and Oroszlan, 1988; Darke et a/., 1988; Henderson et al., 1988; Dreyer et al., 1989) and are not recognized by cellular PRs or those of distantly related viruses supports the notion that the in vitro proteolytic activity observed in lysed BIV virions on BIV Pr53gag is mediated by the viral PR. Definitive studies to resolve the precursor-processing enigma await the development of monospecific sera and monoclonal antibodies for analyzing the gag precursor cleavage products. PR cleavage sites in the baculovirus-expressed BIV Pr53g”g are recognized by the BIV PR and partially by the EIAV PR. BIV and EIAV are members of the lentivirus subfamily of retroviruses; thus, we anticipate with further analysis that BIV and EIAV will be found to share some PR cleavage sites. Interestingly, measurements of evolutionary relatedness (based on sequences in the most conserved domain of the pal gene of BIV, EIAV, and other lentiviruses) suggest a closer phylogenetic relationship for BIV and EIAV, although BIV appears to be more related to the primate lentiviruses when other segments of the genome are analyzed and the overall genomrc organization of BIV is taken into account (Garvey et a/., 1990). BIV VLPs are not infectious. Nevertheless, antigenic determinants on the recombinant Pr53g”g are immunogenic and are serologically recognized by antisera to HIV-1 and BIV. In vitro, Pr53g”g can be processed by theviral PR Into subunits similar in size to the intermediate and mature viral proteins found in BIV-infected cells and/or virions. Taken together, the morphologic, serologic, and proteolytic processing data suggest that the recombinant BIV Pr53g”g expressed in insect cells behaves like authentic BIV gag precursor. The expression of VLPs by AcNPV-BIVga9 in insect cells appears to be very efficient; we have observed as much as 15-50 Fg of BIV VLP protein per milliliter of supernatant. The BIV VLPs are amenable to purification by sucrose gradient sedimentation. The cleavage of recombinant Pr53 gagby virion PR is inhibited by pepstatin A, which implies that the BIV PR is of the aspartyl type, like the HIV-1 PR (Seelmeier et al., 1988). The baculovrrus-expressed Pr53g”g appears to be an excellent source of BIV gag antigen, which may enable the

OF

BIV gag

GENE

449

development of assays to detect aspartyl-type PR inhibitors. X-ray crystallographic analysis of the HIV-1 PR has provided the detailed information necessary for the design of PR inhibitors (Miller et a/., 1989). Nevertheless, structural studies of the natural HIV-1 PR substrate (gag precursor) have not been performed. Studies of the location and events of proteolytic processing of gag precursors may lead to the rational development of PR inhibitors such as antiviral drugs for use against lentivirus infections (Haseltine, 1989; Dreyer et al., 1989). The production and purification of large amounts of BIV gag precursor and PR synthesized in the baculovirus-insect expression system will be useful in this respect; furthermore, this system may yteld information on enzyme-substrate interactions that will be of significance for the study of HIV-1 ACKNOWLEDGMENTS l-he authors thank M. S. Oberste and L. Henderson for helpful drsL. 0. Arthur and 1. Bess for purrfred retrovrrus preparations and HIV-1 antiserum, J Greenwood, M Hu, D Chrsholm, L. Graham, and D. Krell for therr sktlled technical assrstance, and J. Hopkrns for help in preparing the manuscript. We are also grateful to M. Sum mers for provrdrng Autographa cabfornica nuclear polyhedrosrs virus and baculovrrus transfer vectors for these studies. Thus ProJect has been funded at least In part with federal funds from the Department of Health and Human Servrces under Contract NOl-CO-74102 wrth Program Resources, lnc The content of thrs pubitcatron dOeS not necessarrly reflect the views or poltcres of the Department of Health and Human Senxes, nor does mention of trade names, commercial products, or organrzatrons Imply endorsement bv the U.S. Government.

CUSSIO~S,

REFERENCES BENTON, C. V., tiorxit, H. M., and FINE, D. L. (1 Y 78). Comparative large-scale propagation of retrovtruses from Old World (Mason Pfizer monkey virus) and New World (squrrrel monkey virus) pn mates, ln t&o 14, 192-l 99 BOOTHE, A D., and VAN DER MAATEN, studies of a vrsna-lrke syncytaproducrng lymphocytosrs J. Vrol. 13, 197 204

M. 1 (1974). Ultrastructural virus from cattle wrth

BRAUN, M. J., LAHN, S., BOYD, A L., KOST, 7. A., NAGASHIMA, K., and GONDA, M A. (1988). Molecular clonrng of brologrcally acttve prove ruses of bovine immunodeficrency+ke vrrus. L+o/ogy 167, 5 I 5 523. BROWN, M., and FAULKNER, P. (1977) Aplaqueassaytornuclearpoly hedrosrs vrruses using a soled overlay / Gen. Viroi. 36, 361 -364 BURNETTE, W N. (I 981). “Western biottrng” Electrophoretrc transter of proteins from sodium dodecyl sulfate- polyacrylamrde gels to unmodified nrtrocellulose and radroachve detectron with antibody and radrotodrnated protein A. Anai. B/o&em 112, I 95-293 CASEY, 1. M., KIM, Y., ANDERSEN, P. R., WAISON, K. F., Fox, J. DEvARE, S G. (1985). Human T-cell lymphotroprc vtrus type mUnOlOglC charactenzatron and primary structure analysrs maior Internal protein, ~24. /. Viroi 55, 41 7 423 COPELAND. T D , and ORCISZLAN, S (1988) Genetic locus,

L., and II/ Im of the primary

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RASMUSSEN vi-

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