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In the production of monoclonal antibodies (mAbs) intended for use in humans, it is a global regulatory requirement that the manufacturing process includes unit operations that are proven to inactivate or remove adventitious agents to ensure viral safety. Viral inactivation by low pH hold (LPH) is typically used to ensure this viral safety in the purification process of mAbs and other biotherapeutics derived from mammalian cell lines. To ascertain the effectiveness of the LPH step, viral clearance studies have evaluated LPH under worst-case conditions of pH above the manufacturing set point and hold duration at or below the manufacturing minimum. Highly acidic conditions (i.e., pH < 3.60) provide robust and effective enveloped virus inactivation but may lead to reduced product quality of the therapeutic protein. However, when viral inactivation is operated above pH 3.60 to ensure product stability, effective (>4 log 10 reduction factor) viral inactivation may not be observed under these worst-case pH conditions in viral clearance studies. A multivariate design of experiments was conducted to further characterize the operating space for low pH viral inactivation of a model retrovirus, xenotropic murine leukemia virus (X-MuLV). The statistically designed experiment evaluated the effect of mAb isotype, pH, temperature, acid titrant, sodium chloride (NaCl) concentration, virus spike timing, and post-spike filtration on X-MuLV inactivation. Data from the characterization study were used to generate predictive models to identify conditions that reliably achieve effective viral inactivation at pH ≥ 3.60. Results of the study demonstrated that NaCl concentration has the greatest effect on virus inactivation in the range studied, and pH has a large effect when the load material has no additional NaCl. Overall, robust and effective inactivation of X-MuLV at pH 3.65-3.80 can be achieved by manipulating either the pH or the NaCl concentration of the load material. This study contributes to the understanding of ionic strength as an influential parameter in low pH viral inactivation studies., (© 2023 Wiley Periodicals LLC.)

Multi-column capture chromatography (MCC) has gained increased attention lately due to the significant economic and process-related advantages it offers compared to traditional batch mode chromatography. However, for wide adoption of this technology in the clinical and commercial space, it requires scalable models for viral validation. In this study, additional viral validation studies were conducted under cGLP guidelines to assess retro-(X-MuLV) and parvo-virus (minute virus of mice) clearance across twin-column continuous capture chromatography (CaptureSMB) to supplement work previously performed. A surrogate model was validated using standard batch mode chromatography equipment based on flow path modifications to mimic the loading strategy employed in CaptureSMB. In addition, aged resin was used in this surrogate format to assess the impact of resin lifetime on viral clearance during continuous capture operation. The impact of column loading was also explored to shed light on the viral clearance mechanisms of protein A chromatography in overloading conditions. The proposed approach greatly simplifies MCC virus validation studies, and provides a robust strategy for regulatory filing of continuous biomanufacturing processes., (© 2021 Wiley Periodicals LLC.)

We evaluated filtration behavior and virus removal capability for a monoclonal antibodies (mAb) and plasma IgG under constant flow rate directly following flow-through column chromatography in an integrated process. mAb solution with quantified host cell protein (HCP) content processed in flow-through mode on in-series mixed-mode AEX and modified CEX columns connected to the Planova BioEX filter (pool-less) achieved HCP logarithmic reduction value (LRV) of 2.3 and 93.9% protein recovery, demonstrating comparable or higher HCP LRV with high protein recovery compared to previous reports. For 5-15 mg/ml plasma IgG run to 100 L/m 2 , similar filtration behavior was achieved for flux of 10-100 LMH, and lower flux runs remained well below the maximum operating pressure, suggesting that higher throughput in continuous processing is achievable. Comparison of fit of plasma IgG and mAb filtration behavior to four blocking models showed little differences but slightly better fit to the cake filtration model. Viral clearance of the filtration step tested by in-line spiking X-MuLV or MVM into purified plasma IgG following the chromatography step showed robust removal at low flux. Integrating the Planova BioEX filter into continuous processes with column chromatography can achieve efficient downstream processing with reduced footprint and process time., (© 2021 The Authors. Biotechnology and Bioengineering Published by Wiley Periodicals LLC.)

Multicolumn capture chromatography is gaining increased attention lately due to the significant economic and process advantages it offers compared with traditional batch mode chromatography. However, for wide adoption of this technology in clinical and commercial space, it requires scalable models for executing viral validation studies. In this study, viral validation studies were conducted under cGLP guidelines to assess retro- (X-MuLV) and parvo-virus (MVM) clearance across twin-column continuous capture chromatography (CaptureSMB). A surrogate model was also developed using standard batch mode chromatography based on flow path modifications to mimic the loading strategy used in CaptureSMB. The results show that a steady state was achieved by the second cycle for both antibody binding and virus clearance and that the surrogate model using batch mode chromatography equipment provided impurity clearance that was comparable to that obtained during cyclical operation of CaptureSMB. Further, the log reduction values (LRVs) achieved during CaptureSMB were also comparable to the LRVs obtained using standard batch capture chromatography. This was expected since the mode of virus separation during protein A chromatography is primarily based on removal during the flow through and wash steps. Finally, this study also presents assessments on the resin cleaning strategy during continuous chromatography and how the duration of clean-in-place solution exposure impacts virus carryover., (© 2019 Wiley Periodicals, Inc.)

Retroviruses assemble and bud from infected cells in an immature form and require proteolytic maturation for infectivity. The CA (capsid) domains of the Gag polyproteins assemble a protein lattice as a truncated sphere in the immature virion. Proteolytic cleavage of Gag induces dramatic structural rearrangements; a subset of cleaved CA subsequently assembles into the mature core, whose architecture varies among retroviruses. Murine leukemia virus (MLV) is the prototypical γ-retrovirus and serves as the basis of retroviral vectors, but the structure of the MLV CA layer is unknown. Here we have combined X-ray crystallography with cryoelectron tomography to determine the structures of immature and mature MLV CA layers within authentic viral particles. This reveals the structural changes associated with maturation, and, by comparison with HIV-1, uncovers conserved and variable features. In contrast to HIV-1, most MLV CA is used for assembly of the mature core, which adopts variable, multilayered morphologies and does not form a closed structure. Unlike in HIV-1, there is similarity between protein-protein interfaces in the immature MLV CA layer and those in the mature CA layer, and structural maturation of MLV could be achieved through domain rotations that largely maintain hexameric interactions. Nevertheless, the dramatic architectural change on maturation indicates that extensive disassembly and reassembly are required for mature core growth. The core morphology suggests that wrapping of the genome in CA sheets may be sufficient to protect the MLV ribonucleoprotein during cell entry., Competing Interests: The authors declare no conflict of interest., (Copyright © 2018 the Author(s). Published by PNAS.)

The assembly of immature retroviral particles is initiated in the cytoplasm by the binding of the structural polyprotein precursor Gag with viral genomic RNA. The protein interactions necessary for assembly are mediated predominantly by the capsid (CA) and nucleocapsid (NC) domains, which have conserved structures. In contrast, the structural arrangement of the CA-NC connecting region differs between retroviral species. In HIV-1 and Rous sarcoma virus, this region forms a rod-like structure that separates the CA and NC domains, whereas in Mason-Pfizer monkey virus, this region is densely packed, thus holding the CA and NC domains in close proximity. Interestingly, the sequence connecting the CA and NC domains in gammaretroviruses, such as murine leukemia virus (MLV), is unique. The sequence is called a charged assembly helix (CAH) due to a high number of positively and negatively charged residues. Although both computational and deletion analyses suggested that the MLV CAH forms a helical conformation, no structural or biochemical data supporting this hypothesis have been published. Using an in vitro assembly assay, alanine scanning mutagenesis, and biophysical techniques (circular dichroism, NMR, microcalorimetry, and electrophoretic mobility shift assay), we have characterized the structure and function of the MLV CAH. We provide experimental evidence that the MLV CAH belongs to a group of charged, E(R/K)-rich, single α-helices. This is the first single α-helix motif identified in viral proteins., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)

Retroviral vectors gained popularity toward other viral vectors as they integrate their genome into hosts' genome, a characteristic required for the modification of stem cells. However, the production of viable particles for gene therapy is hampered by the low ratio of infectious to non-infectious viral particles after purification, low titers and limited number of competent viral receptors. We have developed de novo two fully synthetic triazine-based ligands that can selectively bind retroviral particles pseudotyped with amphotropic murine leukemia virus envelope (AMPHO4070A). A 78-membered library of triazine-based ligands was designed in silico and was virtually screened against the modeled structure of the AMPHO4070A protein. Ligands displaying the highest energy of binding were synthesized on cross-linked agarose and experimentally tested. Adsorbents containing ligands A5A10 and A10A11 showed selectivity toward viral particles containing the target protein (VLP-AMPHO), binding 19 ± 5 μg/g support and 47 ± 13 μg/g support, respectively. The elution conditions for both ligands were mild and with high recovery yields (80-100%), in comparison with common purification practices. These results were based on a lab-scale experimental setting with VLP integrity being confirmed through TEM. In particular, the elution buffer containing 12 mM imidazole allowed the recovery of intact amphotropic viral particles., (Copyright © 2016 Elsevier B.V. All rights reserved.)

HIV-1 Nef, a protein important for the development of AIDS, has well-characterized effects on host membrane trafficking and receptor downregulation. By an unidentified mechanism, Nef increases the intrinsic infectivity of HIV-1 virions in a host-cell-dependent manner. Here we identify the host transmembrane protein SERINC5, and to a lesser extent SERINC3, as a potent inhibitor of HIV-1 particle infectivity that is counteracted by Nef. SERINC5 localizes to the plasma membrane, where it is efficiently incorporated into budding HIV-1 virions and impairs subsequent virion penetration of susceptible target cells. Nef redirects SERINC5 to a Rab7-positive endosomal compartment and thereby excludes it from HIV-1 particles. The ability to counteract SERINC5 was conserved in Nef encoded by diverse primate immunodeficiency viruses, as well as in the structurally unrelated glycosylated Gag from murine leukaemia virus. These examples of functional conservation and convergent evolution emphasize the fundamental importance of SERINC5 as a potent anti-retroviral factor.

HIV-1 Nef and the unrelated mouse leukaemia virus glycosylated Gag (glycoGag) strongly enhance the infectivity of HIV-1 virions produced in certain cell types in a clathrin-dependent manner. Here we show that Nef and glycoGag prevent the incorporation of the multipass transmembrane proteins serine incorporator 3 (SERINC3) and SERINC5 into HIV-1 virions to an extent that correlates with infectivity enhancement. Silencing of both SERINC3 and SERINC5 precisely phenocopied the effects of Nef and glycoGag on HIV-1 infectivity. The infectivity of nef-deficient virions increased more than 100-fold when produced in double-knockout human CD4(+) T cells that lack both SERINC3 and SERINC5, and re-expression experiments confirmed that the absence of SERINC3 and SERINC5 accounted for the infectivity enhancement. Furthermore, SERINC3 and SERINC5 together restricted HIV-1 replication, and this restriction was evaded by Nef. SERINC3 and SERINC5 are highly expressed in primary human HIV-1 target cells, and inhibiting their downregulation by Nef is a potential strategy to combat HIV/AIDS.

Virus filtration (VF) is a key step in an overall viral clearance process since it has been demonstrated to effectively clear a wide range of mammalian viruses with a log reduction value (LRV) > 4. The potential to achieve higher LRV from virus retentive filters has historically been examined using bacteriophage surrogates, which commonly demonstrated a potential of > 9 LRV when using high titer spikes (e.g. 10(10) PFU/mL). However, as the filter loading increases, one typically experiences significant decreases in performance and LRV. The 9 LRV value is markedly higher than the current expected range of 4-5 LRV when utilizing mammalian retroviruses on virus removal filters (Miesegaes et al., Dev Biol (Basel) 2010;133:3-101). Recent values have been reported in the literature (Stuckey et al., Biotech Progr 2014;30:79-85) of LRV in excess of 6 for PPV and XMuLV although this result appears to be atypical. LRV for VF with therapeutic proteins could be limited by several factors including process limits (flux decay, load matrix), virus spike level and the analytical methods used for virus detection (i.e. the Limits of Quantitation), as well as the virus spike quality. Research was conducted using the Xenotropic-Murine Leukemia Virus (XMuLV) for its direct relevance to the most commonly cited document, the International Conference of Harmonization (ICH) Q5A (International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use, Geneva, Switzerland, 1999) for viral safety evaluations. A unique aspect of this work is the independent evaluation of the impact of retrovirus quality and virus spike level on VF performance and LRV. The VF studies used XMuLV preparations purified by either ultracentrifugation (Ultra 1) or by chromatographic processes that yielded a more highly purified virus stock (Ultra 2). Two monoclonal antibodies (Mabs) with markedly different filtration characteristics and with similar levels of aggregate (<1.5%) were evaluated with the Ultra 1 and Ultra 2 virus preparations utilizing the Planova 20 N, a small virus removal filter. Impurities in the virus preparation ultimately limited filter loading as measured by determining the volumetric loading condition where 75% flux decay is observed versus initial conditions (V75). This observation occurred with both Mabs with the difference in virus purity more pronounced when very high spike levels were used (>5 vol/vol %). Significant differences were seen for the process performance over a number of lots of the less-pure Ultra 1 virus preparations. Experiments utilizing a developmental lot of the chromatographic purified XMuLV (Ultra 2 Development lot) that had elevated levels of host cell residuals (vs. the final Ultra 2 preparations) suggest that these contaminant residuals can impact virus filter fouling, even if the virus prep is essentially monodisperse. Process studies utilizing an Ultra 2 virus with substantially less host cell residuals and highly monodispersed virus particles demonstrated superior performance and an LRV in excess of 7.7 log10 . A model was constructed demonstrating the linear dependence of filtration flux versus filter loading which can be used to predict the V75 for a range of virus spike levels conditions using this highly purified virus. Fine tuning the virus spike level with this model can ultimately maximize the LRV for the virus filter step, essentially adding the LRV equivalent of another process step (i.e. protein A or CEX chromatography)., (© 2014 American Institute of Chemical Engineers.)

Recombinant retroviruses provide highly efficient gene delivery and the potential for sustained gene expression, but suffer from significant disadvantages including low titer, expensive production, poor stability and limited flexibility for modification of tropism. In contrast, polymer-based vectors are more robust and allow cell- and tissue-specific deliveries via conjugation of ligands, but are comparatively inefficient. The design of hybrid gene delivery agents comprising both virally derived and synthetic materials (nanobiovectors) represents a promising approach to development of safe and efficient gene therapy vectors. Non-infectious murine leukemia virus-like particles (M-VLPs) were electrostatically complexed with chitosan (χ) to replace the function of the viral envelope protein. At optimal fabrication conditions and compositions, ranging from 6 to 9μg chitosan/10(9) M-VLPs at 10×10(9)M-VLPs/ml to 40μg chitosan/10(9) M-VLPs at 2.5×10(9)M-VLPs/ml, χ/M-VLPs were ~300-350nm in diameter and exhibited efficient transfection similar to amphotropic MLV vectors. In addition, these nanobiovectors were non-cytotoxic and provided sustained transgene expression for at least three weeks in vitro. This combination of biocompatible synthetic agents with inactive viral particles to form a highly efficient hybrid vector is a significant extension in the development of novel gene delivery platforms., (Copyright © 2014 Elsevier B.V. All rights reserved.)

We have developed nanoparticles based on Murine Leukemia Virus virus-like-particles (VLPs) that efficiently deliver therapeutic bioactive proteins in their native state into target cells. Nuclear transcription factors and toxic proteins were incorporated into the VLPs from stable producer cells without transducing viral-encoded genetic material. Delivery of nuclear transcription factors required incorporation of nuclear export signals (NESs) into the vector backbone for the efficient formation of VLPs. In the presence of an appropriate targeting Env glycoprotein, transcription factors delivered and activated nuclear transcription in the target cells. Additionally, we show delivery of the bacterial toxin, MazF, which is an ACA-specific mRNA interferase resulted in the induction of cell death. The stable producer cells are protected from the toxin through co-expression of the anti-toxin MazE and continuously released MazF incorporating VLPs. This highly adaptable platform can be harnessed to alter and regulate cellular processes by bioactive protein delivery., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

The ability of an anion exchange membrane to purify a γ-retrovirus was assessed and optimised with respect to different loading and wash buffers. Recoveries of infectious virus greater than 50% were consistently obtained, while specific titre was increased up to one thousand fold when compared to the material loaded. Specific proteins removed and retained by this optimised process were identified by mass spectrometry. It was possible to successfully bind and elute the equivalent of 1.27 × 10(8) Ifu/ml of ion exchange membrane. This could then be highly concentrated, with infectious virus concentrated to a maximum of 420-fold compared to the load., (Copyright © 2014 The Authors. Published by Elsevier B.V. All rights reserved.)

N-terminal myristoylation of retroviral matrix proteins is essential for the targeting of the Gag polyproteins to the plasma membrane. To investigate the effect of the myristoylation on the structure and membrane binding ability of the matrix proteins, it is necessary to prepare their myristoylated forms. We present purification of myristoylated matrix proteins of the mouse mammary tumor virus and murine leukemia virus, two morphogenetically distinct retroviruses. The proteins were expressed in Escherichia coli coexpressing a yeast N-myristoyltransferase. This E. coli expression system yielded a mixture of myristoylated and nonmyristoylated matrix proteins. We established efficient one-step metal affinity purification that enabled to obtain pure myristoylated matrix proteins suitable for structural and functional studies., (Copyright © 2013 Elsevier Inc. All rights reserved.)

To better understand how detergents disrupt enveloped viruses, we monitored the biophysical stability of murine leukemia virus (MLV) virus-like particles (VLPs) against a panel of commonly used detergents using real-time biosensor measurements. Although exposure to many detergents, such as Triton X-100 and Empigen, results in lysis of VLP membranes, VLPs appeared resistant to complete membrane lysis by a significant number of detergents, including Tween 20, Tween 80, Lubrol, and Saponin. VLPs maintained their structural integrity after exposure to Tween 20 at concentrations up to 500-fold above its CMC. Remarkably, VLPs containing immature cores composed of unprocessed (uncleaved) Gag polyprotein were significantly more resistant to detergent lysis than VLPs with mature cores. Although the maturity of retroviral Gag is known to influence the stability of the protein core structure itself, our studies suggest that the maturity of the Gag core also influences the stability of the lipid bilayer surrounding the core., (Copyright © 2012 Elsevier Inc. All rights reserved.)

We have recently shown that reverse transcriptases (RTs) perform template switches when there is a very short (two-nucleotide) complementarity between the 3' ends of the primer (donor) strand and the DNA or RNA template acceptor strands [Oz-Gleenberg et al. (2011) Nucleic Acids Res 39, 1042-1053]. These dinucleotide pairs are stabilized by RTs that are capable of 'clamping' together the otherwise unstable duplexes. This RT-driven stabilization of the micro-homology sequence promotes efficient DNA synthesis. In the present study, we have examined several factors associated with the sequence and structure of the DNA substrate that are critical for the clamp activity of RTs from human immunodeficiency virus type 1 (HIV-1), murine leukemia virus (MLV), bovine immunodeficiency virus (BIV) and the long terminal repeat retrotransposon Tf1. The parameters studied were the minimal complementarity length between the primer and functional template termini that sustains stable clamps, the effects of gaps between the two template strands on the clamp activity of the tested RTs, the effects of template end phosphorylations on the RT-associated clamp activities, and clamp activity with a long 'hairpin' double-stranded primer comprising both the primer and the complementary non-functional template strands. The results show that the substrate conditions for clamp activity of HIV-1 and MLV RTs are more stringent, while Tf1 and BIV RTs show clamp activity under less rigorous substrate conditions. These differences shed light on the dissimilarities in catalytic activities of RTs, and suggest that clamp activity may be a potential new target for anti-retroviral drugs., (© 2012 The Authors Journal compilation © 2012 FEBS.)

The human immunodeficiency virus (HIV) is an enveloped virus constituted by two monomeric RNA molecules that encode for 15 proteins. Among these are the structural proteins that are translated as the gag polyprotein. In order to become infectious, HIV must undergo a maturation process mediated by the proteolytic cleavage of gag to give rise to the isolated structural protein matrix, capsid (CA), nucleocapsid as well as p6 and spacer peptides 1 and 2. Upon maturation, the 13 N-terminal residues from CA fold into a β-hairpin, which is stabilized mainly by a salt bridge between Pro1 and Asp51. Previous reports have shown that non-formation of the salt bridge, which potentially disrupts proper β-hairpin arrangement, generates noninfectious virus or aberrant cores. To date, however, there is no consensus on the role of the β-hairpin. In order to shed light in this subject, we have generated mutations in the hairpin region to examine what features would be crucial for the β-hairpin's role in retroviral mature core formation. These features include the importance of the proline at the N-terminus, the amino acid sequence, and the physical structure of the β-hairpin itself. The presented experiments provide biochemical evidence that β-hairpin formation plays an important role in regard to CA protein conformation required to support proper mature core arrangement. Hydrogen/deuterium exchange and in vitro assembly reactions illustrated the importance of the β-hairpin structure, its dynamics, and its influence on the orientation of helix 1 for the assembly of the mature CA lattice., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Murine leukemia viruses (MuLVs) encode two forms of Gag polyprotein: the precursor for the viral core proteins (Pr65(gag) for Moloney MuLV [M-MuLV]) and a longer glycosylated form (glyco-gag, or gPr80(gag)). gPr80(gag) is translated from the same unspliced viral RNA as Pr65(gag), from an upstream in-frame CUG initiation codon. As a result, gPr80(gag) contains 88 unique N-terminal amino acids that include a signal peptide that conducts gPr80(gag) into the rough endoplasmic reticulum, where it is glycosylated, exported to the cell surface, and cleaved into two proteins of 55 and 40 kDa. The amino-terminal 55-kDa protein remains cell associated with the 88 unique amino acids exposed to the cytosol. We previously showed that gPr80(gag) facilitates efficient M-MuLV release through lipid rafts. In this report, we found that the unique N-terminal domain of gPr80(gag) is sufficient to facilitate enhanced M-MuLV particle release from transfected 293T cells. A search for cellular proteins involved in gPr80(gag) function led to cellular La protein. Overexpression of mouse or human La enhanced M-MuLV particle release in the absence of glyco-gag, and the released virus had a reduced buoyant density characteristic of increased cholesterol content. Moreover, small interfering RNA (siRNA) knockdown of human La abolished glyco-gag enhancement of M-MuLV release. These results implicate La as a cellular protein involved in M-MuLV glyco-gag function. We also found that overexpression of mouse or human La could enhance HIV-1 release in the absence of gPr80(gag). Therefore, M-MuLV and HIV-1 may share a pathway for release through lipid rafts involving La., (Copyright © 2011 Nitta et al.)

A mass spectrometry (MS) approach was used to analyze viral core proteins of the murine leukemia virus (MuLV)-based gene delivery vector. The retroviral particles produced by traditional methods were concentrated and purified by ultracentrifugation and spin column for matrix-assisted laser desorption ionization (MALDI) and electrospray ionization (ESI) MS. MALDI application detected all core MuLV proteins, partial degradation of p10, phosphorylation of p12, as well as the previously unknown formation of a polymeric supramolecular complex between p15 and p30 core proteins. ESI provided information on the post-translational modifications of MuLV core proteins. Data suggest myristoylation of p15 and oxidation of methionine residues in both p12 and p30, whereas cysteine residues in p10, p15 and p30 were not oxidized. The current study demonstrates that MALDI and ESI are efficient tools for viral core protein analysis and can be used as analytical tools in virology and biotechnology of gene delivery vectors., (Copyright © 2010. Published by Elsevier B.V.)

Background: 'Intrinsic' resistance to retroviral infection was first recognised with the Friend virus susceptibility gene (Fv1), which determines susceptibility to murine leukaemia virus (MLV) infection in different murine species. Similarly, the tripartite motif (TRIM) family of proteins determine lentiviral restriction in a primate host-species specific manner. For example rhesus TRIM5α (rhTRIM5α) can potently restrict HIV-1 infection while human TRIM5α (huTRIM5α) only has a mild effect on SIVmac and HIV-1 infectivity (Lv1). Human TRIM5α is able to restrict MLV-N virus replication, but is ineffective against MLV-B or MLV-NB virus infection. Lv2 restriction of some HIV-2 viruses is seen in human cells. Like Lv1, Lv2 is a post-entry restriction factor, whose viral determinants have been mapped to the viral capsid (CA). Unlike Lv1, however, Lv2 is determined by envelope (Env) in addition to CA. Here we present evidence of a novel Env determined post entry restriction to infection in human cells of pseudotyped MLV-B and MLV-NB cores., Results: We generated retroviral vectors pseudotyped with various gamma and lentiviral Envs on MLV-B and -NB CAs containing a green fluorescent protein (GFP) reporter. Flow cytometry was used to determine transduction efficiencies in NP2/CD4/CXCR4 (glioma cell line stably transduced with the HIV receptors) and HeLa/CD4 cell lines. The HeLa/CD4 cell line restricted both MLV CAs in an Env dependent manner, compared to NP2/CD4/CXCR4 cells. Quantitative polymerase chain reaction (QT-PCR) analysis of reverse transcription (RT) transcripts demonstrates that this restriction occurs at a post entry and RT level. siRNA knockdown of huTRIM5α ruled out a direct role for this cellular component in mediating this restriction. We describe a previously unobserved Env determined restriction of MLV-B and MLV-NB CAs in HeLa/CD4 cells when pseudotyped with HIV-2 and RD114 Envs, but not gibbon ape leukaemia virus (GALV), HIV-1 or Amphotrophic (Ampho) Envs., Conclusions: Our data further demonstrate the variability of Env and CA mediated susceptibility to post entry host cell restriction. We discuss the relevance of these findings in light of the growing evidence supporting the complexities involved in innate host immunity to retroviral infection.

Retroviruses acquire a lipid envelope during budding from the membrane of their hosts. Therefore, the composition of this envelope can provide important information about the budding process and its location. Here, we present mass spectrometry analysis of the lipid content of human immunodeficiency virus type 1 (HIV-1) and murine leukemia virus (MLV). The results of this comprehensive survey found that the overall lipid content of these viruses mostly matched that of the plasma membrane, which was considerably different from the total lipid content of the cells. However, several lipids are enriched in comparison to the composition of the plasma membrane: (i) cholesterol, ceramide, and GM3; and (ii) phosphoinositides, phosphorylated derivatives of phosphatidylinositol. Interestingly, microvesicles, which are similar in size to viruses and are also released from the cell periphery, lack phosphoinositides, suggesting a different budding mechanism/location for these particles than for retroviruses. One phosphoinositide, phosphatidylinositol 4,5-bisphosphate [PI(4,5)P(2)], has been implicated in membrane binding by HIV Gag. Consistent with this observation, we found that PI(4,5)P(2) was enriched in HIV-1 and that depleting this molecule in cells reduced HIV-1 budding. Analysis of mutant virions mapped the enrichment of PI(4,5)P(2) to the matrix domain of HIV Gag. Overall, these results suggest that HIV-1 and other retroviruses bud from cholesterol-rich regions of the plasma membrane and exploit matrix/PI(4,5)P(2) interactions for particle release from cells.

Virus-cell and cell-cell fusion events are affected by various properties of the fusogenic Env protein on the cell surface. The G541R mutation within the TM ectodomain of murine leukemia virus (MuLV) 4070A arose by positive selection in viral passage and results in a reduction of cell-cell fusion events while maintaining viral titer. Size exclusion chromatography shows that the multimerization properties are similar among expressed wild-type and mutant ectodomain peptides. Circular dichroism measurements reveal decreased thermal stability of the G541R mutant as compared to wild type. The G541R mutant also renders the peptide more susceptible to Lys-C protease cleavage. The 42-114 monoclonal antibody does not bind to the G541R mutant peptides, suggesting a structural difference from wild type. These altered physical properties result in productive viral infection of G541R bearing virus with decreased syncytia.

Owing to their small size, synthetic nanoparticles show unprecedented biophysical and biochemical properties which may foster novel advances in life-science research. Using flame-spray synthesis technology we have produced non-coated aluminum-, calcium-, cerium-, and zirconium-derived inorganic metal oxide nanoparticles which not only exhibit high affinity for nucleic acids, but can sequester such compounds from aqueous solution. This non-covalent DNA-binding capacity was successfully used to transiently transfect a variety of mammalian cells including human, reaching transfection efficiencies which compared favorably with classic calcium phosphate precipitation (CaP) procedures and lipofection. In this straightforward protocol, transfection was enabled by simply mixing nanoparticles with DNA in solution prior to addition to the target cell population. Transiently transfected cells showed higher production levels of the human secreted glycoprotein SEAP compared to isogenic populations transfected with established technologies. Inorganic metal oxide nanoparticles also showed a high binding capacity to human-pathogenic viruses including adenovirus, adeno-associated virus and human immunodeficiency virus type 1 and were able to clear these pathogens from aqueous solutions. The DNA transfection and viral clearance capacities of inorganic metal oxide nanoparticles may provide cost-effective biopharmaceutical manufacturing and water treatment in developing countries.

The cytoplasmic tail of the murine leukemia virus (MuLV) envelope (Env) protein is known to play an important role in regulating viral fusion activity. Upon removal of the C-terminal 16 amino acids, designated as the R peptide, the fusion activity of the Env protein is activated. To extend our understanding of the inhibitory effect of the R peptide and investigate the specificity of inhibition, we constructed chimeric influenza virus-MuLV hemagglutinin (HA) genes. The influenza virus HA protein is the best-studied membrane fusion model, and we investigated the fusion activities of the chimeric HA proteins. We compared constructs in which the coding sequence for the cytoplasmic tail of the influenza virus HA protein was replaced by that of the wild-type or mutant MuLV Env protein or in which the cytoplasmic tail sequence of the MuLV Env protein was added to the HA cytoplasmic domain. Enzyme-linked immunosorbent assays and Western blot analysis showed that all chimeric HA proteins were effectively expressed on the cell surface and cleaved by trypsin. In BHK21 cells, the wild-type HA protein had a significant ability after trypsin cleavage to induce syncytium formation at pH 5.1; however, neither the chimeric HA protein with the full-length cytoplasmic tail of MuLV Env nor the full-length HA protein followed by the R peptide showed any syncytium formation. When the R peptide was truncated or mutated, the fusion activity was partially recovered in the chimeric HA proteins. A low-pH conformational-change assay showed that similar conformational changes occurred for the wild-type and chimeric HA proteins. All chimeric HA proteins were capable of promoting hemifusion and small fusion pore formation, as shown by a dye redistribution assay. These results indicate that the R peptide of the MuLV Env protein has a sequence-dependent inhibitory effect on influenza virus HA protein-induced membrane fusion and that the inhibitory effect occurs at a late stage in fusion pore enlargement.

Human TRIM5alpha (TRIM5alpha(hu)) potently restricts N-tropic (N-MLV), but not B-tropic, murine leukemia virus in a manner dependent upon residue 110 of the viral capsid. Rhesus monkey TRIM5alpha (TRIM5alpha(rh)) inhibits N-MLV only weakly. The study of human-monkey TRIM5alpha chimerae revealed that both the v1 and v3 variable regions of the B30.2/SPRY domain contain potency determinants for N-MLV restriction. These variable regions are predicted to be surface-exposed elements on one face of the B30.2 domain. Acidic residues in v3 complement basic residue 110 of the N-MLV capsid. The results support recognition of the retroviral capsid by the TRIM5alpha B30.2 domain.

Retroviruses use endosomal machinery to bud out of infected cells, and various Gag proteins recruit this machinery by interacting with either of three cellular factors as follows: ubiquitin ligases of the Nedd4 family, Tsg101, or Alix/Aip1. Here we show that the murine leukemia virus Gag has the unique ability to interact with all three factors. Small interfering RNAs against Tsg101 or Alix and dominant-negative forms of Nedd4 can all reduce production of virus-like particles. However, inactivating the Nedd4-binding site abolishes budding, whereas disrupting Tsg101 or Alix binding has milder effects. Nedd4 ubiquitin ligases are therefore essential, and Tsg101 and Alix play auxiliary roles. Most interestingly, overexpression of Alix can stimulate the release of Gag, and this occurs independently of most Alix partners Tsg101, Cin85, Alg-2, and endophilins. In addition, Gag mutants that do not bind Tsg101 or Alix concentrate on late endosomes and become very sensitive to dominant-negative forms of Nedd4 that do not conjugate ubiquitin. This suggests that the direct interaction of Gag with Tsg101 and Alix favors budding from the plasma membrane and relieves a requirement for ubiquitination by Nedd4.1. Other Nedd4-dependent Gag proteins also contain binding sites for Tsg101 or Alix, suggesting that this could be a common feature of retroviruses.

The human APOBEC3G protein exhibits broad antiretroviral activity against a variety of retroviruses. It is packaged into viral particles and executes its antiviral function in the target cell. The packaging of APOBEC3G into different viral particles requires a mechanism that confers this promiscuity. Here, APOBEC3G incorporation into murine leukemia virus (MLV) was studied using retroviral vectors. APOBEC3G uptake did not require either its cytidine deaminase activity or the presence of a retroviral vector genome. Results from immunoprecipitation and co-localization studies of APOBEC3G with a MLV Gag-CFP (cyan fluorescent protein) fusion protein imply an interaction between both proteins. RNase A treatment did not inhibit the co-precipitation of Gag-CFP and APOBEC3G, suggesting that the interaction is RNA independent. Like human immunodeficiency virus (HIV) Gag, the MLV Gag precursor protein appears to interact with APOBEC3G, indicating that Gag contains conserved structures which are used to encapsidate APOBEC3G into different retroviral particles.

Heat shock protein 70 (Hsp70) is incorporated within the membrane of primate lentiviral virions. Here we demonstrate that Hsp70 is also incorporated into oncoretroviral virions and that it remains associated with membrane-stripped human immunodeficiency virus type 1 (HIV-1) virion cores. To determine if Hsp70 promotes virion infectivity, we attempted to generate Hsp70-deficient virions with gag deletion mutations, Hsp70 transdominant mutants, or RNA interference, but these efforts were confounded, largely because they disrupt virion assembly. Given that polypeptide substrates are bound and released by Hsp70 in an ATP-hydrolytic reaction cycle, we supposed that incubation of HIV-1 virions with ATP would perturb Hsp70 interaction with substrates in the virion and thereby decrease infectivity. Treatment with ATP or ADP had no observable effect, but ATPgammaS and GTPgammaS, nucleotide triphosphate analogues resistant to Hsp70 hydrolysis, dramatically reduced the infectivity of HIV-1 and murine leukemia virus virions. ATPgammaS-treated virions were competent for fusion with susceptible target cells, but viral cDNA synthesis was inhibited to an extent that correlated with the magnitude of decrease in infectivity. Intravirion reverse transcription by HIV-1, simian immunodeficiency virus, or murine leukemia virus was also inhibited by ATPgammaS. The effects of ATPgammaS on HIV-1 reverse transcription appeared to be indirect, resulting from disruption of virion core morphology that was evident by transmission electron microscopy. Consistent with effects on capsid conformation, ATPgammaS-treated viruslike particles failed to saturate host antiviral restriction activity. Our observations support a model in which the catalytic activity of virion-associated Hsp70 is required to maintain structural integrity of the virion core.

Murine leukemia viruses (MLVs) can be lymphomagenic and bone pathogenic. In this work, the possible roles of two distinct proviral enhancer nuclear factor 1 (NF1) binding sites in osteopetrosis and tumor induction by B-lymphomagenic Akv1-99 MLV were investigated. Akv1-99 and mutants either with NF1 site 1, NF1 site 2 or both sites disrupted induced tumors (plasma cell proliferations by histopathology) with remarkably similar incidence and mean latency in inbred NMRI mice. Clonal immunoglobulin gene rearrangement detection, by Southern analysis, confirmed approximately half of the tumors induced by each virus to be plasmacytomas while the remaining lacked detectable clonally rearranged Ig genes and were considered polyclonal; a demonstration that enhancer NF1 sites are dispensable for plasmacytoma induction by Akv1-99. In contrast, X-ray analysis revealed significant differences in osteopetrosis induction by the four viruses strongly indicating that NF1 site 2 is critical for viral bone pathogenicity, whereas NF1 site 1 is neutral or moderately inhibitory. In conclusion, enhancer NF1 sites are major determinants of osteopetrosis induction by Akv1-99 without significant influence on viral oncogenicity.

Retroviruses are the aetiological agents of a range of human diseases including AIDS and T-cell leukaemias. They follow complex life cycles, which are still only partly understood at the molecular level. Maturation of newly formed retroviral particles is an essential step in production of infectious virions, and requires proteolytic cleavage of Gag polyproteins in the immature particle to form the matrix, capsid and nucleocapsid proteins present in the mature virion. Capsid proteins associate to form a dense viral core that may be spherical, cylindrical or conical depending on the genus of the virus. Nonetheless, these assemblies all appear to be composed of a lattice formed from hexagonal rings, each containing six capsid monomers. Here, we describe the X-ray structure of an individual hexagonal assembly from N-tropic murine leukaemia virus (N-MLV). The interface between capsid monomers is generally polar, consistent with weak interactions within the hexamer. Similar architectures are probably crucial for the regulation of capsid assembly and disassembly in all retroviruses. Together, these observations provide new insights into retroviral uncoating and how cellular restriction factors may interfere with viral replication.

Retroviruses, such as human and simian immunodeficiency viruses (HIV and SIV), and murine leukemia viruses (MuLV), harbor a tyrosine-based motif in the intracytoplasmic domain of their envelope glycoprotein. This motif can act as an endocytosis signal or as a targeting signal, restricting viral budding at specific cell surface membrane domains. In the present study, proviral DNA of the ecotropic Cas-Br-E strain of MuLV was modified by substitution or deletion of the critical tyrosine residue. Mutant viruses lost basolateral targeting in polarized MDCK epithelial cells while expression level of the glycoprotein at the cell surface was not affected. This suggests that the tyrosine-based motif in MuLV does not act as an endocytosis signal. Only a small delay in the appearance of disease was observed in inoculated mice. In contrast, a striking change in the pathology was observed with enlarged thymus and lymph nodes in animals inoculated with mutant viruses.

The membrane fusion activity of murine leukaemia virus Env is carried by the transmembrane (TM) and controlled by the peripheral (SU) subunit. We show here that all Env subunits of the virus form disulphide-linked SU-TM complexes that can be disrupted by treatment with NP-40, heat or urea, or by Ca(2+) depletion. Thiol mapping indicated that these conditions induced isomerization of the disulphide-bond by activating a thiol group in a Cys-X-X-Cys (CXXC) motif in SU. This resulted in dissociation of SU from the virus. The active thiol was hidden in uninduced virus but became accessible for alkylation by either Ca(2+) depletion or receptor binding. The alkylation inhibited isomerization, virus fusion and infection. DTT treatment of alkylated Env resulted in cleavage of the SU-TM disulphide-bond and rescue of virus fusion. Further studies showed that virus fusion was specifically inhibited by high and enhanced by low concentrations of Ca(2+). These results suggest that Env is stabilized by Ca(2+) and that receptor binding triggers a cascade of reactions involving Ca(2+) removal, CXXC-thiol exposure, SU-TM disulphide-bond isomerization and SU dissociation, which lead to fusion activation.

Background: The mouse Fv1 (friend virus) susceptibility gene inhibits the development of the murine leukaemia virus (MLV) by interacting with its capsid (CA) protein. As no structures are available for these proteins we have constructed molecular models based on distant sequence similarity to other retroviral capsid proteins., Results: Molecular models were constructed for the amino terminal domains of the probable capsid-like structure for the mouse Fv1 gene product and the capsid protein of the MLV. The models were based on sequence alignments with a variety of other retrovirus capsid proteins. As the sequence similarity of these proteins with MLV and especially Fv1 is very distant, a threading method was employed that incorporates predicted secondary structure and multiple sequence information. The resulting models were compared with equivalent models constructed using the sequences of the capsid proteins of known structure., Conclusions: These comparisons suggested that the MLV model should be accurate in the core but with significant uncertainty in the loop regions. The Fv1 model may have some additional errors in the core packing of its helices but the resulting model gave some support to the hypothesis that it adopts a capsid-like structure.

Retroviruses encapsidate two copies of full-length viral RNA molecules linked together as a dimeric genome. RNA stem loop structures harboring palindromic (or "kissing") loop sequences constitute important cis-elements for viral dimerization known as dimer initiation sites (DIS). In murine leukemia virus (MLV), a 10-mer and a 16-mer palindrome (DIS-1 and DIS-2, respectively) located in the viral leader region mediate dimerization in vitro and affect dimer stability of vector RNA in vivo. We have investigated the effect on viral replication of introducing deletions or nucleotide substitutions within these palindromes in a full-length MLV genome. Our results demonstrate that viruses modified at the dimer initiation site regions are viable and show wild-type levels of RNA encapsidation. One mutant lacking the DIS-1 palindrome was severely impaired and displayed an increased cellular ratio of spliced versus genomic RNA that most likely contributes to the inefficient replication. The implications for development of DIS-modified retrovirus-based vectors are discussed.

RNA molecules have been much less studied by atomic force microscopy (AFM) than have DNA molecules. In this paper, AFM imaging is presented for two different RNA molecules able to self-assemble into complex supramolecular architectures. The first one is a molecular dimer of a 230-nt RNA fragment coming from the RNA genome of a murine leukaemia virus. The monomeric RNA fragment, which appears by AFM as an elongated structure with a mean aspect ratio of 1.4, assembles into a dimer of elongated structures through the formation of a 'kissing-loop' RNA interaction. The second one is a large supramolecular fibre formed of artificial self-assembling RNA molecular units called tectoRNA. The fibre lengths by AFM suggest that there are 50-70 tectoRNA units per fibre. Some methods and limitations are presented for measuring molecular volumes from AFM images.

Many mammalian species express restriction factors that confer host resistance to retroviral infection. Here we show that HIV-1 sensitivity to restriction factors is modulated by cyclophilin A (CypA), a host cell protein that binds the HIV-1 capsid protein (CA). In certain nonhuman primate cells, the CA-CypA interaction is essential for restriction: HIV-1 infectivity is increased >100-fold by cyclosporin A (CsA), a competitive inhibitor of the interaction, or by an HIV-1 CA mutation that disrupts CypA binding. Conversely, disruption of CA-CypA interaction in human cells reveals that CypA protects HIV-1 from the Ref-1 restriction factor. These findings suggest that HIV-1 has co-opted a host cell protein to counteract restriction factors expressed by human cells and that this adaptation can confer sensitivity to restriction in unnatural hosts. Manipulation of HIV-1 CA recognition by restriction factors promises to advance animal models and new therapeutic strategies for HIV-1 and AIDS.

The cytoplasmic domains of viral glycoproteins influence the trafficking and subcellular localization of the glycoproteins and their incorporation into virions. They also promote correct virus morphology and viral budding. The cytoplasmic domains of murine-leukemia-virus envelope-protein TM subunits regulate membrane fusion. During virion maturation the carboxy-terminal 16 amino acid residues of the TM protein are removed by the retroviral protease. Deletion of these residues activates envelope-protein-mediated membrane fusion. Our quantitative analysis of the effects of Moloney murine leukemia virus TM mutations on envelope-protein function support the proposition that a trimeric coiled coil in the TM cytoplasmic domain inhibits fusion. The data demonstrate that cleavage of the TM cytoplasmic domain is not required for viral entry and provide evidence for a model in which fusogenic and nonfusogenic conformations of the envelope protein exists in an equilibrium that is regulated by the cytoplasmic domain. In addition, a conserved tyrosine residue in the TM cytoplasmic domain was shown to play an important role in envelope-protein incorporation into retroviral particles.

The structure and function of the C-terminal domain of the murine leukemia virus Surface protein (MuLV SU) is not well defined. Passage of chimeric ecotropic-amphotropic MuLV viruses with junctions within the SU C-terminus results in the selection of specific point mutations which improve virus viability and Env function. Point mutations were characterized that alter the conformation of the SU/TM heterodimers on the viral particles. Mutation of position E311 within the Moloney MuLV SU protein alters the conformation of the TM protein and its recognition by antibody 42-114 in immunoprecipitation reactions. Mutation of either G541R in the amphotropic 4070A TM, V421M in the 4070A SU, or deletion of S39 and P40 at the N-terminus of the M-MuLV SU results in an irreversible cold-sensitive phenotype at 4 degrees C. This loss of viral titer can be restored by incorporating V421M plus G541R or del S39 P40 plus G541R in cis within the SU/TM.

A series of murine leukemia viruses (MuLVs) with chimeric envelope proteins (Env) was generated to map functional interactions between the N- and the C-terminal domains of surface proteins (SU). All these chimeras have the 4070A amphotropic receptor-binding region flanked by various lengths of Moloney ecotropic N- and C-terminal Env. A charged residue, E49 (E16 on the mature protein), was identified at the N-terminals of Moloney MuLV SU that is important for the interaction with the C-terminal domain of the SU. The region that interacts with E49 was localized between junction 4 (R265 of M-MuLV Env) and junction 6 (L374 of M-MuLV Env) of SU. Sequencing the viable chimeric Env virus populations identified residues within the SU protein that improved the replication kinetics of the input chimeric Env viruses. Mutations in the C-domain of SU (G387E/R, L435I, L442P) were found to improve chimera IV4, which displayed a delayed onset of replication. The replication of AE6, containing a chimeric junction in the SU C-terminus, was improved by mutations in the N-domain (N40H, E80K), the proline-rich region (Q252R), or the transmembrane protein (L538N). Altogether, these observations provide insights into the structural elements required for Env function.

Viral safety is a predominant concern for monoclonal antibodies (mAbs) and other recombinant proteins (RPs) with pharmaceutical applications. Certain commercial purification modules, such as nanofiltration and low-pH inactivation, have been observed to reliably clear greater than 4 log(10) of large enveloped viruses, including endogenous retrovirus. The concept of "bracketed generic clearance" has been proposed for these steps if it could be prospectively demonstrated that viral log(10) reduction value (LRV) is not impacted by operating parameters that can vary, within a reasonable range, between commercial processes. In the case of low-pH inactivation, a common step in mAb purification processes employed after protein A affinity chromatography, these parameters would include pH, time and temperature of incubation, the content of salts, protein concentration, aggregates, impurities, model protein pI, and buffer composition. In this report, we define bracketed generic clearance conditions, using a prospectively defined bracket/matrix approach, where low-pH inactivation consistently achieves >or=4.6 log(10) clearance of xenotropic murine leukemia virus (X-MLV), a model for rodent endogenous retrovirus. The mechanism of retrovirus inactivation by low-pH treatment was also investigated., (Copyright 2003 Wiley Periodicals, Inc.)

To delineate parameters contributing to the extracellular lifetime of retroviral vectors, we carried out stability tests of retroviruses derived from cell lines of different origin and kept under different cultivation conditions. Results show that amphotropic mouse retroviruses (MLV-A) derived from human and hamster cells exhibit 2- to 3-fold higher half-lives compared to retroviruses from mouse cells. Cultivation at 32 degrees C has been reported to yield high virus titers. However, the benefit of virus production in mouse cells at 32 degrees C is controversial. In our hands the cultivation temperature affected, hitherto not noticed, the half-life time of MLV-A. The 37/32 degrees C shift resulted in a 3-fold decrease of viral half-lifes compared to MLV-A released from mouse cells at 37 degrees C. Thus, MLV-A released at 37 degrees C is phenotypically different from MLV-A synthesized at 32 degrees C. Increased virus stability was inversely correlated with the level of cholesterol in the viral membrane. Finally, depletion of viral cholesterol in vitro resulted in intact virus with increased thermal stability. Thus, retrovirus lability depends on the host cell and parallels the cholesterol amount in the viral lipid shell.

Envelope glycoproteins (Env) of retroviruses are trimers of SU (surface) and TM (transmembrane) heterodimers and are expressed on virions in fusion-competent forms that are likely to be metastable. Activation of the viral receptor-binding domain (RBD) via its interaction with a cell surface receptor is thought to initiate a cascade of events that lead to refolding of the Env glycoprotein into its stable fusion-active conformation. While the fusion-active conformation of the TM subunit has been described in detail for several retroviruses, little is known about the fusion-competent structure of the retroviral glycoproteins or the molecular events that mediate the transition between the two conformations. By characterizing Env chimeras between the ecotropic and amphotropic murine leukemia virus (MLV) SUs as well as a set of point mutants, we show that alterations of the conformation of the SU glycoprotein strongly elevate Env fusogenicity by disrupting the stability of the Env complex. Compensatory mutations that restored both Env stability and fusion control were also identified, allowing definition of interactions within the Env complex that maintain the stability of the native Env complex. We show that, in the receptor-unbound form, structural interactions between the N terminus of the viral RBD (NTR domain), the proline-rich region (PRR), and the distal part of the C-terminal domain of the SU subunit maintain a conformation of the glycoprotein that is fusion inhibitory. Additionally, we identified mutations that disrupt this fusion-inhibitory conformation and allow fusion activation in the absence of viral receptors, provided that receptor-activated RBD fragments are added in trans during infection. Other mutations were identified that allow fusion activation in the absence of receptors for both the viral glycoprotein and the trans-acting RBD. Finally, we found mutations of the SU that bypass in cis the requirement for the NTR domain in fusion activation. All these different mutations call for a critical role of the PRR in mediating conformational changes of the Env glycoprotein during fusion activation. Our results suggest a model of MLV Env fusion activation in which unlocking of the fusion-inhibitory conformation is initiated by receptor binding of the viral RBD, which, upon disruption of the PRR, allows the NTR domain to promote further events in Env fusion activation. This involves a second type of interaction, in cis or in trans, between the receptor-activated RBD and a median segment of the freed C-terminal domain.

Activation of the murine leukaemia virus (MLV) envelope protein (Env) requires proteolytic cleavage of the R-peptide, a 16 amino acid C-terminal part of the cytoplasmic tail (C-tail) of Env. This paper demonstrates the presence of R-peptides in Env proteins of C-type retroviruses of simian, avian and porcine origin. Sequence alignment with the MLV C-tail led to the identification of a conserved hydrophobic protease cleavage motif located in the centre of retroviral Env protein C-tails. Expression of Env proteins, truncated at the predicted cleavage sites, of spleen necrosis virus (SNV), gibbon ape leukaemia virus and porcine endogenous retroviruses resulted in cell-cell fusion as monitored by microscopy and reporter gene fusion assays. Western blot analysis of MLV particles pseudotyped with the SNV Env protein demonstrated proteolytic cleavage of the SNV R-peptide by the MLV protease. Our data suggest that activation of membrane fusion by R-peptide cleavage is a common mode in C-type retroviruses.

A subclone of the NS0 murine myeloma cell line, frequently used to produce recombinant monoclonal antibodies, was found by a transmission electron microscopy method to express a surprisingly high titer of 10(11) retroviral particles per ml of culture supernatant. Infectivity assays showed a very low infectious titer with the restricted host range expected for a murine amphotropic retrovirus. A Western blot assay for the viral capsid protein was developed to confirm the high titer values and provide a means for monitoring batch consistency and virus removal during the purification process. Mass spectrometry of several of the viral Gag proteins demonstrated that the cell line appeared to produce at least two closely related retroviruses. N-terminal sequencing of three of the Gag proteins demonstrated that these retroviruses were members of the murine leukemia retroviral family. Western blot detection with an antibody for the capsid protein gave a linear standard curve over the range of 0.1-3 ng per lane. This allows the detection of viral titers as low as 6x10(7) virions per ml without the need to concentrate the sample. The Western blot method has higher throughput and less variability than transmission electron microscopy methods and has potential for monitoring viral titer and clearance during development of manufacturing processes.

We have previously shown that vesicles containing the spike glycoprotein of the vesicular stomatitis virus (VSV-G) can associate efficiently with immature, non-infectious, envelope-deficient retrovirus-like particles assembled by packaging cells to produce infectious, pseudotyped viruses in cell-free conditions in vitro. We have also previously reported that VSV-G can enhance DNA lipofection efficiency by interacting with liposomes to form fusogenic, serum-stable liposomes with enhanced transfection properties. Here, we report that VSV-G can form a complex directly with naked plasmid DNA in the absence of a lipofection reagent and can thereby enhance the transfection efficiency of the naked plasmid vector. Sucrose gradient sedimentation analysis demonstrated that VSV-G can also associate with plasmid DNA and murine leukemia virus (MLV) gag-pol particles to form ternary complexes that co-sediment with high DNA transfecting activity. The increased transfection efficiency with VSV-G was dependent on the presence of the polycation (Polybrene) in the culture medium during transfection. Enhanced transfection was abolished by a neutralizing antibody to VSV-G. These results may be useful in the study of retrovirus assembly, in the further design of hybrid DNA-based retrovirus-like vectors, and in the full in vitro, cell-free assembly of infectious virus-like particles from component parts.

Two types of endogenous ecotropic murine leukemia viruses (MuLVs), termed AKV- and Cas-E-type MuLVs, differ in nucleotide sequence and distribution in wild mouse subspecies. In contrast to AKV-type MuLV, Cas-E-type MuLV is not carried by common laboratory mice. Wild mice of Mus musculus (M. m.) castaneus carry multiple copies of Cas-E-type endogenous MuLV, including the Fv-4(r) gene that is a truncated form of integrated MuLV and functions as a host's resistance gene against ecotropic MuLV infection. Our genetic cross experiments showed that only the Fv-4(r) gene was associated with resistance to ecotropic F-MuLV infection. Because the spontaneous expression of infectious virus was not detected in M. m. castaneus, we generated mice that did not carry the Fv-4(r) gene but did carry a single or a few endogenous MuLV loci. In mice not carrying the Fv-4(r) gene, infectious MuLVs were isolated in association with three of six Cas-E-type endogenous MuLV loci. The isolated viruses showed a weak syncytium-forming activity for XC cells, an interfering property of ecotropic MuLV, and a slight antigenic variation. Two genomic DNAs containing endogenous Cas-E-type MuLV were cloned and partially sequenced. All of the Cas-E-type endogenous MuLVs were closely related, hybrid-type viruses with an ecotropic env gene and a xenotropic long terminal repeat. Duplications and a deletion were found in a restricted region of the hypervariable proline-rich region of Env glycoprotein.

A single retroviral protein, Gag, is sufficient for virus particle assembly. While Gag is capable of specifically packaging the genomic RNA into the particle, this RNA species is unnecessary for particle assembly in vivo. In vitro, nucleic acids profoundly enhance the efficiency of assembly by recombinant Gag proteins, apparently by acting as "scaffolding" in the particle. To address the participation of RNA in retrovirus assembly in vivo, we analyzed murine leukemia virus particles that lack genomic RNA because of a deletion in the packaging signal of the viral RNA. We found that these particles contain cellular mRNA in place of genomic RNA. This result was particularly evident when Gag was expressed by using a Semliki Forest virus-derived vector: under these conditions, the Semliki Forest virus vector-directed mRNA became very abundant in the cells and was readily identified in the retroviral virus-like particles. Furthermore, we found that the retroviral cores were disrupted by treatment with RNase. Taken together, the data strongly suggest that RNA is a structural element in retrovirus particles.

Retrovirus infection is initiated by receptor-dependent fusion of the envelope to the cell membrane. The modular organization of the envelope protein of C type retroviruses has been exploited to investigate how binding of the surface subunit (SU) to receptor triggers fusion mediated by the transmembrane (TM) subunit. We show that deletion of the receptor-binding domain (RBD) from SU of Friend murine leukemia virus (Fr-MLV) abolishes infection that is restored by supplying RBD as a soluble protein. Infection by this mechanism remains dependent on receptor expression. When membrane attachment of the virus lacking RBD is reestablished by inserting the hormone erythropoietin, infection remains dependent on the RBD/receptor complex. However, infection increases 50-fold to 5 x 10(5) units/ml on cells that also express the erythropoietin receptor. Soluble RBD from Fr-MLV also restores infection by amphotropic and xenotropic MLVs in which RBD is deleted. These experiments demonstrate that RBD has two functions: mediating virus attachment and activating the fusion mechanism. In addition, they indicate that receptor engagement triggers fusion by promoting a subgroup-independent functional interaction between RBD and the remainder of SU and/or TM.

Recently, significant progress has been made in identifying specific tumor-associated antigens recognized by T cells and defining the specific peptide epitopes within these proteins that are processed and presented on class I major histocompatibility antigens. Most of these antigens have been identified in human melanoma, where many of them appear to be tissue-specific, nonmutated proteins expressed by melanoma and normal melanocytes but not by other tissues. There has been much less progress in identifying the tumor antigens on murine tumors that are recognized by T cells, and this has restricted the development of preclinical animal models for immunotherapy. The authors previously described a method for generating tumor-reactive T cells from murine tumors (tumor infiltrating lymphocytes) that are CD8+ T cells recognizing autologous tumor and that can inhibit established tumor on adoptive transfer. Here the authors show that the envelope protein of an endogenous murine retrovirus of the AKV family, found in the germline of the C57BL/6 mouse, is recognized by tumor-infiltrating lymphocytes from two histologically different tumors syngeneic to that mouse strain. Furthermore, the authors identify the specific 9-amino acid peptide from the p15E transmembrane component of this envelope protein that is recognized in the context of major histocompatibility complex Kb, show that it is naturally presented and recognized on several other H-2b tumors, and that cytotoxic T lymphocytes specific for this epitope are therapeutic for these antigen-expressing tumors on adoptive transfer.

Because mutations in envelope glycoproteins of retroviruses or in their cell surface receptors can eliminate function by multiple mechanisms, it has been difficult to unambiguously identify sites for their interactions by site-directed mutagenesis. Recently, we developed a gain-of-function approach to overcome this problem. Our strategy relies on the fact that feline leukemia virus subgroup B (FeLV-B) and amphotropic murine leukemia virus (A-MLV) have closely related gp70 surface envelope glycoproteins and use related Na(+)-dependent phosphate symporters, Pit1 and Pit2, respectively, as their receptors. We previously observed that FeLV-B/A-MLV envelope glycoprotein chimeras spliced between the variable regions VRA and VRB were unable to use Pit1 or Pit2 as a receptor but could efficiently use specific Pit1/Pit2 chimeras. The latter study suggested that the VRA of A-MLV and FeLV-B functionally interact with the presumptive extracellular loops 4 and 5 (ECL4 and -5) of their respective receptors, whereas VRB interacts with ECL2. We also found that FeLV-B gp70 residues F60 and P61 and A-MLV residues Y60 and V61 in the first disulfide-bonded loop of VRA were important for functional interaction with the receptor's ECL4 or -5. We have now extended this approach to identify additional VRA and VRB residues that are involved in receptor recognition. Our studies imply that FeLV-B VRA residues F60 and P61 interact with the Pit1 ECL5 region, whereas VRA residues 66 to 78 interact with Pit1 ECL4. Correspondingly, A-MLV VRA residues Y60 and V61 interact with the Pit2 ECL5 region, whereas residues 66 to 78 interact with Pit2 ECL4. Similar studies that focused on the gp70 VRB implicated residues 129 to 139 as contributing to specific interactions with the receptor ECL2. These results identify three regions of gp70 that interact in a specific manner with distinct portions of their receptors, thereby providing a map of the functionally interacting surfaces.