Gag Release Research Articles

Therapeutic effects of PG201, an ethanol extract from herbs, through cartilage protection on collagenase-induced arthritis in rabbits

In order to assess the therapeutic effects of PG201 (an ethanol extract from herbs) on osteoarthritis, we investigated whether PG201 could suppress the disease progression of collagenase-induced arthritis (CNIA) in rabbits. The right knees of rabbits were injected intra-articularly with collagenase, and the rabbits were orally treated with distilled water (DW), PG201 (200 mg/kg) or diclofenac (DCF, 10 mg/kg) once a day for 8 weeks. Oral administration of PG201 significantly suppressed the stiffness and bone space narrowing. Cartilage erosion and GAG release ( p < 0.01) were considerably reduced in the knee joints. As well, the mRNA expression of matrix degradation enzymes including MMP-1, -3, and -13 was decreased. On the contrary, the concentrations of TIMP-2 in the synovial fluids were considerably amplified in the PG201 treated group ( p < 0.01), but not in the DCF treated group. The pathologic inflammatory molecules involved in cartilage destruction such as IL-1β, PGE 2, and NO were also diminished by PG201. Taken together, these results indicate that PG201 has therapeutic effects on CNIA through the prominent protection of cartilage. PG201 indeed has great potential as a form of treatment for osteoarthritis.

Role of Nedd4 and Ubiquitination of Rous Sarcoma Virus Gag in Budding of Virus-Like Particles from Cells

Rous sarcoma virus (RSV) budding requires an interaction of the L domain within the p2b region of Gag with cellular Nedd4-family E3 ubiquitin protein ligases. Members of our laboratories previously demonstrated that overexpression of a fragment of the chicken Nedd4-like protein (LDI-1 WW) inhibits Gag release in a dominant-negative manner (A. Kikonyogo, F. Bouamr, M. L. Vana, Y. Xiang, A. Aiyar, C. Carter, and J. Leis, Proc. Natl. Acad. Sci. USA 98:11199-11204, 2001). We have now identified the complete 3' end of LDI-1 and determined that it has a C-terminal ubiquitin ligase HECT domain, similar to other Nedd4 family members. While overexpression of the full-length LDI-1 clone (LDI-1 FL) had little effect on Gag budding, an LDI-1 FL mutant with a substitution in the HECT domain catalytic site blocked Gag release, similar to LDI-1 WW. The coexpression of Gag and hemagglutinin-tagged ubiquitin (HA-Ub) resulted in the detection of mono- and polyubiquitinated forms of Gag in cells and mostly monoubiquitinated Gag in virus-like particles (VLPs). When the Nedd4-binding site (L domain) was deleted, ubiquitinated Gag was not detected. Interestingly, the release of Gag with ubiquitin covalently linked to the C terminus (Gag-Ub) was still blocked by LDI-1 WW. To understand the mechanism of this inhibition, we examined cells expressing Gag and LDI-1 WW by electron microscopy. In the presence of LDI-1 WW, VLPs were found in electron-dense inclusion bodies in the cytoplasm of transfected cells. In contrast, when cells that coexpressed Gag-Ub and LDI-1 WW were examined, inclusion bodies were detected but did not contain VLPs. These results indicate that the ubiquitination of Gag is dependent upon Nedd4 binding to the L domain and suggest that Nedd4 has additional functions during RSV release besides the ubiquitination of Gag.

ADAMTS‐4 and ADAMTS‐5 sequestration and activity in chondrocyte‐agarose cultures

Introduction A primary event in the destruction of cartilage in arthritic diseases is the loss of aggrecan from the extracellular matrix of articular cartilage. During aggrecan breakdown, cleavage sites are utilized, which reside within the IGD of the aggrecan core protein. The Asn341–Phe342 bond is cleaved by members of the MMP family, whereas the second of the two cleavage sites, the Glu373–Ala374 bond, is cleaved by the aggrecanases which are all members of the A disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS) family. Both ADAMTS‐4 and ‐5 have been shown to readily cleave aggrecan at this so‐called aggrecanase site (Tortorella et al. 1999; Abbaszade et al. 1999; Sandy et al. 2000). An in vitro model of cartilage degradation has also shown that these enzymes are responsible for the loss of aggrecan from explant cultures of articular cartilage stimulated with IL‐1‐a or TNF‐a (Tortorella et al. 2001). Both ADAMTS‐4 and ‐5 are thought to be synthesized as inactive zymogens that are activated via removal of their propeptide domain by the Golgi enzyme furin. The secreted active ADAMTS‐4 and ‐5 have predicted molecular weights of 67.9 and 73.6 kDa, respectively. The objective of this study was to monitor ADAMTS‐4 and ‐5 secretion and sequestration in the extracellular matrix of chondrocyte‐agarose cultures.Methods Porcine articular chondrocytes were isolated and embedded in agarose (Hughes et al. 1997) before preculture in DMEM + 50 µg/ml gent. with 10% FBS and 25 µg/ml Phos.C for 21 days. The plates were washed, then cultured in serum‐free DMEM with or without IL‐1‐α for 96 h. GAG release to the medium was measured using the DMMB assay. Media samples were analysed by Western blotting for aggrecan metabolites using mAb BC‐3 to recognize the aggrecanase‐generated neoepitope ARGSV. The presence of ADAMTS‐4 in the media was analysed using the mAb anti‐TS‐4N, which recognizes the metalloproteinase domain, and commercially available polyclonal antibodies to the pro‐ and spacer domains of ADAMTS‐4. The presence of ADAMTS‐5 was detected using commercially available polyclonal antibodies to the pro‐ and spacer domains of ADAMTS‐5. Agarose plugs were extracted in detergent buffer and analysed by Western blotting for ADAMTS‐4 and ‐5 using the same monoclonal and polyclonal antibodies.Results In control cultures, only 20–30% of the total GAG was released into the medium after 96 h of culture. In contrast, 80–90% of the total GAG was released in cultures exposed to IL‐1. Western blot analysis showed aggrecanase‐generated aggrecan metabolites in the IL‐1‐treated cultures but none in the control cultures. Sequestered forms of both ADAMTS‐4 and ‐5 are present in the matrix prior to treatment in serum‐free conditions, and following treatment with or without IL‐1 for 96 h, there are no differences in the high molecular weight isoforms of the enzymes sequestered in the matrix. Western blots of partially purified media samples showed no differences in the zinc chelator‐bound isoforms of either ADAMTS‐4 or ‐5 between control and IL‐1‐treated cultures. However, the predominant heparin sepharose‐bound isoforms of ADAMTS‐4 and ‐5 co‐migrate at approximately 37 kDa. Each of the heparin‐bound 37‐kDa isoforms of ADAMTS‐4 and ‐5 are detected in increased amounts in IL‐1α‐treated cultures compared to controls.Discussion The increased amounts of the 37‐kDa isoforms of both ADAMTS‐4 and ‐5 in the IL‐1‐treated cultures suggest a role for these smaller isoforms in the increased aggrecanase activity seen in the IL‐1‐treated cultures compared to controls. This study has identified multiple isoforms of putative aggrecanase activity that could be responsible for increased aggrecan catabolism that leads to cartilage degradation in arthritis.

Tsg101 control of human immunodeficiency virus type 1 Gag trafficking and release.

The structural precursor polyprotein of human immunodeficiency virus type 1, Pr55(gag), contains a proline-rich motif (PTAP) called the "late domain" in its C-terminal p6 region that directs release of mature virus-like particles (VLPs) from the plasma membranes of gag-transfected COS-1 cells. The motif binds Tsg101 (vacuolar protein-sorting protein 23, or Vps23), which functions in endocytic trafficking. Here, we show that accumulation of the wild-type (wt) Gag precursor in a fraction of COS-1 cytoplasm enriched in multivesicular bodies and small particulate components of the plasma membrane (P100) is p6 dependent. Cleavage intermediates and mature CA mainly partitioned with more rapidly sedimenting larger material enriched in components of lysosomes and early endosomes (P27), and this also was p6 dependent. Expression of truncated or full-length Tsg101 proteins interfered with VLP assembly and Gag accumulation in the P100 fraction. This correlated with reduced accumulation of Gag tagged with green fluorescent protein (Gag-GFP) at the plasma membrane and colocalization with the tagged Tsg101 in perinuclear early endosomes, as visualized by confocal microscopy. Fractionation analysis and confocal examination both indicated that the N-terminal region of Tsg101, which contains binding sites for PTAP and ubiquitin (Ub), was required for Gag trafficking to the plasma membrane. Expression of FLAG-tagged Tsg101 with a deletion in the Ub-binding pocket inhibited VLP release almost completely and to a significantly greater extent than expression of the wt tagged Tsg101 protein or Tsg101-FLAG containing a deletion in the PTAP-binding region. The results demonstrate that Gag associates with endosomal trafficking compartments and indicate that efficient release of virus particles from the plasma membrane requires both the PTAP- and Ub-binding functions of Tsg101 to recruit the cellular machinery required for budding.

Equine infectious anemia virus utilizes host vesicular protein sorting machinery during particle release.

A final step in retrovirus assembly, particle release from the cell, is modulated by a small motif in the Gag protein known as a late domain. Recently, human immunodeficiency virus type 1 (HIV-1) and Moloney murine leukemia virus (M-MuLV) were shown to require components of the cellular vacuolar protein sorting (VPS) machinery for efficient viral release. HIV-1 interacts with the VPS pathway via an association of HIV-1 Gag with TSG101, a component of the cellular complexes involved in VPS. Equine infectious anemia virus (EIAV) is unique among enveloped viruses studied to date because it utilizes a novel motif, YPDL in Gag, as a late domain. Our analysis of EIAV assembly demonstrates that EIAV Gag release is blocked by inhibition of the VPS pathway. However, in contrast to HIV-1, EIAV Gag release is insensitive to TSG101 depletion and EIAV particles do not contain significant levels of TSG101. Finally, we demonstrate that fusing EIAV Gag directly with another cellular component of the VPS machinery, VPS28, can restore efficient release of an EIAV Gag late-domain mutant. These results provide evidence that retroviruses can interact with the cellular VPS machinery in several different ways to accomplish particle release.

Susceptibility to physiological concentrations of IL-1β varies in cartilage at different anatomical locations on human osteoarthritic knee joints

Objectives To determine whether cartilage biopsies from specific regions of osteoarthritic knee joints differ in susceptibility to the degradative effects of the amounts of interleukin-1β (IL-1β; 1–10pg/ml) found in osteoarthritic joints. To establish whether biopsies are sensitive to the effects of either IL-1β or TNFα or both catabolic cytokines.Methods Cartilage from specified regions of 22 osteoarthritic knee joints was examined. Biopsies were incubated for 14 days without or with IL-1β or TNFα at physiological concentrations and GAG release into supernatants assessed.Results Variation was observed in susceptibility to the effects of 1–10pg/ml IL-1β in biopsies from different sites within the same joints and the same site in different joints. The number of regions responding to the cytokine increased significantly (P< 0.0063, Chi square test) with concentration: only 10% (2/21) of all regions tested were susceptible to the effects of 1pg/ml IL-1β, whereas 45% (9/20) were susceptible to the effects of 5pg/ml and 56% (10/18) to the effects of 10pg/ml IL-1β. Significantly fewer regions (4%, 2/47) responded to both IL-1β and TNFα (P< 0.047, Chi square test); biopsies from some patients responded to neither cytokine.Conclusions IL-1β, at the low concentrations detected in joints, can degrade cartilage from susceptible locations. Susceptibility of some cartilage biopsies to the effects of either IL-1β and TNFα, but not both, suggests the signalling receptors for the two major catabolic cytokines are not usually expressed concurrently. The fact that some biopsies respond to neither cytokine suggests that in some patients the local concentration of inhibitors may be high or that other catabolic stimuli predominate. These results could have important implications for pharmacological intervention strategies.

Accumulation of advanced glycation endproducts reduces chondrocyte-mediated extracellular matrix turnover in human articular cartilage

Objective The prevalence of osteoarthritis (OAs) increases with age and coincides with the accumulation of advanced glycation endproducts (AGEs) in articular cartilage, suggesting that accumulation of glycation products may be involved in the development of OA. This study was designed to examine the effects of accumulation of AGEs on the turnover of the extracellular matrix of human articular cartilage.Design Chondrocyte mediated cartilage degradation (GAG release, colorimetric) was measured in human articular cartilage of donors aged 19–82 years (N=30, 4-day culture). In addition, to mimic the age-related increase in AGE levels in vitro, cartilage was cultured in the absence or presence of glucose, ribose or threose. Cartilage degradation and proteoglycan synthesis (35SO2−4 incorporation) were measured and related to the degree of cartilage AGE levels (fluorescence at 360/460nm).Results Chondrocyte-mediated degradation of articular cartilage (i.e. GAG release) decreased with increasing age of the cartilage donor (r=−0.43, P< 0.02). In vitro incubation of cartilage with glucose, ribose or threose resulted in a range of AGE levels that was highly correlated to the chondrocyte-mediated cartilage degradation (r=−0.77, P< 0.001, N=26). In addition, in these in vitro glycated cartilage samples, a decrease in proteoglycan synthesis was observed at increasing AGE levels (r=−0.54, P< 0.005,N =25).Conclusions This study shows that an increase in AGE levels negatively affects the proteoglycan synthesis and degradation of articular cartilage. In combination, these two effects reduce the turnover of the cartilage and thereby the maintenance and repair capacity of the tissue. By this mechanism, the age-related increase in cartilage AGE levels may contribute to the development of OA.

A role for the proteasome in HIV infection

The central role of the proteasome–ubiquitin system in cellular metabolism suggests that it might play a role in viral replication; indeed, this has been shown for a number of viruses. Recent research has shown how the system is involved in HIV replication, and Schubert and colleagues1xProteasome inhibition interferes with Gag polyprotein processing, release, and maturation of HIV-1 and HIV-2. Schubert, U. et al. Proc. Natl. Acad. Sci. USA. 2000; 97: 13057–13062Crossref | PubMed | Scopus (267)See all References1 now reveal that an active proteasome–ubiquitin system is necessary for the release and maturation of infectious HIV particles.Proteins in the cytosol that are damaged or unwanted for any reason are disposed of via the proteasome–ubiquitin system. The proteins are tagged for disposal by oligomers of four or more ubiquitin molecules, whereupon the multicatalytic, multisubunit proteasomes degrade the ubiquitin–protein complexes.HIV particles leave one cell to infect the next by a process of budding: particles leave the cell surrounded by a ‘bud’ of that cell's membrane, which then provides an envelope to fuse with the membrane of a neighbouring cell. HIV particles, in common with all retroviruses, are synthesized as three polyproteins that produce either the inner virion core (Gag), the viral enzymes (Pol) or the glycoproteins of the virion envelope (Env). HIV particles bud off as immature noninfectious viruses, which mature following proteolytic cleavage of the Gag protein.Schubert's team studied the proteasome–ubiquitin system in HIV infected CD4+ T cells. By inhibiting the proteasomal system in these cells, the team found that reinfection by the virus was greatly impeded; some particles were unable to leave the cells at all, while those that did often failed to undergo the proteolytic maturational changes that are necessary for reinfection.By inhibiting the proteasomal system, reinfecton was greatly impeded.The proteasomal blockade was shown to interfere with the processing of Gag polyproteins, a process that occurred independently of the virus release function of the HIV-1 specific accessory protein Vpu (which supports virus release by ion channel activity) but depended on the integrity of the viral protease and C-terminal Gag domain (p6gag). In addition, proteasomal inhibition led to a reduced level of free ubiquitin in the HIV infected cells, and prevented the linkage of mono-ubiquitin to p6gag, which — in contrast to linkage with ubiquitin oligomers — can be necessary for the regulation of protein function.Deficiencies in HIV budding and maturation have been linked to the late assembly (L) domain genes of the virus. The team found that in proteasome-blocked cells, some L-domain proteins lacked ubiquitin. It is possible that L-domain proteins are not able to assemble correctly without a single ubiquitin molecule, a hypothesis that Schubert is currently investigating.The proteasome–ubiquitin system plays such an important role in almost every cellular function that therapies designed to block it, unless they can be designed to be sufficiently specific, are unlikely to be useful. However, understanding these mechanisms ofviral infection should hopefully lead to novel therapeutic strategies in the future.

Differential Budding Efficiencies of Human T-Cell Leukemia Virus Type I (HTLV-I) Gag and Gag-Pro Polyproteins from Insect and Mammalian Cells

In this study, we examined the ability of human T-cell leukemia virus type I (HTLV-I) Gag and Gag-Pro to assemble immature virus-like particles (VLPs) and bud from insect and mammalian cells. Transmission electron microscopy of insect cells infected with a recombinant baculovirus carrying the entire gag gene revealed that Pr53Gag is targeted to the plasma membrane, where it extensively accumulates and forms electron-dense evaginations. However, no particles could be detected either inside the cells or in the culture supernatants. With the Gag-Pro-expressing construct, we observed HTLV-I-specific cytoplasmic proteolysis of the Gag precursor, but again no particle released in the culture supernatants. Transmission electron microscopic analysis of insect cells expressing Gag-Pro polyprotein revealed large vacuoles in the cytoplasm and no budding particles at the plasma membrane. In contrast, human immunodeficiency virus type 1 Gag polyprotein expressed in insect cells is able to release VLPs. These data showed that unlike other retroviruses, Pr53Gag is unable to be released as immature VLPs from insect cells. To determine whether the block in particle budding and release is due to an intrinsic property of Pr53Gag or the absence of essential cellular factors in insect cells, we expressed Gag and Gag-Pro polyproteins in human 293 cells. The results indicate that Pr53Gag and p24 capsid are released within particles into the culture supernatants of human 293 cells. We found that the myristylation of the N-terminal glycine residue is essential for Gag release. Altogether, these results strongly suggest that the proper assembly of HTLV-I particles is dependent on mammalian host cell factors.

Proteasome inhibition interferes with gag polyprotein processing, release, and maturation of HIV-1 and HIV-2.

Retrovirus assembly and maturation involve folding and transport of viral proteins to the virus assembly site followed by subsequent proteolytic cleavage of the Gag polyprotein within the nascent virion. We report that inhibiting proteasomes severely decreases the budding, maturation, and infectivity of HIV. Although processing of the Env glycoproteins is not changed, proteasome inhibitors inhibit processing of Gag polyprotein by the viral protease without affecting the activity of the HIV-1 viral protease itself, as demonstrated by in vitro processing of HIV-1 Gag polyprotein Pr55. Furthermore, this effect occurs independently of the virus release function of the HIV-1 accessory protein Vpu and is not limited to HIV-1, as proteasome inhibitors also reduce virus release and Gag processing of HIV-2. Electron microscopy analysis revealed ultrastructural changes in budding virions similar to mutants in the late assembly domain of p6(gag), a C-terminal domain of Pr55 required for efficient virus maturation and release. Proteasome inhibition reduced the level of free ubiquitin in HIV-1-infected cells and prevented monoubiquitination of p6(gag). Consistent with this, viruses with mutations in PR or p6(gag) were resistant to detrimental effects mediated by proteasome inhibitors. These results indicate the requirement for an active proteasome/ubiquitin system in release and maturation of infectious HIV particles and provide a potential pharmaceutical strategy for interfering with retrovirus replication.

Metabolic and mitogenic activities of insulin-like growth factor-1 in interleukin-1-conditioned equine cartilage.

To determine response of interleukin-1alpha (IL-1alpha)-conditioned equine articular cartilage explants to insulin-like growth factor-1 (IGF-1). Sample Population-Cartilage from the trochlea and condyles of the femur of a clinically normal 4-year-old horse. Effects of IGF-1 (0 to 500 ng/ml) after addition of IL-1alpha were evaluated by assessing matrix responses, using a sulfated glycosaminoglycan (GAG) assay, matrix 35SO4 GAG incorporation, and release of GAG. Mitogenic response was assessed by 3H-thymidine incorporation into DNA and fluorometric assay of total DNA concentration. Human recombinant IL-1alpha (40 ng/ml) increased the amount of labeled GAG released and decreased labeled and total GAG remaining in explants, and IL-1alpha decreased mitogenic response. Addition of IGF-1 counteracted effects seen with IL-1alpha alone. In general, IGF-1 decreased total and labeled GAG released into the medium, compared with IL-1alpha-treated explants (positive-control sample). Values for these variables did not differ significantly from those for negative-control explants. A significant increase in total and newly synthesized GAG in the explants at termination of the experiment was observed with 500 ng of IGF-1/ml. Labeled GAG remaining in explants was greater with treatment at 50 ng of IGF-1/ml, compared with treatment with IL-1alpha alone. Concentrations of 200 ng of IGF-1/ml abolished actions of IL-1alpha and restored DNA synthesis to values similar to those of negative-control explants. IGF-1 at 500 ng/ml was best at overcoming detrimental effects associated with IL-1alpha in in vitro explants. These beneficial effects may be useful in horses with osteoarthritis.

Matrix degradation by chondrocytes cultured in alginate: IL-1β induces proteoglycan degradation and proMMP synthesis but does not result in collagen degradation

Objective: To determine the role of interleukin-1β (IL-1β) in the degradation of proteoglycans and collagen by articular chondrocytes.Design: Chondrocytes were cultured in alginate beads for 2 weeks to produce extracellular matrix, followed by the addition of IL-1β for 1 or 2 days. Breakdown of extracellular matrix (with and without activation of pro-matrix metalloproteinases (MMPs) by APMA) was monitored by release of glycosaminoglycans (GAG, proteoglycans) and hydroxyproline (collagen) from the beads into the medium, and by the amount of damaged collagen in the bead. Levels of (pro)MMPs in the beads were assayed by zymography and their activity was quantified fluorometrically.Results: IL-1β induced a profound GAG release (∽80% after 2 days at 20 ng/ml IL-1β) that was both time and IL-1β concentration dependent. Under these conditions no increase in collagen release or damaged collagen in the bead was detected. Zymography demonstrated that the synthesis of a variety of proMMPs was induced by IL-1β, without a detectable increase of MMP-activity as measured in the activity assay. After activation of the proMMPs by APMA, a time and IL-1β concentration-dependent increase in MMP-activity was found, which resulted in almost complete deterioration of collagen already after 18 h of incubation. In the presence of APMA, GAG release from IL-1β treated beads was significantly increased from 24 to 31%.Conclusions: Our data suggest that proteoglycan and collagen degradation are regulated through different mechanisms: IL-1β induces the synthesis of active enzymes that degrade proteoglycans, such as ‘aggrecanase’, and inactive proMMPs. Thus, IL-1β alone is not sufficient to result in collagen-degrading MMPs. Once activated, MMPs may account for up to a quarter of the aggrecan degradation in this model.

Mutational analysis of the human immunodeficiency virus type 1 Vpu transmembrane domain that promotes the enhanced release of virus-like particles from the plasma membrane of mammalian cells.

Human immunodeficiency virus type 1 Vpu is a multifunctional phosphoprotein composed of the N-terminal transmembrane (VpuTM) and C-terminal cytoplasmic domains. Each of these domains regulates a distinct function of the protein; the transmembrane domain is critical in virus release, and phosphorylation of the cytoplasmic domain is necessary for CD4 proteolysis. We carried our experiments to identify amino acids in the VpuTM domain that are important in the process of virus-like particle (VLP) release from HeLa cells. VLPs are released from the plasma membrane of HeLa cells at constitutive levels, and Vpu expression enhanced the release of VLPs by a factor of 10 to 15. Deletion of two to five amino acids from both N- and C-terminal ends or the middle of the VpuTM domain generated mutant Vpu proteins that have lost the ability to enhance VLP release. These deletion mutants have not lost the ability to associate with the wild-type or mutant Vpu proteins and formed complexes with equal efficiency. They were also transported normally to the Golgi complex. Furthermore, a Vpu protein having the CD4 transmembrane and Vpu cytoplasmic domains was completely inactive, and Vpu proteins harboring hybrid Vpu-CD4 TM domains were also defective in the ability to enhance the release of VLPs. When tested for functional complementation in cotransfected cells, two inactive proteins were not able to reconstitute Vpu activity that enhances the release of Gag particles. Coexpression of functional CD4/Vpu hybrids or wild-type Vpu with inactive mutant CD4/Vpu proteins revealed that mutations in the VpuTM domain could dominantly interfere with Vpu activity in Gag release. Taken together, these results demonstrated that the structural integrity of the VpuTM domain is critical for Vpu activity in the release of VLPs from the plasma membrane of mammalian cells.

The HIV-1 Matrix Domain of Gag Is Required for Vpu Responsiveness during Particle Release

HIV-1 viral protein U (Vpu) facilitates virus particle release. To determine whether Gag is sufficient for generation of a target for Vpu-mediated particle release, we expressed HIV-1 Gag protein in the absence of the other viral genes. The resulting particles were still Vpu responsive. Mutational analysis of Gag indicated that the matrix domain (MA) is required for Vpu responsiveness. However, additional mutations in other domains of Gag, which affect the formation of stable virus particles, also abrogate Vpu responsiveness on total Gag release. Coexpression of the wild-typegaggene and agagmutant lacking the MA domain renders the MA−mutant Vpu responsive. This indicates that Gag molecules lacking MA are still incorporated into particles through association with wild-type Gag molecules and that the resulting composite particles are sufficient for Vpu-mediated exit.

Human immunodeficiency virus type 1 MA deletion mutants expressed in baculovirus-infected cells: cis and trans effects on the Gag precursor assembly pathway

The role of the matrix protein (MA) of human immunodeficiency virus type 1 in intracellular transport, assembly, and extracellular release of Gag polyprotein precursor (Pr55gag) was investigated by deletion mutagenesis of the MA domain of recombinant Gag precursor expressed in baculovirus-infected cells. In addition, three carboxy-terminally truncated forms of the Gag precursor, representing mainly the MA, were constructed. One corresponded to an MA with a deletion of its last 12 residues (amb120), while the others corresponded to the entire MA with an additional sequence from the N-terminal portion of the CA (amb143 and och180). Deletions within the MA central region (residues 41 to 78) appeared to be detrimental to Gag particle assembly and budding from the plasma membrane. A slightly narrower domain, between amino acids 41 and 68, was found to be critical for soluble Gag secretion. Mutations which totally or partially deleted one or the other of the two polybasic signals altered the transport of N-myristylated Gag precursor to the plasma membrane. In coexpression with wild-type Gag precursor, a discrete trans-dominant negative effect on wild-type Gag particle assembly and release was observed with deletion mutants located in the central MA region (residues 41 to 78). A more significant negative effect was obtained with the two recombinant proteins of amb120 and och180, which redirected the Gag particle assembly pathway from the plasma membrane compartment to intracellular vesicles (amb120) and to the nuclear compartment (och180).

Substitution Mutations Affecting a Small Region of the Moloney Murine Leukemia Virus MA Gag Protein Block Assembly and Release of Virion Particles

A series of substitution mutations affecting the Moloney murine leukemia virus MA protein were introduced into a cloned proviral DNA, and the mutant DNAs were tested for their biological activity in NIH/3T3 cells and COS cells. Many of the mutant viruses were viable and replicated with kinetics indistinguishable from the wild type. Seven mutants with alterations in a small region (residues 7-14 from the amino terminus) were replication-defective. These mutants were blocked in assembly end release of the virion particles in NIH/3T3 cells and were defective in both gag and gag-pol gene function. The results suggest that this very small region near the amino terminus of both proteins is required for their membrane targeting or self-association. Three of the defective mutant DNAs were able to induce virion particle formation when present at high copy number in COS cells.

Phenotypic characterization of insertion mutants of the human immunodeficiency virus type 1 Gag precursor expressed in recombinant baculovirus-infected cells.

A panel of 28 insertion mutants of the human immunodeficiency virus type 1 (HIV-1) Gag precursor (Pr55Gag) was constructed by linker-insertion mutagenesis and expressed in recombinant baculovirus-infected insect cells. One set of 14 mutants carried the normal N-myristylation signal; the other set constituted their non-N-myristylated counterparts. The mutants were characterized with respect to (i) assembly and extracellular release of membrane-enveloped budding Gag particles, (ii) intracellular assembly and nuclear transport of Gag cores, (iii) specific processing of Pr55Gag by HIV-1 protease in vivo, and (iv) binding of Pr55Gag to an HIV-1 genomic RNA probe in Northwestern blotting. Insertions within the region between amino acid residues 209 and 334 in the CA domain appeared to be the most detrimental to Gag particle assembly and release of Gag into the external medium, whereas a narrower window, between residues 209 and 241, was found to be critical for secretion of soluble Pr55Gag. Differences in Pr55Gag processing in vivo and RNA binding in vitro between N-myristylated and non-N-myristylated Gag mutants suggested a major conformational role for the myristylated N terminus of Gag precursor. In coinfection experiments using wild-type Gag- and mutant Gag-expressing recombinants, a transdominant negative effect on Gag particle assembly and release was observed for insertions located in two separate domains, the matrix and nucleocapsid.

Mutations in the Protease Gene of Human Immunodeficiency Virus Type 1 Affect Release and Stability of Virus Particles

The expression of the pol gene of human immunodeficiency virus type 1 (HIV-1) occurs by a ribosomal frameshift between the gag and the pol genes. The Gag-Pol polyprotein is produced at levels of 5 to 10% of that of the Gag protein, and is incorporated into virions to provide the viral protease, reverse transcriptase, and integrase which are essential for replication. The mechanism(s) by which the Gag-Pol polyprotein are targeted to the HIV virion is unknown, although it is believed to be via an interaction with the Gag protein. To further explore the mechanism by which the Gag-Pol polyprotein is incorporated into virions, we have constructed a mutation which changes an aspartic acid in the protease active site to asparagine (pHXB2pro-); a four-amino-acid insertion into the protease gene (pHXB2Smal); and insertion of translational termination codons in the protease gene following the gag gene (pHXB55). Transfection of these proviral genomes into COS-1 cells resulted in intracellular expression of only Pr55 gag, demonstrating the inactivation of the viral protease. The expression of Pr55 gag was evident in cells transfected with pHXB2pro- during a short pulse and first 3 hr of chase period, whereas at later times the intracellular levels of Pr55gag were greatly reduced. In contrast, the intracellular Pr55 gag expressed from transfection of pHXB2Smal or pHXB55 were evident even after 6- or 12-hr chase times. To ascertain the effects of the mutations on the assembly and release of viruslike particles, the supernatants from the transfected cells were analyzed for the presence of Pr55 gag. The release of Pr55 gag from cells transfected with pHXB2pro- occurred as early as 1 hr following chase period, and increased for up to 3 hr. In contrast, reduced levels of Pr55 gag were detected in the medium from cells transfected with pHXB2Smal or pHXB55. Subcellular fractionation studies demonstrated that the Pr55 gag expressed from transfection of pHXB2pro- was rapidly targeted to intracellular membranes, while the majority of the Pr55 gag expressed from transfection of pHXB2Smal or pHXB55 was distributed evenly between the cytoplasm and membrane fractions. Finally, the released viruslike particles obtained from the transfection of proviral genome pHXB2pro- were stable to mild detergent treatment, whereas particles obtained from transfection of pHXB2Smal and pHXB55 were relatively unstable. These results demonstrate that subtle changes in the Gag-Pol polyprotein of HIV-1 can have significant effects on the assembly and physical stability of the released virus.

Expression and extracellular release of human immunodeficiency virus type 1 Gag precursors by recombinant baculovirus-infected cells.

The level of synthesis and extracellular release of human immunodeficiency virus type 1 Gag by insect cells was analyzed, using eight different recombinants of Autographa californica nuclear polyhedrosis virus harboring various constructs of the gag gene, cloned under the polyhedrin promoter. The results obtained suggested that gag expression was mainly regulated at the transcriptional level and was not significantly influenced by posttranslational events, e.g., Gag self-assembly, nuclear transport, or extracellular release. Two different forms of Gag were found in the culture medium of recombinant-infected cells. One form consisted of membrane-enveloped, corelike particles released by budding at the plasma membrane; the other of nonparticulate, soluble Gag polyprotein molecules. Both forms coexisted in recombinants expressing Gag with an intact N-terminal myristylation signal, whereas recombinants expressing nonmyristylated Gag released solely the soluble form. This suggested that myristylation of the N terminus was not a prerequisite for efficient extracellular release of Gag by insect cells, which could proceed via two independent but simultaneous mechanisms.

Transport and assembly of gag proteins into Moloney murine leukemia virus

We have studied the process of Moloney murine leukemia virus (M-MuLV) assembly by characterization of core (gag) protein mutants and analysis of wild-type (wt) gag proteins produced by cells in the presence of the ionophore monensin. Our genetic studies involved examination of linker insertion mutants of a Gag-beta-galactosidase (Gag-beta-gal) fusion protein, GBG2051, which is incorporated into virus particles when expressed in the presence of wt viral proteins. Analysis indicated that the amino-terminal two-thirds of the gag matrix domain is essential for targeting of proteins to the plasma membrane; mutant proteins localized to the cytoplasm or were trapped on intracellular membranes. Mutations through most of the coding region of the gag capsid domain generated proteins which were released from cells in membrane vesicles but not in virions. In contrast, linker insertions into p12gag or carboxy-terminal portions of the matrix or capsid coding regions did not affect assembly of fusion proteins into virus particles. Monensin, which blocks vesicular transport, inhibited gag protein intracellular transport and release from cells. Our results suggest that a significant proportion of M-MuLV myristylated gag proteins travel via vesicles to the cell surface. Specific matrix protein polypeptide regions and myristic acid modification are both necessary for appropriate gag protein transport, while capsid protein interactions appear to mediate the final phase of virion formation.