Supplemental Data GGA and Arf Proteins Modulate Retrovirus Assembly and Release (original) (raw)
Related papers
Journal of Virology, 2003
The envelope (Env) protein of Moloney murine leukemia virus (MoMuLV) is a homotrimeric complex whose monomers consist of linked surface (SU) and transmembrane (TM) proteins cleaved from a precursor protein by a cellular protease. In addition, a significant fraction of virion-associated TM is further processed by the viral protease to remove the C-terminal 16 amino acids of the cytoplasmic domain, the R peptide. This cleavage greatly enhances the fusogenicity of the protein and is necessary for the formation of a fully functional Env protein complex. We have previously proposed that R peptide cleavage enhances fusogenicity by altering the conformation of the ectodomain of the protein (Y. Zhao et al., J. Virol. 72:5392-5398, 1998). Using a series of truncation and point mutants of MoMuLV Env, we now provide direct biochemical and immunological evidence that the cytoplasmic tail and the membrane-spanning region of Env can influence the overall structure of the ectodomain of the protein and alter the strength of the SU-TM interaction. The R-peptidetruncated form of the protein, in particular, exhibits a markedly different conformation than the full-length protein.
Antibody-Directed Targeting of Retroviral Vectors via Cell Surface Antigens
Journal of Virology, 2001
Targeted stable transduction of specific cells is a highly desirable goal for gene therapy applications. We report an efficient and broadly applicable approach for targeting retroviral vectors to specific cells. We find that the envelope of the alphavirus Sindbis virus can pseudotype human immunodeficiency virus type 1-and murine leukemia virus-based retroviral vectors. When modified to contain the Fc-binding domain of protein A, this envelope gives a significant enhancement in specificity in combination with antibodies specific for HLA and CD4 relative to that without antibody. Unlike previous targeting strategies for retroviral transduction, the virus titers are relatively high and stable and can be further increased by ultracentrifugation. This study provides proof of principle for a targeting strategy that would be generally useful for many gene therapy applications.
Identification of a Cytoplasmic Targeting/Retention Signal in a Retroviral Gag Polyprotein
1999
Retroviral capsid assembly can occur by either of two distinct morphogenic processes: in type C viruses, the capsid assembles and buds at the plasma membrane, while in type B and D viruses, the capsid assembles within the cytoplasm and is then transported to the plasma membrane for budding. We have previously reported that a single-amino-acid substitution of a tryptophan for an arginine in the matrix protein (MA) of Mason-Pfizer monkey virus (MPMV) converts its capsid assembly from that of a type D retrovirus to that of the type C viruses (S. S. Rhee and E. Hunter, Cell 63:77-86, 1990). Here we identify a region of 18 amino acids within the MA of MPMV that is responsible for type D-specific morphogenesis. Insertion of these 18 amino acids into the MA of type C Moloney murine leukemia virus causes it to assemble an immature capsid in the cytoplasm. Furthermore, fusion of the MPMV MA to the green fluorescent protein resulted in altered intracellular targeting and a punctate accumulation of the fusion protein in the cytoplasm. These 18 amino acids, which are necessary and sufficient to target retroviral Gag polyproteins to defined sites in the cytoplasm, appear to define a novel mammalian cytoplasmic targeting/retention signal.
Nature of Nonfunctional Envelope Proteins on the Surface of Human Immunodeficiency Virus Type 1
Journal of Virology, 2006
Human immunodeficiency virus type 1 (HIV-1) neutralizing antibodies are thought be distinguished from nonneutralizing antibodies by their ability to recognize functional gp120/gp41 envelope glycoprotein (Env) trimers. The antibody responses induced by natural HIV-1 infection or by vaccine candidates tested to date consist largely of nonneutralizing antibodies. One might have expected a more vigorous neutralizing response, particularly against virus particles that bear functional trimers. The recent surprising observation that nonneutralizing antibodies can specifically capture HIV-1 may provide a clue relating to this paradox. Specifically, it was suggested that forms of Env, to which nonneutralizing antibodies can bind, exist on virus surfaces. Here, we present evidence that HIV-1 particles bear nonfunctional gp120/gp41 monomers and gp120-depleted gp41 stumps. Using a native electrophoresis band shift assay, we show that antibody-trimer binding predicts neutralization and that the nonfunctional forms of Env may account for virus capture by nonneutralizing antibodies. We hypothesize that these nonfunctional forms of Env on particle surfaces serve to divert the antibody response, helping the virus to evade neutralization.
Journal of Virology, 2007
The membrane-proximal external region (MPER) of human immunodeficiency virus type 1 (HIV-1) gp41 bears the epitopes of two broadly neutralizing antibodies (Abs), 2F5 and 4E10, making it a target for vaccine design. A third Ab, Fab Z13, had previously been mapped to an epitope that overlaps those of 2F5 and 4E10 but only weakly neutralizes a limited set of primary isolates. Here, libraries of Fab Z13 variants displayed on phage were engineered and affinity selected against an MPER peptide and recombinant gp41. A high-affinity variant, designated Z13e1, was isolated and found to be ϳ100-fold improved over the parental Fab not only in binding affinity for the MPER antigens but also in neutralization potency against sensitive HIV-1. Alanine scanning of MPER residues 664 to 680 revealed that N671 and D674 are crucial for peptide recognition as well as for the neutralization of HIV-1 by Z13e1. Ab competition studies and truncation of MPER peptides indicate that Z13e1 binds with high affinity to an epitope between and overlapping with those of 2F5 and 4E10, with the minimal peptide epitope WASLWNWFDITN. Still, Z13e1 remained about an order of magnitude less potent than 4E10 against several isolates of pseudotyped HIV-1. The sum of our molecular analyses with Z13e1 suggests that the segment on the MPER of gp41 between the 2F5 and 4E10 epitopes is exposed on the functional envelope trimer but that access to the specific Z13e1 epitope within this segment is limited. Thus, the ability of MPER-bearing immunogens to elicit potent HIV-1-neutralizing Abs may depend in part on recapitulating the particular constraints that the functional envelope trimer imposes on the segment of the MPER to which Z13e1 binds.
Gene Therapy, 1997
Various methods for determining the expression of the b-gene transfer. It could be demonstrated that GFP is genergalactosidase (b-gal) gene after retroviral transduction ally superior to b-gal in terms of sensitivity, speed and nonwere compared as a means to assess retroviral titre. To invasiveness of assay, allowing easy direct FACS sorting allow better comparison, different retroviral vectors were of populations of transduced cells. This opens the possiconstructed carrying two mutants of the green fluorescent bility of enrichment by sorting of ex vivo transduced cells protein and assessed as sensitive markers of retroviral in gene therapy protocols.
Journal of Virology, 2003
The presence of porcine endogenous retroviruses presents a potential risk of transmission of infectious diseases (xenozoonosis) if tissues and organs from genetically modified pigs are to be used in xenotransplantation. Here, we report that intracellular expression of a llama single-domain antibody against p15, the matrix domain protein of the porcine endogenous retrovirus Gag polyprotein, blocks retrovirus production, providing the possibility of eliminating the risk of infection in xenotransplantation. MATERIALS AND METHODS Antigen preparation and immunization. PERV-B gag cDNA (AJ1293657) was amplified from PK15 cell RNA with the forward primer Gag forward/Asp (5Ј-ATAGGTACCATGGGACAGACAGTGACTACC-3Ј) and reverse primer Gag reverse/Hind (5Ј-ATAAGCTTGTCCGAACCCCGTCTCCCCTA-3Ј). The 1.6-kb gag cDNA was cloned into the pET-30a expression vector (Novagen, Breda, The Netherlands) and overexpressed upon isopropyl--D-thiogalactopyranoside (IPTG) induction in Escherichia coli BL21 DE3(pLysS). Purified 60-kDa Gag protein was used for immunization of a New Zealand rabbit that yielded a polyclonal rabbit antiserum against the PERV's Gag. The same protein was used for the immunization of a young adult male Lama glama. The immunization schedule was as previously described by van der Linden et al. (40). Immunoelectron microscopy. PK15 cells were fixed in 4% paraformaldehyde and prepared for the ultracryotome as previously described (37). Ultrathin cryosections (75 nm) were immunolabeled with llama polyclonal antiserum against Gag (1:250) followed by the goat anti-llama immunoglobulin G antibody (1:250; Bethyl Laboratories, Inc.) and rabbit anti-goat antibody conjugated with 10-nm colloidal gold particles (1:20; Aurion, Wageningen, The Netherlands) as described by Geuze et al. (13). Cloning and expression of Gag cleavage products. DNA encoding p27 capsid protein was cloned as a SmaI fragment in the pTRCB expession vector (Invitrogen). The fragment was amplified from gag cDNA with primers P27 forward/Sma (5Ј-TCCCCCGGGATCCGCTGCGCACCTATGGCCCT-3Ј) and p27 reverse/ Sma (5Ј-TCCCCCGGGAAAATCTCTCTCTCTCTCCCT-3Ј). DNA encoding p15 matrix protein was obtained by EagI fragment removal from the pET-30a-gag construct containing the full-length gag cDNA. DNA encoding p12 was cloned as an NcoI fragment in pET-30a. The primers used for its amplification were P12 forward/Nco (5Ј-CATGCCATGGAGATCG AGGAGCCGCCGATC-3Ј) and P12 reverse/Nco (5Ј-CATGCCATGGGCCAT AGGTGCGAGCGGTAA-3Ј). P10 nucleocapsid DNA was cloned as an NcoI fragment in pET-30a. The primers used for its amplification were P10 forward/Nco (5Ј-CATGCCATGGC CGCACTGGTTGAAGGGAAG-3Ј) and P10 reverse/Nco (5Ј-CATGCCATGG ACCCCGTCTCCCCTAATCTT-3Ј). All clones were transformed into E. coli BL21 DE3(pLysS), in which Gag cleavage products were overexpressed upon IPTG induction. Library construction and screening. Total RNA was isolated from peripheral lymphocytes of the immunized llama with the Ultraspec RNA isolation system (Biotecx laboratories, Inc., Houston, Tex.). After purification of polyadenylated RNA (Oligotex 70022; Qiagen), cDNA was made with oligo(dT). DNA fragments
Journal of Virology, 2001
The early steps of adeno-associated virus (AAV) infection involve attachment to a variety of cell surface receptors (heparan sulfate, integrins, and fibroblast growth factor receptor 1) followed by clathrin-dependent or independent internalization. Here we have studied the subsequent intracellular trafficking of AAV particles from the endosomal compartment to the nucleus. Human cell lines were transduced with a recombinant AAV (rAAV) carrying a reporter gene (luciferase or green fluorescent protein) in the presence of agents that affect trafficking. The effects of bafilomycin A 1 , brefeldin A, and MG-132 were measured. These drugs act at the level of endosome acidification, early-to-late endosome transition, and proteasome activity, respectively. We observed that the transducing virions needed to be routed as far as the late endosomal compartment. This behavior was markedly different from that observed with adenovirus particles. Antiproteasome treatments with MG-132 led to a 50-fold enhancement in transduction efficiency. This effect was accompanied by a 10-fold intracellular accumulation of single-stranded DNA AAV genomes, suggesting that the mechanism of transduction enhancement was different from the one mediated by a helper adenovirus, which facilitates the conversion of the rAAV single-stranded DNA genome into its replicative form. MG-132, a drug currently in clinical use, could be of practical use for potentializing rAAV-mediated delivery of therapeutic genes.
The murine AIDS virus Gag precursor protein binds to the SH3 domain of c-Abl
Journal of …, 1997
The Pr60 gag protein of the murine AIDS (MAIDS) defective virus promotes the proliferation of the infected target B cells and is responsible for inducing a severe immunodeficiency disease. Using the yeast two-hybrid system, we identified the SH3 domain of c-Abl as interacting with the proline-rich p12 domain of Pr60 gag. The two proteins were shown to associate in vitro and in vivo in MAIDS virus-infected B cells. Overexpression of Pr60 gag in these cells led to a detectable increase of the levels of c-Abl protein and to its translocation at the membrane. These results suggest that this viral protein serves as a docking site for signaling molecules and that c-Abl may be involved in the proliferation of infected B cells. Murine AIDS (MAIDS) is a severe immunodeficiency disease characterized by lymphadenopathy, splenomegaly, hypergammaglobulinemia, T-and B-cell dysfunctions, and late appearance of B-cell lymphomas and opportunistic infections (19, 23). This severe immune disease is caused by a defective strain of murine leukemia virus (MuLV) (2, 4). The main target cells of this defective virus appear to be peripheral B lymphocytes which are induced to proliferate after infection (17). The defective viral genome encodes a single Gag precursor (Pr60 gag) protein (15) whose p12 region is highly divergent from those of the p12 proteins of other helper MuLVs. Mutational analyses have confirmed that the Pr60 gag is necessary and sufficient for disease induction (14, 20, 25). In addition, myristylation-negative mutant MAIDS viruses were found to be nonpathogenic, indicating that myristylation and tight membrane association are required for Pr60 gag to be pathogenic (16). These results suggested that intact Pr60 gag may interact with some cellular effectors and possibly serve as a docking site at the membrane to initiate target cell expansion and pathogenesis. We searched for some proteins interacting with Pr60 gag and found that the c-Abl protein is one of these interacting proteins. MATERIALS AND METHODS Construction of plasmids for yeast two-hybrid screening. The construction of the clone encoding the complete Pr60 gag fused to the LexA DNA-binding domain (DB) (pBTM/DuGAG) was made by PCR site-directed mutagenesis using oligonucleotides 5Ј-CCGGAATTCATGGGACAGACCGTAACCACTC-3Ј (sense) and 5Ј-AGTACCATCTAGTGGCCACC-3Ј (antisense). An EcoRI site was introduced at nucleotide (nt) 970 from Du5H (2). The amplified fragment was digested with EcoRI and AatII (nt 1025) and subcloned into pBS-SK together with an AatII (nt 1025)-HindIII (nt 3264) fragment from plasmid pDu5Hneo (2) to generate Du5H R1-970 , which was sequenced. Digestion with EcoRI and SalI (of pBS-SK) from Du5H R1-970 generated a fragment which was cloned into pBTM116 to generate pBTM/DuGAG. Plasmids pBTM/DuGAG/⌬CA, pBTM/DuGAG/⌬12, and pBTM/DuGAG/⌬MA were constructed by swapping the AatII-HindIII fragment of plasmid Du5H R1-970 with those of plasmids pDu5H-A, pDu5H-B, and pDu5H-C, encoding Pr60 gag deletion mutants described previously (14), before subcloning into pBTM116. The G6T2 fusion was made by replacement of the BstEII-HindIII fragment of G6T2 (26) in Du5H R1-970 to generate pBTM/G6T2. The pGAL4-DB-DuGAG clone, which encodes the complete Pr60 gag fused with the GAL4 DB, was constructed by subcloning the EcoRI-SalI fragment of plasmid pBTM/DuGAG into plasmid pGBT9 (12). These GAL4 DB fusion constructs were tested in Saccharomyces cerevisiae YSF526 (MATa his3 leu2 trp1 URA3::GAL1-lacZ), and the LexA DB constructs were tested in strain L40. Yeast two-hybrid screening. To identify proteins that interact with Pr60 gag , we used a partial 10.5-day-old mouse embryo (41) cDNA library fused to the VP16 acidic activation domain. A clone of S. cerevisae L40 (MATa his3 trp1 leu2 LYS2::lexA-HIS3 URA3::lexA-lacZ) which contained pBTM/DuGAG was transformed with this cDNA library as described previously (35). An estimated 30 ϫ 10 6 transformants were grown for 16 h in synthetic medium lacking leucine and tryptophan to maintain selection for the Pr60 gag and library plasmids, respectively, and to allow expression of the HIS3 reporter gene. Thereafter, the transformants were plated onto synthetic medium lacking histidine, leucine, tryptophan, uracil, and lysine. After 3 days at 30ЊC, His ϩ colonies were picked, grown again for 3 days on plates lacking leucine, tryptophan, and uracil, and then assayed for -galactosidase activity by a filter assay (3). To test for specificity, the 100 His ϩ LacZ ϩ colonies were tested by using a mating protocol with yeast strain AMR70 (MAT␣ his3 lys2 trp1 leu2 URA3::(lexAop) 8-lacZ) as described previously (41) against the nonspecific bait lamin (pLexALamin). Plasmids from specific His ϩ LacZ ϩ colonies were extracted and retransformed into yeast strain L40 with either pLexALamin, pBTM116, pLexDA (encoding the Drosophila daughterless protein), or pBTM/DuGAG to further confirm their strict Pr60 gag requirement for interaction. Three clones strongly transactivated the reporter in a Pr60 gag-dependent manner, and these plasmids were recovered. GST fusion constructs. Plasmid pGST/c-Abl-SH3-SH2 was constructed by inserting the c-Abl NotI fragment of pVP16-Abl into plasmid pGEX-4T1. pGST/ c-Abl-SH3 was constructed by subcloning the c-Abl BamHI-HincII fragment from pVP16-Abl into plasmid pGEX-3X. Plasmid pGST/Abl SH2 was obtained by inserting the c-Abl HincII-EcoRI fragment from pVP16-Abl into SmaI-EcoRI-digested pGEX-3X. Glutathione S-transferase (GST)-p12 was generated by PCR amplification of a BglII subclone of the MAIDS gag region (nt 691 to 2228) with oligonucleotide 5Ј-CGGGGGATCCCCTTTTCCTTTATCGACACT T-3Ј and the M13 universal primer. The PCR product was purified and digested with BamHI (included in the oligonucleotide) and XhoI before ligation into pGEX-4T1. The GST-p12⌬NotI and GST-p12⌬SmaI deletion mutants were constructed like the wild-type GST-p12 plasmid except that the PCR product was cleaved with BamHI-NotI and BamHI-SmaI before subcloning in pGEX-4T1. To introduce a point mutation in the proline of the SH3-binding sites at residues 134, 158, and 181, the p12 region was amplified from GST-p12 with primers 5Ј-CCGGATCCCGGTCTGACCTTTACAC-3Ј (sense), 5Ј-TATCGCCTCTAAA CCTCCTAA-3Ј (P134A) (antisense), 5Ј-CCTCGCTAACCTTCCTCC-3Ј (P158A) (antisense), and 5Ј-TTCCGCCCCTAATCCCATG-3Ј (P181A) (antisense). These PCR fragments were then used as megaprimers. The mutant DNAs were cloned back into pGEX-4T1. GST fusions were purified as described previously (38). Biotinylation of proteins. Purified GST fusion proteins were biotinylated in 100 mM sodium borate (pH 8.8) with biotinamidocaproate N-hydroxysuccinimide ester at a ratio of 50 g of ester per mg of protein (22). The reaction was blocked with NH 4 Cl, and excess biotin was removed by extensive dialysis against phosphate-buffered saline.