Enveloped Retroviruses Research Articles

The ESCRT Machinery: Remodeling, Repairing, and Sealing Membranes.

The ESCRT machinery is an evolutionarily conserved membrane remodeling complex that is used by the cell to perform reverse membrane scission in essential processes like protein degradation, cell division, and release of enveloped retroviruses. ESCRT-III, together with the AAA ATPase VPS4, harbors the main remodeling and scission function of the ESCRT machinery, whereas early-acting ESCRTs mainly contribute to protein sorting and ESCRT-III recruitment through association with upstream targeting factors. Here, we review recent advances in our understanding of the molecular mechanisms that underlie membrane constriction and scission by ESCRT-III and describe the involvement of this machinery in the sealing and repairing of damaged cellular membranes, a key function to preserve cellular viability and organellar function.

Defining biological and biophysical properties of SARS-CoV-2 genetic material in wastewater

SARS-CoV-2 genetic material has been detected in raw wastewater around the world throughout the COVID-19 pandemic and has served as a useful tool for monitoring community levels of SARS-CoV-2 infections. SARS-CoV-2 genetic material is highly detectable in a patient's feces and the household wastewater for several days before and after a positive COVID-19 qPCR test from throat or sputum samples. Here, we characterize genetic material collected from raw wastewater samples and determine recovery efficiency during a concentration process. We find that pasteurization of raw wastewater samples did not reduce SARS-CoV-2 signal if RNA is extracted immediately after pasteurization. On the contrary, we find that signal decreased by approximately half when RNA was extracted 24–36 h post-pasteurization and ~90% when freeze-thawed prior to concentration. As a matrix control, we use an engineered enveloped RNA virus. Surprisingly, after concentration, the recovery of SARS-CoV-2 signal is consistently higher than the recovery of the control virus leading us to question the nature of the SARS-CoV-2 genetic material detected in wastewater. We see no significant difference in signal after different 24-hour temperature changes; however, treatment with detergent decreases signal ~100-fold. Furthermore, the density of the samples is comparable to enveloped retrovirus particles, yet, interestingly, when raw wastewater samples were used to inoculate cells, no cytopathic effects were seen indicating that wastewater samples do not contain infectious SARS-CoV-2. Together, this suggests that wastewater contains fully intact enveloped particles.

Vehicles of intercellular communication: exosomes and HIV-1.

The terms extracellular vesicles, microvesicles, oncosomes, or exosomes are often used interchangeably as descriptors of particles that are released from cells and comprise a lipid membrane that encapsulates nucleic acids and proteins. Although these entities are defined based on a specific size range and/or mechanism of release, the terminology is often ambiguous. Nevertheless, these vesicles are increasingly recognized as important modulators of intercellular communication. The generic characterization of extracellular vesicles could also be used as a descriptor of enveloped viruses, highlighting the fact that extracellular vesicles and enveloped viruses are similar in both composition and function. Their high degree of similarity makes differentiating between vesicles and enveloped viruses in biological specimens particularly difficult. Because viral particles and extracellular vesicles are produced simultaneously in infected cells, it is necessary to separate these populations to understand their independent functions. We summarize current understanding of the similarities and differences of extracellular vesicles, which henceforth we will refer to as exosomes, and the enveloped retrovirus, HIV-1. Here, we focus on the presence of these particles in semen, as these are of particular importance during HIV-1 sexual transmission. While there is overlap in the terminology and physical qualities between HIV-1 virions and exosomes, these two types of intercellular vehicles may differ depending on the bio-fluid source. Recent data have demonstrated that exosomes from human semen serve as regulators of HIV-1 infection that may contribute to the remarkably low risk of infection per sexual exposure.

The Botanical Glycoside Oleandrin Inhibits Human T-cell Leukemia Virus Type-1 Infectivity and Env-Dependent Virological Synapse Formation.

At present, there are no antiretroviral drugs that inhibit incorporation of the envelope glycoprotein into newly-synthesized virus particles. The botanical glycoside, oleandrin, derived from extracts of Nerium oleander, has previously been shown to reduce the levels of the gp120 envelope glycoprotein on human immunodeficiency virus type-1 (HIV-1) particles and inhibit HIV-1 infectivity in vitro. We therefore tested whether oleandrin or an extract from N. oleander could also inhibit the infectivity of the human T-cell leukemia virus type-1 (HTLV-1): A related enveloped retrovirus and emerging tropical infectious agent. The treatment of HTLV-1+ lymphoma T-cells with either oleandrin or a N. oleander extract did not significantly inhibit viral replication or the release of p19Gag-containing particles into the culture supernatants. However, the collected virus particles from treated cells exhibited reduced infectivity on primary human peripheral blood mononuclear cells (huPBMCs). Unlike HIV-1, extracellular HTLV-1 particles are poorly infectious and viral transmission typically occurs via direct intercellular interactions across a virological synapse. We therefore investigated whether oleandrin or a N. oleander extract could inhibit virus transmission from a GFP-expressing HTLV-1+ lymphoma T-cell-line to huPBMCs in co-culture assays. These results demonstrated that both oleandrin and the crude phytoextract inhibited the formation of virological synapses and the transmission of HTLV-1 in vitro. Importantly, these findings suggest oleandrin may have broad antiviral activity against enveloped viruses by reducing the incorporation of the envelope glycoprotein into mature particles, a stage of the infection cycle not targeted by modern HAART.

The Inhibition of HIV-1 Entry Imposed by Interferon Inducible Transmembrane Proteins Is Independent of Co-Receptor Usage.

Interferon inducible transmembrane proteins (IFITMs) are one of several IFN-stimulated genes (ISGs) that restrict entry of enveloped viruses, including flaviviruses, filoviruses and retroviruses. It has been recently reported that in U87 glioblastoma cells IFITM proteins inhibit HIV-1 entry in a co-receptor-dependent manner, that is, IFITM1 is more inhibitory on CCR5 tropic HIV-1 whereas IFITM2/3 confers a greater suppression of CXCR4 counterparts. However, how entry of HIV-1 with distinct co-receptor usage is modulated by different IFITM orthologs in physiologically relevant CD4+ T cells and monocytes/macrophages has not been investigated in detail. Here, we report that overexpression of IFITM1, 2 and 3 in human CD4+ HuT78 cells, SupT1 cells, monocytic THP-1 cells and U87 cells expressing CD4 and co-receptor CCR5 or CXCR4, suppressed entry of CXCR4 tropic viruses NL4.3 and HXB2, CCR5 tropic viruses AD8 and JRFL, dual tropic 89.6 virus, as well as a panel of 32 transmitted founder (T/F) viruses, with a consistent order of potency, that is, IFITM3 > IFITM2 > IFITM1. Consistent with previous reports, we found that some CCR5-using HIV-1 isolates, such as AD8 and JRFL, were relatively resistant to inhibition by IFITM2 and IFITM3, although the effect can be cell-type dependent. However, in no case have we observed that IFITM1 had a stronger inhibition on entry of any HIV-1 strains tested, including those of CCR5-using T/Fs. We knocked down the endogenous IFITMs in peripheral blood mononuclear cells (PBMCs) and purified CD4+ T cells and observed that, while this treatment did greatly enhance the multiple-round of HIV-1 replication but had modest effect to rescue the single-round HIV-1 infection, reinforcing our previous conclusion that the predominant effect of IFITMs on HIV-1 infection is in viral producer cells, rather than in target cells to block viral entry. Overall, our results argue against the idea that IFITM proteins distinguish co-receptors CCR5 and CXCR4 to inhibit entry but emphasize that the predominant role of IFITMs on HIV-1 is in producer cells that intrinsically impair the viral infectivity.

D-101 CryoEM Studies of HIV-1 Envelope Glycoprotein

HIV-1 is an enveloped retrovirus that is the causative agent of AIDS. To date there is no effective vaccine that prevents acquisition of the virus, and therefore represents a significant burden on worldwide health. Structures of the HIV-1 envelope glycoprotein (Env) trimer in complex with neutralizing antibodies are a key component of ongoing rational vaccine design efforts aimed at creating stable immunogens that mimic Env. Thus, we have created a cryoEM pipeline to solve a number of such structures that are actively being used to design new immunogens for testing in animals. I will describe the types of information generated by cryoEM and the methods used to solve a diversity of structures that are representative of Env across multiple clades. By understanding the molecular determinants of trimer formation and quaternary epitope presentation across different Env sequences we can start to build a consensus for the creation of new and exciting immunogens.

A Unique N-Terminal Sequence in the Carnation Italian ringspot virus p36 Replicase-Associated Protein Interacts with the Host Cell ESCRT-I Component Vps23

Like most positive-strand RNA viruses, infection by plant tombusviruses results in extensive rearrangement of specific host cell organelle membranes that serve as the sites of viral replication. The tombusvirus Tomato bushy stunt virus (TBSV) replicates within spherules derived from the peroxisomal boundary membrane, a process that involves the coordinated action of various viral and cellular factors, including constituents of the endosomal sorting complex required for transport (ESCRT). ESCRT is comprised of a series of protein subcomplexes (i.e., ESCRT-0 -I, -II, and -III) that normally participate in late endosome biogenesis and some of which are also hijacked by certain enveloped retroviruses (e.g., HIV) for viral budding from the plasma membrane. Here we show that the replication of Carnation Italian ringspot virus (CIRV), a tombusvirus that replicates at mitochondrial membranes also relies on ESCRT. In plant cells, CIRV recruits the ESCRT-I protein, Vps23, to mitochondria through an interaction that involves a unique region in the N terminus of the p36 replicase-associated protein that is not conserved in TBSV or other peroxisome-targeted tombusviruses. The interaction between p36 and Vps23 also involves the Vps23 C-terminal steadiness box domain and not its N-terminal ubiquitin E2 variant domain, which in the case of TBSV (and enveloped retroviruses) mediates the interaction with ESCRT. Overall, these results provide evidence that CIRV uses a unique N-terminal sequence for the recruitment of Vps23 that is distinct from those used by TBSV and certain mammalian viruses for ESCRT recruitment. Characterization of this novel interaction with Vps23 contributes to our understanding of how CIRV may have evolved to exploit key differences in the plant ESCRT machinery. Positive-strand RNA viruses replicate their genomes in association with specific host cell membranes. To accomplish this, cellular components responsible for membrane biogenesis and modeling are appropriated by viral proteins and redirected to assemble membrane-bound viral replicase complexes. The diverse pathways leading to the formation of these replication structures are poorly understood. We have determined that the cellular ESCRT system that is normally responsible for mediating late endosome biogenesis is also involved in the replication of the tombusvirus Carnation Italian ringspot virus (CIRV) at mitochondria. Notably, CIRV recruits ESCRT to the mitochondrial outer membrane via an interaction between a unique motif in the viral protein p36 and the ESCRT component Vps23. Our findings provide new insights into tombusvirus replication and the virus-induced remodeling of plant intracellular membranes, as well as normal ESCRT assembly in plants.

Temporal and Spatial Remodeling of Host Cell Plasma Membrane during HIV Assembly Revealed by Quantitative Superresolution Microscopy

The budding of enveloped retrovirus, such as HIV, involves oligomerization of the structural protein Gag at the plasma membrane and the incorporation of a patch of the host cell plasma membrane in the viral particle. Previous studies indicate that certain host proteins and viral envelope proteins are enriched in the membrane of virus particles. Since the viral membrane is derived from the host membrane, such specialized composition of viral membrane suggests viral assembly and budding involve differentiation of the host cell plasma-membrane at the assembly site. However, the mechanism of the membrane remodeling process during viral assembly is not understood. using a combination of quantitative superresolution microscopy (PALM and STORM) and diffraction-limited TIRF microscopy, we have investigated the spatial and temporal differentiation of the host cell plasma-membrane during budding of HIV. We find that in absence of Gag, the viral envelope protein is randomly distributed in the plasma membrane. However, oligomerization of Gag at the membrane leads to dramatic redistribution of the envelope proteins at the site of Gag assembly. We also see enrichment of GPI-anchored protein such as CD59 at the assembly site, while a model transmembrane protein EGFP-GT46 is actively excluded from the membrane patch. Gag is anchored to the plasma membrane via myristoylated lipid anchor and electrostatic interactions. Interestingly, we find that myristoylated inner leaflet anchored proteins are enriched while proteins with farnesyl and geranyl lipid anchors are depleted at the assembly site. Taken together, our results indicate that the oligomerization of HIV Gag at the plasma-membrane creates a specialized microenvironment, which leads to the differentiation of the local plasma membrane. This provides new insights about the temporal and spatial remodeling of plasma membrane during assembly and budding of retroviruses.

Virus Maturation

We examine virus maturation of selected nonenveloped and enveloped single-stranded RNA viruses, retroviruses, bacteriophages, and herpesviruses. Processes associated with maturation in the RNA viruses range from subtle (nodaviruses and picornaviruses) to dramatic (tetraviruses and togaviruses). The elaborate assembly and maturation pathway of HIV is discussed in contrast to the less sophisticated but highly efficient processes associated with togaviruses. Bacteriophage assembly and maturation are discussed in general terms, with specific examples chosen for emphasis. Finally the herpesviruses are compared with bacteriophages. The data support divergent evolution of nodaviruses, picornaviruses, and tetraviruses from a common ancestor and divergent evolution of alphaviruses and flaviviruses from a common ancestor. Likewise, bacteriophages and herpesviruses almost certainly share a common ancestor in their evolution. Comparing all the viruses, we conclude that maturation is a convergent process that is required to solve conflicting requirements in biological dynamics and function.

Rendezvous with Tat: Transactivator of Transcription during Human Immunodeficiency Virus Pathogenesis

Acquired immune deficiency syndrome is a deadly disease caused by HIV, declared as a global catastrophe engulfing millions of lives per year. It took ~1.8 million lives in the year 2010 a lone, as per reports of UNAIDS. HIV-1 is an enveloped retrovirus, having genome composed of two copies of ssRNA. It attacks the T-lymphocytes of the hosts, c auses several multifaceted immune failures and fina lly leads to fatality. Our articles focuses on small re gulatory tat (transactivator of transcription) gene which is responsible for the initiation and elongation of vi ral transcription through the LTR transactivation. It consists of two exons combining to form protein of 101 amino acids that enhances the efficiency of viral transcription. Tat exon 1(1-72) is important for viral transcripti on and Tat exon 2 (73-101) is important for cell adhesion and several activities like cellular uptake of the exogenous Tat, apoptosi s, improvement of HIV-1 replication and IL-2 super ind uction in HIV-1-infected T cells. Tat plays vital role in LTR transcription, reverse transcriptions, Kaposi's sarcoma, neuropathogenesis, immunosuppression, concluding its importance. Eventually in future approaches, tat gene can be targeted for gene based drug targeting as Tat exon 1 is quite conserved and Tat exon 2 has important conserved motifs, which can behave as potent drug t argets. Our review recommends to focus over tat gene, which might prove as a lead in HIV research, which should be treated with ssense of urgency.

The PI3K/Akt pathway is involved in early infection of some exogenous avian leukosis viruses

Avian leukosis virus (ALV) is an enveloped and oncogenic retrovirus. Avian leukosis caused by the members of ALV subgroups A, B and J has become one of the major problems challenging the poultry industry in China. However, the cellular factors such as signal transduction pathways involved in ALV infection are not well defined. In this study, our data demonstrated that ALV-J strain NX0101 infection in primary chicken embryo fibroblasts or DF-1 cells was correlated with the activity and phosphorylation of Akt. Akt activation was initiated at a very early stage of infection independently of NX0101 replication. The specific phosphatidylinositol 3-kinase (PI3K) inhibitors LY294002 or wortmannin could suppress Akt phosphorylation, indicating that NX0101-induced Akt phosphorylation is PI3K-dependent. ALV-A strain GD08 or ALV-B strain CD08 infection also demonstrated a similar profile of PI3K/Akt activation. Treatment of DF-1 cells with the drug 5-(N, N-hexamethylene) amiloride that inhibits the activity of chicken Na(+)/H(+) exchanger type 1 significantly reduced Akt activation induced by NX0101, but not by GD08 and CD08. Akt activation triggered by GD08 or CD08 was abolished by clathrin-mediated endocytosis inhibitor chlorpromazine. Receptor-mediated endocytosis inhibitor dansylcadaverine had a negligible effect on all ALV-induced Akt phosphorylation. Moreover, viral replication of ALV was suppressed by LY294002 in a dose-dependent manner, which was due to the inhibition of virus infection by LY294002. These data suggest that the activation of the PI3K/Akt signalling pathway by exogenous ALV infection plays an important role in viral entry, yet the precise mechanism remains under further investigation.

Probing HIV-1 Membrane Liquid Order by Laurdan Staining Reveals Producer Cell-dependent Differences

Viruses acquire their envelope by budding from a host cell membrane, but viral lipid composition may differ from that of the budding membrane. We have previously reported that the HIV-1 membrane is highly enriched in cholesterol, sphingolipids, and other raft lipids, suggesting that the virus may bud from pre-existing or virus-induced lipid rafts. Here, we employed the environmentally sensitive fluorescent dye Laurdan to study the membrane lateral structure of HIV-1 derived from different cell lines. Differences in viral membrane order detected by Laurdan staining were shown by mass spectrometry to be due to differences in lipid composition. Isogenic viruses from two different cell lines were both strongly enriched in raft lipids and displayed a liquid-ordered membrane, but these effects were significantly more pronounced for HIV-1 from the T-cell line MT-4 compared with virus from 293T cells. Host-dependent differences in the lipidomes predominantly affected the ratio of sphingomyelins (including dihydrosphingomyelin) to phosphatidylcholine, whereas cholesterol contents were similar. Accordingly, treatment of infectious HIV-1 with the sphingomyelin-binding toxins Equinatoxin-II or lysenin showed differential inhibition of infectivity. Liposomes consisting of lipids that had been extracted from viral particles exhibited slightly less liquid order than the respective viral membranes, which is likely to be due to absence of membrane proteins and to loss of lipid asymmetry. Synthetic liposomes consisting of a quaternary lipid mixture emulating the viral lipids showed a liquid order similar to liposomes derived from virion lipids. Thus, Laurdan staining represents a rapid and quantitative method to probe viral membrane liquid order and may prove useful in the search for lipid active drugs.

No strings attached: the ESCRT machinery in viral budding and cytokinesis

Since the initial discovery of the endosomal sorting complex required for transport (ESCRT) pathway, research in this field has exploded. ESCRT proteins are part of the endosomal trafficking system and play a crucial role in the biogenesis of multivesicular bodies by functioning in the formation of vesicles that bud away from the cytoplasm. Subsequently, a surprising role for ESCRT proteins was defined in the budding step of some enveloped retroviruses, including HIV-1. ESCRT proteins are also employed in this outward budding process, which results in the resolution of a membranous tether between the host cell and the budding virus particle. Remarkably, it has recently been described that ESCRT proteins also have a role in the topologically equivalent process of cell division. In the same way that viral particles recruit the ESCRT proteins to the site of viral budding, ESCRT proteins are also recruited to the midbody - the site of release of daughter cell from mother cell during cytokinesis. In this Commentary, we describe recent advances in the understanding of ESCRT proteins and how they act to mediate these diverse processes.

Investigation of HIV‐1 Protein‐Lipid Interactions During Assembly at the Plasma Membrane

Human immunodeficiency virus (HIV‐1) is an enveloped retrovirus that infects T‐helper lymphocytes and leads to acquired immune deficiency syndrome (AIDS) through destruction of this crucial arm of the human immune system. HIV‐1 replicates through virion assembly and budding at the plasma membrane of T‐helper lymphocytes, leading to virus proliferation and lymphocyte depletion. Several virus‐encoded proteins direct this process, including the p17 matrix domain (MA). This 17‐kDa protein binds to the plasma membrane and is essential for membrane localization, assembly, and budding of nascent virions. Recent structural, computational, and cell studies have provided insight into how HIV‐1 MA is targeted to the plasma membrane. This study supplements previous research by providing biophysical data on the membrane binding properties of MA. We demonstrate the lipid affinity, specificity, and membrane penetration of MA in addition to showing how phosphorylation events may mediate membrane targeting. Together, these data help elucidate the mechanism of MA targeting to the plasma membrane and the role matrix‐lipid interactions play in the assembly and budding of HIV‐1 virions. Further examination of matrix‐membrane interactions will provide a greater understanding of the role of lipids in the viral life cycle and of their potential as targets for exciting and novel therapeutic strategies.

The ESCRT machinery: new functions in viral and cellular biology

The ESCRT (endosomal sorting complex required for transport) machinery consists of a number of cytosolic proteins that make up three functional subcomplexes: ESCRT-I, ESCRT-II and ESCRT-III. These proteins function in multivesicular body formation and cell division and are co-opted by enveloped retroviruses to facilitate viral egress. Analysis of these functions may help illuminate conserved mechanisms of ESCRT function.

Discrimination between exosomes and HIV-1: Purification of both vesicles from cell-free supernatants

Although enveloped retroviruses bud from the cell surface of T lymphocytes, they use the endocytic pathway and the internal membrane of multivesicular bodies for their assembly and release from macrophages and dendritic cells (DCs). Exosomes, physiological nanoparticles produced by hematopoietic cells, egress from this same pathway and are similar to retroviruses in terms of size, density, the molecules they incorporate and their ability to activate immune cells. Retroviruses are therefore likely to contaminate in vitro preparations of exosomes and vice versa and sucrose gradients are inefficient at separating them. However, we have found that their sedimentation velocities in an iodixanol (Optiprep™) velocity gradient are sufficiently different to allow separation and purification of both vesicles. Using acetylcholinesterase as an exosome marker, we demonstrate that Optiprep™ velocity gradients are very efficient in separating exosomes from HIV-1 particles produced on 293T cells, primary CD4 + T cells, macrophages or DCs, with exosomes collecting at 8.4–12% iodixanol and HIV-1 at 15.6%. We also show that immunodepletion with an anti-acetylcholinesterase antibody rapidly produces highly purified preparations of HIV-1 or exosomes. These findings have applications in fundamental research on exosomes and/or AIDS, as well as in clinical applications where exosomes are involved, more specifically in tumour therapy or in gene therapy using exosomes generated from DCs genetically modified by transfection with virus.

A Novel Requirement for C. elegans Alix/ALX-1 in RME-1-Mediated Membrane Transport

Alix/Bro1p family proteins have recently been identified as important components of multivesicular endosomes (MVEs) and are involved in the sorting of endocytosed integral membrane proteins, interacting with components of the ESCRT complex, the unconventional phospholipid LBPA, and other known endocytosis regulators. During infection, Alix can be co-opted by enveloped retroviruses, including HIV, providing an important function during virus budding from the plasma membrane. In addition, Alix is associated with the actin cytoskeleton and might regulate cytoskeletal dynamics. Here we demonstrate a novel physical interaction between the only apparent Alix/Bro1p family protein in C. elegans, ALX-1, and a key regulator of receptor recycling from endosomes to the plasma membrane, called RME-1. The analysis of alx-1 mutants indicates that ALX-1 is required for the endocytic recycling of specific basolateral cargo in the C. elegans intestine, a pathway previously defined by the analysis of rme-1 mutants. The expression of truncated human Alix in HeLa cells disrupts the recycling of major histocompatibility complex class I, a known Ehd1/RME-1-dependent transport step, suggesting the phylogenetic conservation of this function. We show that the interaction of ALX-1 with RME-1 in C. elegans, mediated by RME-1/YPSL and ALX-1/NPF motifs, is required for this recycling process. In the C. elegans intestine, ALX-1 localizes to both recycling endosomes and MVEs, but the ALX-1/RME-1 interaction appears to be dispensable for ALX-1 function in MVEs and/or late endosomes. This work provides the first demonstration of a requirement for an Alix/Bro1p family member in the endocytic recycling pathway in association with the recycling regulator RME-1.

HIV interaction with endosomes in macrophages and dendritic cells

The human immunodeficiency virus type 1 (HIV-1) is an enveloped retrovirus that undergoes assembly at specific sites in infected cells. In macrophages, at least, this assembly occurs primarily on a subset of endocytic organelles that contain some of the markers found in late endosomes or multivesicular bodies (MVBs), in particular CD63. The budding of virus particles into endosomes has many features in common with the formation of exosomes and some limited biochemical comparison of HIV-1 particles produced from macrophages with exosomes suggests that the two have similar cellular origins. Here we show that macrophages infected with HIV contain large intracellular pools of infectious virus that can be released by homogenisation of intact cells. Immunoprecipitation experiments indicate this virus has a similar complement of cellular membrane proteins to viruses that can be recovered from the extracellular medium, further suggesting that viruses released from macrophages initially bud into endosomal organelles and are then released by fusion of these organelles with the plasma membrane.

469. Induction of Donor Specific Tolerance Using Gene Therapy and Sublethal Conditioning

Top of pageAbstract Introduction: Induction of molecular chimerism following genetic engineering of autologous hematopoietic stem cells can be used to induce transplantation tolerance following reconstitution of myeloablated hosts. In this study we set out to determine the minimum conditioning requirements to induce molecular chimerism and achieve donor-specific tolerance. Methods: B10.AKM (H-2Kk, Ik, Dq) mice received either 3, 6 or 10 Gy of whole body irradiation and were reconstituted the next day with either 4|[times]|106 mock or VSV-Kb (VSV-G enveloped retroviruses carrying the gene encoding H-2Kb (Kb)) transduced syngeneic bone marrow. In addition, recipient mice received 0.5 mg of anti-CD154 mAb (MR1) on the day of reconstitution and a second dose 3 days later. Reconstituted mice were then examined to determine expression of Kb on bone marrow derived cells and whether donor specific tolerance was induced by analyzing Kb-specific T cell responses using cytokine ELISPOT and CTL assays and by grafting with both donor-type and third party skin. Results: sixty percent of mice treated with 6 Gy of irradiation alone became chimeric and expressed Kb on 1.8 |[plusmn]| 1.7% of their peripheral blood leukocytes (PBL). The addition of anti-CD154 mAb allowed stable, multi-lineage chimerism to develop with as little as 3 Gy of irradiation, and increased both the percentage of mice that became chimeric (100%) as well as the percentage of Kb expression in the PBL (5.3 |[plusmn]| 2.6 %). T cells from mice conditioned with 3 Gy, anti-CD154 and VSV-Kb transduced bone marrow were unable to lyse targets expressing Kb, and contained substantially fewer Kb-reactive IL-2 and IFN-|[gamma]| producing CD4 T cells. Furthermore, this sublethal conditioning regimen induced donor specific tolerance in chimeric recipients as evidenced by the permanent survival of MHC class I disparate B10.MBR (H-2Kb, Ik, Dq) skin grafts. All mice promptly rejected third party BALB/c (H-2Kd) skin grafts. Conclusions: Sublethal conditioning and costimulation molecule blockade combined with genetic manipulation of autologous bone marrow is a new and potent way to induce donor specific tolerance. The use of genetically modified autologous bone marrow eliminates the possibility of inducing graft versus host disease which is encountered in allogeneic bone marrow transplants.

Affinity recovery of Moloney Murine Leukaemia Virus

Lipid enveloped retroviruses such as Moloney Murine Leukaemia Virus (MoMuLV) are commonly used gene therapy vectors. Downstream processing protocols used for their purification are time consuming and a potentially generic, single step capture method for the recovery of retroviral particles is proposed that exploits streptavidin–biotin affinity chromatography. The ability of four conventional adsorbent solid phases, Fractogel ®, Sepharose, Magnespheres ® and STREAMLINE immobilised with streptavidin, to capture and recover biotinylated Moloney Murine Leukaemia Virus was studied. MoMuLV can be biotinylated whilst retaining infectivity and the biotinylated virus can be adsorbed to Streptavidin Magnespheres yielding a 2298-fold increase in titre. For optimal virus biotinylation purification using Fractogel ® streptavidin can yield a 1896-fold increase in cfu/mg of protein and a 1191-fold decrease in DNA/cfu. Infectious virus can be recovered from Fractogel ® streptavidin with a maximum recovery of 16.7%.