Release Of Virus-like Particles Research Articles

M protein is sufficient for assembly and release of Peste des petits ruminants virus-like particles.

Peste des petits ruminants virus (PPRV), belonging to paramyxoviruses, has six structure proteins (such as matrix protein (M), nucleocapsid proteins (N), fusion protein (F) and hemagglutinin protein (H)) and could cause high morbidity and mortality in sheep and goats. Although a vaccine strain of PPRV has been rescued and co-expression of M and N could yield PPRV-like particles, the roles of structure proteins in virion assembly and release have not been investigated in detail. In this study, plasmids carrying PPRV cDNA sequences encoding the N, M, H, and F proteins were expressed in Vero cells. The co-expression of all four proteins resulted in the release of virus-like particles (VLPs) with similar release efficiency to that of authentic virions. Moreover, the co-expression of M together with F also resulted in efficient VLPs release. In the absence of M protein, the expression of no combination of the other proteins resulted in particle release. In summary, a VLPs production system for PPRV has been established and M protein is necessary for promoting the assembly and release of VLPs, of which the predominant protein is M protein. Further study will be focused on the immunogenicity of the VLPs.

Modeling Ebolavirus Budding with Virus Like Particles.

About 15 years ago, several groups initially described the release of virus like particles (VLPs) upon expression of Ebola virus VP40 in mammalian cells. Further development of the protocol later allowed for the dissection of the Ebola virus budding mechanism and for the identification of critical VP40 residues involved in this process. VLPs are now produced routinely in several laboratories as a tool to study virus entry or egress and have even been proposed as vaccine candidates against Ebola virus disease. Here we described protocols for the production and the analysis of Ebola virus VLP release.

Viral protein requirements for assembly and release of human parainfluenza virus type 3 virus-like particles.

To understand the roles of human parainfluenza virus 3 (HPIV3) proteins in assembly and release, viral proteins were expressed individually and in combination in 293T cells. Expression of the matrix (M) protein triggered release of enveloped, matrix-containing virus-like particles (VLPs) from cells. When M was co-expressed with the nucleocapsid (N), fusion (F) or haemagglutinin-neuraminidase (HN) proteins, VLPs that contained M+N, M+F and M+HN, respectively, were generated, suggesting that M can independently interact with each protein to facilitate assembly and release. Additionally, expression of N protein enabled incorporation of the phosphoprotein (P) into VLPs, likely due to known N-P interactions. Finally, the HPIV3 C protein did not enhance VLP release, in contrast to observations with the related Sendai virus. These findings reinforce the central importance of the M protein in virus assembly and release, but also illustrate the variable roles of other paramyxovirus proteins during these processes.

Regulation of ALIX during Exocytic Vesicle Release and Assembly of ESCRT Proteins on the Plasma Membrane

Endosomal sorting complexes required for transport (ESCRTs) are protein complexes that facilitate the release of budding vesicles into extracellular environment. ESCRTs are essential in most cellular processes requiring fission of budding membrane, including multi-vesicular body formation, envelope virus release, and exosome release. During HIV-1 budding, HIV-1 Gag assembles on the plasma membrane and drives the outward deformation of the membrane toward forming vesicles. HIV-1 virus like particles (VLPs) can be generated by expressing Gag alone in the cytosol, these VLPs are consistent of ∼2000 copies of Gag and form vesicles with an average diameter of 120 nanometers and their assembly on the plasma membrane take ∼20-30 minutes. HIV-1 utilizes ESCRTs extensively for the release of progeny virions into extracellular space and VLPs serve as a good model system for studying ESCRT recruitment on the plasma membrane. Gag has two late domain motifs which interact with TSG101 and ALIX, two of the early ESCRT components. Given the stoichiometry of Gag and its steady accumulation in the forming VLPs, it has been expected that ALIX would be recruited into the forming VLPs along with Gag during the assembly. Using live imaging to observe real time assembly of VLPs in the presence of fluorescently tagged ALIX proteins, we have observed that ALIX is recruited transiently at the end of Gag assembly and is mostly recycled after VLPs release. This observation implies high spatial and temporal regulation for recruitment of ALIX during budding. We have further dissected the essential interactions that govern ALIX recruitment, showed the interaction of ALIX Bro1 domain with CHMP4 is critical for recruitment of ALIX and proposed the emerging regulatory mechanism essential for recruitment of ALIX to the plasma membrane during vesicle budding.

Analysis of Assembly and Budding of Lujo Virus.

The recently identified arenavirus Lujo virus (LUJV) causes fatal hemorrhagic fever in humans. We analyzed its mechanism of viral release driven by matrix protein Z and the cell surface glycoprotein precursor GPC. The L domains in Z are required for efficient virus-like particle release, but Tsg101, ALIX/AIP1, and Vps4A/B are unnecessary for budding. LUJV GPC is cleaved by site 1 protease (S1P) at the RKLM motif, and treatment with the S1P inhibitor PF-429242 reduced LUJV production.

Enhanced enterovirus 71 virus-like particle yield from a new baculovirus design.

ABSTRACTEnterovirus 71 (EV71) is responsible for the outbreaks of hand‐foot‐and‐mouth disease in the Asia‐Pacific region. To produce the virus‐like particle (VLP) vaccine, we previously constructed recombinant baculoviruses to co‐express EV71 P1 polypeptide and 3CD protease using the Bac‐to‐Bac® vector system. The recombinant baculoviruses resulted in P1 cleavage by 3CD and subsequent VLP assembly in infected insect cells, but caused either low VLP yield or excessive VLP degradation. To tackle the problems, here we explored various expression cassette designs and flashBAC GOLD™ vector system which was deficient in v‐cath and chiA genes. We found that the recombinant baculovirus constructed using the flashBAC GOLD™ system was insufficient to improve the EV71 VLP yield. Nonetheless, BacF‐P1‐C3CD, a recombinant baculovirus constructed using the flashBAC GOLDTM system to express P1 under the polh promoter and 3CD under the CMV promoter, dramatically improved the VLP yield while alleviating the VLP degradation. Infection of High FiveTM cells with BacF‐P1‐C3CD enhanced the total and extracellular VLP yield to ≈268 and ≈171 mg/L, respectively, which enabled the release of abundant VLP into the supernatant and simplified the downstream purification. Intramuscular immunization of mice with 5 μg purified VLP induced cross‐protective humoral responses and conferred protection against lethal virus challenge. Given the significantly improved extracellular VLP yield (≈171 mg/L) and the potent immunogenicity conferred by 5 μg VLP, one liter High FiveTM culture produced ≈12,000 doses of purified vaccine, thus rendering the EV71 VLP vaccine economically viable and able to compete with inactivated virus vaccines. Biotechnol. Bioeng. 2015;112: 2005–2015. © 2015 Wiley Periodicals, Inc.

Production of influenza virus-like particles from stably transfected Trichoplusia ni BT1 TN-5B1-4 cells

We investigated the production of influenza A/Korea/01-2-9/2009 (H1N1) virus-like particles (VLPs) containing four structural proteins, matrix protein 1 (M1), matrix protein 2 (M2), neuraminidase (NA) and hemagglutinin (HA), using stably transfected Trichoplusia ni BT1 TN-5B1-4 (TN-5B1-4) cells. Recombinant M1, M2, NA and HA were expressed as bands with molecular weights of 28, 17, 60, and 70 kDa, respectively, in stably transfected TN-5B1-4 (TN-5B1-4/M1-M2-NA-HA) cells. VLPs were purified from the culture medium of the TN-5B1-4/M1-M2-NA-HA cells by pelleting on a sucrose cushion, followed by ultracentrifugation in a sucrose density gradient. The four structural proteins were released together from the TN-5B1-4/M1-M2-NA-HA cells, and were co-purified from the same fractions of the sucrose density gradient, indicating that recombinant M1, M2, NA, and HA self-assembled into VLPs in the TN-5B1-4/M1-M2-NA-HA cells. Recombinant baculoviruses (rBac/NA and rBac/HA) expressing recombinant NA and HA were generated and used to co-infect stably transfected TN-5B1-4 (TN-5B1-4/M1-M2) cells expressing recombinant M1 and M2, resulting in the production of high-molecular-weight VLPs and the release of self-assembled VLPs with diameters of 80 ∼ 120 nm. The production of VLPs was greatly enhanced in the TN-5B1-4/M1-M2 cells co-infected with rBac/NA and rBac/HA, compared with the TN-5B1-4/M1-M2-NA-HA cells. Our results suggest that a stably transfected-insect cell expression system might be useful for producing VLPs for the development of recombinant vaccines against influenza.

Dimerization of matrix protein is required for budding of respiratory syncytial virus.

Respiratory syncytial virus (RSV) infects epithelial cells of the respiratory tract and is a major cause of bronchiolitis and pneumonia in children and the elderly. The virus assembles and buds through the plasma membrane, forming elongated membrane filaments, but details of how this happens remain obscure. Oligomerization of the matrix protein (M) is a key step in the process of assembly and infectious virus production. In addition, it was suggested to affect the conformation of the fusion protein, the major current target for RSV antivirals, in the mature virus. The structure and assembly of M are thus key parameters in the RSV antiviral development strategy. The structure of RSV M was previously published as a monomer. Other paramyxovirus M proteins have been shown to dimerize, and biochemical data suggest that RSV M also dimerizes. Here, using size exclusion chromatography-multiangle laser light scattering, we show that the protein is dimeric in solution. We also crystallized M in two crystal forms and show that it assembles into equivalent dimers in both lattices. Dimerization interface mutations destabilize the M dimer in vitro. To assess the biological relevance of dimerization, we used confocal imaging to show that dimerization interface mutants of M fail to assemble into viral filaments on the plasma membrane. Additionally, budding and release of virus-like particles are prevented in M mutants that fail to form filaments. Importantly, we show that M is biologically active as a dimer and that the switch from M dimers to higher-order oligomers triggers viral filament assembly and virus production. Human respiratory syncytial virus (RSV) is the most frequent cause of infantile bronchiolitis and pneumonia. The enormous burden of RSV makes it a major unmet target for a vaccine and antiviral drug therapy. Oligomerization of the matrix protein is a key step in the process of assembly and production of infectious virus, but the molecular mechanism of RSV assembly is still poorly understood. Here we show that the RSV matrix protein forms dimers in solution and in crystals; the dimer is essential for formation of higher-order oligomers. Destabilizing the dimer interface resulted in the loss of RSV filament formation and a lack of budding of virus-like particles. Importantly, our findings can potentially lead to new structure-based RSV inhibitors targeting the assembly process.

Membrane binding and subcellular localization of retroviral Gag proteins are differentially regulated by MA interactions with phosphatidylinositol-(4,5)-bisphosphate and RNA.

ABSTRACTThe matrix (MA) domain of HIV-1 mediates proper Gag localization and membrane binding via interaction with a plasma-membrane (PM)-specific acidic phospholipid, phosphatidylinositol-(4,5)-bisphosphate [PI(4,5)P2]. HIV-1 MA also interacts with RNA, which prevents Gag from binding to membranes containing phosphatidylserine, a prevalent cellular acidic phospholipid. These results suggest that the MA-bound RNA promotes PM-specific localization of HIV-1 Gag by blocking nonspecific interactions with cellular membranes that do not contain PI(4,5)P2. To examine whether PI(4,5)P2 dependence and RNA-mediated inhibition collectively determine MA phenotypes across a broad range of retroviruses and elucidate the significance of their interrelationships, we compared a panel of Gag-leucine zipper constructs (GagLZ) containing MA of different retroviruses. We found that in vitro membrane binding of GagLZ via HIV-1 MA and Rous sarcoma virus (RSV) MA is both PI(4,5)P2 dependent and susceptible to RNA-mediated inhibition. The PM-specific localization and virus-like particle (VLP) release of these GagLZ proteins are severely impaired by overexpression of a PI(4,5)P2-depleting enzyme, polyphosphoinositide 5-phosphatase IV (5ptaseIV). In contrast, membrane binding of GagLZ constructs that contain human T-lymphotropic virus type 1 (HTLV-1) MA, murine leukemia virus (MLV) MA, and human endogenous retrovirus K (HERV-K) MA is PI(4,5)P2 independent and not blocked by RNA. The PM localization and VLP release of these GagLZ chimeras were much less sensitive to 5ptaseIV expression. Notably, single amino acid substitutions that confer a large basic patch rendered HTLV-1 MA susceptible to the RNA-mediated block, suggesting that RNA readily blocks MA containing a large basic patch, such as HIV-1 and RSV MA. Further analyses of these MA mutants suggest a possibility that HIV-1 and RSV MA acquired PI(4,5)P2 dependence to alleviate the membrane binding block imposed by RNA.

A loop region in the N-terminal domain of Ebola virus VP40 is important in viral assembly, budding, and egress.

Ebola virus (EBOV) causes viral hemorrhagic fever in humans and can have clinical fatality rates of ~60%. The EBOV genome consists of negative sense RNA that encodes seven proteins including viral protein 40 (VP40). VP40 is the major Ebola virus matrix protein and regulates assembly and egress of infectious Ebola virus particles. It is well established that VP40 assembles on the inner leaflet of the plasma membrane of human cells to regulate viral budding where VP40 can produce virus like particles (VLPs) without other Ebola virus proteins present. The mechanistic details, however, of VP40 lipid-interactions and protein-protein interactions that are important for viral release remain to be elucidated. Here, we mutated a loop region in the N-terminal domain of VP40 (Lys127, Thr129, and Asn130) and find that mutations (K127A, T129A, and N130A) in this loop region reduce plasma membrane localization of VP40. Additionally, using total internal reflection fluorescence microscopy and number and brightness analysis we demonstrate these mutations greatly reduce VP40 oligomerization. Lastly, VLP assays demonstrate these mutations significantly reduce VLP release from cells. Taken together, these studies identify an important loop region in VP40 that may be essential to viral egress.

A leucine residue in the C terminus of human parainfluenza virus type 3 matrix protein is essential for efficient virus-like particle and virion release.

Paramyxovirus particles, like other enveloped virus particles, are formed by budding from membranes of infected cells, and matrix (M) proteins are critical for this process. To identify the M protein important for this process, we have characterized the budding of the human parainfluenza virus type 3 (HPIV3) M protein. Our results showed that expression of the HPIV3 M protein alone is sufficient to initiate the release of virus-like particles (VLPs). Electron microscopy analysis confirmed that VLPs are morphologically similar to HPIV3 virions. We identified a leucine (L302) residue within the C terminus of the HPIV3 M protein that is critical for M protein-mediated VLP production by regulating the ubiquitination of the M protein. When L302 was mutated into A302, ubiquitination of M protein was defective, the release of VLPs was abolished, and the membrane binding and budding abilities of M protein were greatly weakened, but the ML302A mutant retained oligomerization activity and had a dominant negative effect on M protein-mediated VLP production. Furthermore, treatment with a proteasome inhibitor also inhibited M protein-mediated VLP production and viral budding. Finally, recombinant HPIV3 containing the M(L302A) mutant could not be rescued. These results suggest that L302 acts as a critical regulating signal for the ubiquitination of the HPIV3 M protein and virion release. Human parainfluenza virus type 3 (HPIV3) is an enveloped virus with a nonsegmented negative-strand RNA genome. It can cause severe respiratory tract diseases, such as bronchiolitis, pneumonia, and croup in infants and young children. However, no valid antiviral therapy or vaccine is currently available. Thus, further elucidation of its assembly and budding will be helpful in the development of novel therapeutic approaches. Here, we show that a leucine residue (L302) located at the C terminus of the HPIV3 M protein is essential for efficient production of virus-like particles (VLPs). Furthermore, we found L302 regulated M protein-mediated VLP production via regulation of M protein ubiquitination. Recombinant HPIV3 containing the M(L302A) mutant is growth defective. These findings provide new insight into the critical role of M protein-mediated VLP production and virion release of a residue that does not belong to L domain and may advance our understanding of HPIV3 viral assembly and budding.

The phosphorylation of HIV-1 Gag by atypical protein kinase C facilitates viral infectivity by promoting Vpr incorporation into virions

BackgroundHuman immunodeficiency virus type 1 (HIV-1) Gag is the main structural protein that mediates the assembly and release of virus-like particles (VLPs) from an infected cell membrane. The Gag C-terminal p6 domain contains short sequence motifs that facilitate virus release from the plasma membrane and mediate incorporation of the viral Vpr protein. Gag p6 has also been found to be phosphorylated during HIV-1 infection and this event may affect virus replication. However, the kinase that directs the phosphorylation of Gag p6 toward virus replication remains to be identified. In our present study, we identified this kinase using a proteomic approach and further delineate its role in HIV-1 replication.ResultsA proteomic approach was designed to systematically identify human protein kinases that potently interact with HIV-1 Gag and successfully identified 22 candidates. Among this panel, atypical protein kinase C (aPKC) was found to phosphorylate HIV-1 Gag p6. Subsequent LC-MS/MS and immunoblotting analysis with a phospho-specific antibody confirmed both in vitro and in vivo that aPKC phosphorylates HIV-1 Gag at Ser487. Computer-assisted structural modeling and a subsequent cell-based assay revealed that this phosphorylation event is necessary for the interaction between Gag and Vpr and results in the incorporation of Vpr into virions. Moreover, the inhibition of aPKC activity reduced the Vpr levels in virions and impaired HIV-1 infectivity of human primary macrophages.ConclusionOur current results indicate for the first time that HIV-1 Gag phosphorylation on Ser487 is mediated by aPKC and that this kinase may regulate the incorporation of Vpr into HIV-1 virions and thereby supports virus infectivity. Furthermore, aPKC inhibition efficiently suppresses HIV-1 infectivity in macrophages. aPKC may therefore be an intriguing therapeutic target for HIV-1 infection.

Inhibition of virus replication and induction of human tetherin gene expression by equine IFN-α1

Type I interferons (IFNs) play important roles in the defense of host cells against viral infection by inducing the expression of a diverse range of antiviral factors. IFNs from different animals likely share similar features with human IFNs, and some of them have cross-species activities. Equine IFN-α was proved effective in both equine and human cells. However, the previous studies mostly focused on the inhibition of virus induced cytopathic effects. In this study, we used virus-specific assays to demonstrate the antiviral activities of equine IFN-α1 in both equine and human cells. Equine IFN-α1 inhibited the expression of viral structural proteins and the production of virions of equine infectious anemia virus (EIAV) and equine arteritis virus (EAV) in equine cells. In addition, equine IFN-α1 inhibited the production of EIAV virus-like particles (VLP) from human 293T cells. An IFN-inducible human gene, tetherin, was induced in 293T cells by equine IFN-α1. Its induction correlated with the inhibition of VLP release from the cell membrane. This result indicates that equine IFN-α1 shares a similar mechanism of action with human IFN-α in regulating antiviral genes expression in human cells.

Polarized release and retroviral subversion of TCR-enriched microvesicles at the T cell immunological synapse (P6155)

Abstract The recognition events that mediate adaptive cellular immunity depend on intercellular contacts between T cells and antigen presenting cells (APC). T cell signaling is initiated at these specialized junctions between T cells and APCs, known as the immunological synapse (IS), when surface-expressed antigen receptors (TCR) recognize peptide fragments of pathogens (pMHC) on APCs. Using high resolution optical and electron microscopy, optical-EM correlation, and electron-tomography, we show that centrally accumulated TCR at the IS is located on the surface of extracellular microvesicles that bud at the IS center and are recognized by APC bearing cognate antigen. An early endosomal-sorting complex required for transport (ESCRT) sorts TCR for inclusion in microvesicles, while terminal ESCRT components mediate scission of microvesicles from the T cell plasma membrane. The HIV polyprotein GAG co-opts this process by displacing TCR from microvesicles, resulting in release of virus-like particles at the antigen-dependent IS. We conclude that post-signaling TCR accumulates at the IS center and is released in extracellular microvesicles by an ESCRT-dependent mechanism triggered by T cell activation. These microvesicles mediate intercellular communication in immune cell collaboration, and can be co-opted by HIV GAG for viral transmission across antigen-dependent synapses.

Foamy Virus Budding and Release

Like all other viruses, a successful egress of functional particles from infected cells is a prerequisite for foamy virus (FV) spread within the host. The budding process of FVs involves steps, which are shared by other retroviruses, such as interaction of the capsid protein with components of cellular vacuolar protein sorting (Vps) machinery via late domains identified in some FV capsid proteins. Additionally, there are features of the FV budding strategy quite unique to the spumaretroviruses. This includes secretion of non-infectious subviral particles and a strict dependence on capsid-glycoprotein interaction for release of infectious virions from the cells. Virus-like particle release is not possible since FV capsid proteins lack a membrane-targeting signal. It is noteworthy that in experimental systems, the important capsid-glycoprotein interaction could be bypassed by fusing heterologous membrane-targeting signals to the capsid protein, thus enabling glycoprotein-independent egress. Aside from that, other systems have been developed to enable envelopment of FV capsids by heterologous Env proteins. In this review article, we will summarize the current knowledge on FV budding, the viral components and their domains involved as well as alternative and artificial ways to promote budding of FV particle structures, a feature important for alteration of target tissue tropism of FV-based gene transfer systems.

Calcium-Mediated Fusion between Endo-Lysosomal Compartments Enhances Virus-Like Particles Release

Assembly of the human immunodeficiency virus (HIV) is governed by the structural polyprotein Gag, which is necessary and sufficient for the release of virus-like particles (VLPs) from the host cell. Although the plasma membrane has been recognized as the major site for the production of VLPs, in some cell-types Gag is targeted to late endosomes/multivesicular bodies (LE/MVBs), where assembly and budding take place. Virus release into the extracellular space then occurs after regulated exocytosis. It is well accepted that the release of VLPs requires participation of different host cell components. In particular, it was shown that induction of a transient rise in cytoplasmic Ca2+ increased the amounts of VLPs in MVBs, and resulted in a dramatic enhancement of VLPs release (Perlman M. et al., 2006). However, although cellular factors have been already proposed as mediators of Ca2+ provision (Ehrlich L. et al., 2010), how Ca2+ can promote the release of VLPs remains to be determined.With FACS analyses on live cells, we could identify variations of intracellular Ca2+ in Gag-expressing cells treated with Ca2+ fluorescent indicators. High-resolution confocal and electron microscopy have confirmed that Gag can assemble and bud into VLPs in lysosomes (Ly) and LE. Furthermore, we could show for the first time that Ca2+ released specifically from those compartments causes formation of Ly/LE hybrid organelles, which in turn fuse with the PM and release VLPs into the extracellular space. This heterotypic fusion process requires components of the SNARE complex and the Ca2+ sensor protein Synaptotagmin VII, which regulates Ly exocytosis. All these elements constitute a productive pathway for virus assembly and release. We believe that the Gag protein itself, or a cellular factor recruited by Gag, might promote the increase of Ca2+ required for this process to function.

Membrane Penetrating Ability of Ebola Matrix Protein, VP40

Ebola from the filoviridae family of viruses causes severe and mostly fatal hemorrhagic fevers in primates and has been listed as a category IV pathogen by the NIH. Viral Protein 40 (VP40), the major matrix protein of Ebola virus, regulates the assembly and budding of the virus and alone harbors the ability to form virus-like particles (VLPs) from human cells. We hypothesize that VP40 is a high affinity lipid binding and membrane curvature-inducing protein with specificity for plasma membrane (PM) lipids. This specificity leads to localization of VP40 to the PS-rich inner leaflet of the PM and formation of VLPs. Using fluorescence spectroscopy to investigate VP40 binding and insertion within lipid vesicles (POPC:POPS) containing brominated lipids. Because the fluorescence of the tryptophan is variably quenched depending on its distance from the bromine atoms on the lipid acyl chain, a tryptophan introduced into the membrane binding interface was utilized as a probe to detect the depth of membrane penetration of VP40. Results were indicative of VP40's high affinity and specificity for PS in a PS-concentration dependent manner demonstrating the robust ability of VP40 to penetrate membranes. Further analysis of VP40 membrane insertion revealed a depth of penetration more than halfway into one monolayer of the membrane. Data also confirmed that VP40 binds with nanomolar affinity to vesicles that recapitulated the PM in comparison to the nuclear membrane. In addition VP40-mutants, which inhibit membrane binding and penetration, obstruct VLP formation and release. Cellular assays confirmed the lipid-binding specificity of VP40 in the PM of different cell lines and also demonstrated that deep membrane penetration is essential for VLP. We predict that these results will elucidate the molecular basis of VP40 induced membrane curvature changes, a prerequisite to the PM deformation required for VLP production.

Defective HIV-1 Particle Assembly in AP-3-Deficient Cells Derived from Patients with Hermansky-Pudlak Syndrome Type 2

Adaptor protein complex 3 (AP-3) is a heterotetramer that is involved in signal-mediated protein sorting to endosomal-lysosomal organelles. AP-3 deficiency in humans, induced by mutations in the AP3B1 gene, which encodes the β3A subunit of the AP-3 complex, results in Hermansky-Pudlak syndrome 2 (HPS2), which is a rare genetic disorder with defective lysosome-related organelles. In a previous study, we identified the AP-3 complex as an important contributor to HIV-1 assembly and release. We hypothesized that cells from patients affected by HPS2 should demonstrate abnormalities of HIV-1 assembly. Here we report that HIV-1 particle assembly and release are indeed diminished in HPS2 fibroblast cultures. Transient or stable expression of the full-length wild-type β3A subunit in HPS2 fibroblasts restored the impaired virus assembly and release. In contrast, virus-like particle release mediated by MA-deficient Gag mutants lacking the AP-3 binding site was not altered in HPS2 cells, indicating that the MA domain serves as the major viral determinant required for the recruitment of the AP-3 complex. AP-3 deficiency decreased HIV-1 Gag localization at the plasma membrane and late endosomes and increased the accumulation of HIV-1 Gag at an intermediate step between early and late endosomes. Blockage of the clathrin-mediated endocytic pathway in HPS2 cells did not reverse the inhibited virus assembly and release imposed by the AP-3 deficiency. These results demonstrate that the intact and stable AP-3 complex is required for HIV-1 assembly and release, and the involvement of the AP-3 complex in late stages of the HIV-1 replication cycle is independent of clathrin-mediated endocytosis.

A facile quantitative assay for viral particle genesis reveals cooperativity in virion assembly and saturation of an antiviral protein

Conventional assays of viral particle assembly and release are time consuming and laborious. We have developed an enzymatic virus-like particle (VLP) genesis assay that rapid and quantitative and is also versatile and applicable to diverse viruses including HIV-1 and Ebola virus. Using this assay, which has a dynamic range of several orders of magnitude, we show that the efficiency of VLP assembly and release, i.e., the fraction of the expressed protein that is assembled into extracellular particles, is dependent on the absolute level of expression of either HIV-1 Gag or Ebola virus VP40. We also demonstrate that the activity of the antiviral factor tetherin is dependent on the level of HIV-1 Gag expression and the numbers of VLPs generated, and appears to become saturated as these parameters are increased.

Intracellular Dynamics of HIV-Gag: The Role of Calcium and the Activation of Phospholipase C

The assembly of the human immunodeficiency virus type 1 occurs at the plasma membrane or within late endosomes/multivesicular bodies, and is determined by the polyprotein Gag. This structural protein is also the only viral component necessary and sufficient for the assembly and the release of virus-like particles (VLPs). It is well known that participation of host cell components is particularly required for any of the many Gag-encoded functions. In particular, as already shown by L.S. Ehrlich et al. 2010, the activation of the phospholipase C and the inositol-(1,4,5)-triphosphate receptor, resulting in an increase of intracellular calcium concentration, are both required for efficient Gag trafficking and VLPs release.So far, it is still unclear whether the Gag protein itself or a cellular factor specifically activates this signaling pathway for the virus release process.We are interested to investigate the activation and the role of the PLC signaling pathway in Gag-expressing cells, and how the intracellular calcium concentration can influence the dynamics of Gag and the VLPs release-process.HeLa cells transfected with Gag fused with a fluorescent protein (e.g. EGFP, EYFP) are our principal tool to investigate the intracellular localization of the viral protein upon activation or inhibition of the PLC signaling pathway. Acetoxymethylester-derivates of fluorescent indicators are used to study variations of the calcium concentration in live cells. Chemical inhibitors and RNAi technology are utilized to turn off the PLC pathway at different steps and time points. Total Internal Reflection Fluorescence microscopy, Forster Resonance Energy Transfer methods and co-immunoprecipitation are applied to study the localization of Gag at the plasma membrane or in the cell-interior, and to identify interactions with specific binding partners of the host cell.