Characterization of ionic strength for X-MuLV inactivation by low pH treatment for monoclonal antibody purification.

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 log10 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.

Analysis of filtration behavior using integrated column chromatography followed by virus filtration.

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/m2 , 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.

Continued insights into virus clearance validation across continuous capture chromatography.

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.

Virus clearance validation across continuous capture chromatography.

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.

Structure and architecture of immature and mature murine leukemia virus capsids.

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.

Functional and Structural Characterization of Novel Type of Linker Connecting Capsid and Nucleocapsid Protein Domains in Murine Leukemia Virus.

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.

Small synthetic ligands for the enrichment of viral particles pseudotyped with amphotropic murine leukemia virus envelope.

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.

SERINC3 and SERINC5 restrict HIV-1 infectivity and are counteracted by Nef.

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.

HIV-1 Nef promotes infection by excluding SERINC5 from virion incorporation.

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.

Limits in virus filtration capability? Impact of virus quality and spike level on virus removal with xenotropic murine leukemia virus.

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).

MLV based viral-like-particles for delivery of toxic proteins and nuclear transcription factors.

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.

Efficient in vitro gene delivery by hybrid biopolymer/virus nanobiovectors.

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.

Optimised concentration and purification of retroviruses using membrane chromatography.

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.

One-step separation of myristoylated and nonmyristoylated retroviral matrix proteins.

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.

Maturation of the Gag core decreases the stability of retroviral lipid membranes.

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.

Substrate variations that affect the nucleic acid clamp activity of reverse transcriptases.

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.

A retroviral chimeric capsid protein reveals the role of the N-terminal ß-hairpin in mature core assembly.

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 ß-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 ß-hairpin arrangement, generates noninfectious virus or aberrant cores. To date, however, there is no consensus on the role of the ß-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 ß-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 ß-hairpin itself. The presented experiments provide biochemical evidence that ß-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 ß-hairpin structure, its dynamics, and its influence on the orientation of helix 1 for the assembly of the mature CA lattice.

The cellular protein La functions in enhancement of virus release through lipid rafts facilitated by murine leukemia virus glycosylated Gag.

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.

A novel envelope mediated post entry restriction of murine leukaemia virus in human cells is Ref1/TRIM5α independent.

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.

Mass spectrometry of murine leukemia virus core proteins.

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.