Virus removal capacity at varying ionic strength during nanofiltration of AlphaNine® SD.

Nanofiltration is incorporated into the manufacturing processes of many protein biopharmaceuticals to enhance safety by providing the capacity to retain pathogens while allowing protein drugs to pass through the filter. Retention is mainly a function of size; however, the shape of the pathogen may also influence retention. The ability of the Viresolve(®) Pro nanofilter to remove different sized viruses during the manufacture of a Coagulation Factor IX (Alphanine(®) SD) was studied at varying ionic strength, a process condition with the potential to affect virus shape and, hence, virus retention. Eight viruses were tested in a scale-down of the nanofiltration process. Five of the viruses (EMCV, Reo, BVDV, HIV, PRV) were nanofiltered at normal sodium processing conditions and three (PPV, HAV and WNV) were nanofiltered at higher and lower sodium. Representative Reduction Factors for all viruses were ≥4.50 logs and removal was consistent over a wide range of ionic strength.

Protein sieving characteristics of sub-20-nm pore size filters at varying ionic strength during nanofiltration of Coagulation Factor IX.

Nanofiltration assures that protein therapeutics are free of adventitious agents such as viruses. Nanofilter pores must allow passage of protein drugs but be small enough to retain viruses. Five nanofilters have been evaluated to identify those that can be used interchangeably to yield a high purity Coagulation Factor IX product. When product preparations prior to nanofiltration were analyzed using electrophoresis, Western blot, liquid chromatography - tandem mass spectrometry and size exclusion HPLC, factor IX, inter - α - trypsin inhibitor and C4b binding protein (C4BP) were observed. C4BP was removed from product by all five nanofilters when nanofiltration was performed at physiological ionic strength. However, at high ionic strength, C4BP was removed by only two nanofilters. HPLC indicated that the Stokes radius of C4BP was larger at low ionic strength than at high ionic strength. The results suggest that C4BP exists in an open conformation at physiological ionic strength and is removed by nanofiltration whereas, at high ionic strength, the protein collapses to an extent that allows passage through some nanofilters. Manufacturers should be aware that protein contaminants in other nanofiltered protein drugs could behave similarly and conditions of nanofiltration must be evaluated to ensure consistent product purity.

The splicing factor proline-glutamine rich (SFPQ/PSF) is involved in influenza virus transcription.

The influenza A virus RNA polymerase is a heterotrimeric complex responsible for viral genome transcription and replication in the nucleus of infected cells. We recently carried out a proteomic analysis of purified polymerase expressed in human cells and identified a number of polymerase-associated cellular proteins. Here we characterise the role of one such host factors, SFPQ/PSF, during virus infection. Down-regulation of SFPQ/PSF by silencing with two independent siRNAs reduced the virus yield by 2-5 log in low-multiplicity infections, while the replication of unrelated viruses as VSV or Adenovirus was almost unaffected. As the SFPQ/PSF protein is frequently associated to NonO/p54, we tested the potential implication of the latter in influenza virus replication. However, down-regulation of NonO/p54 by silencing with two independent siRNAs did not affect virus yields. Down-regulation of SFPQ/PSF by siRNA silencing led to a reduction and delay of influenza virus gene expression. Immunofluorescence analyses showed a good correlation between SFPQ/PSF and NP levels in infected cells. Analysis of virus RNA accumulation in silenced cells showed that production of mRNA, cRNA and vRNA is reduced by more than 5-fold but splicing is not affected. Likewise, the accumulation of viral mRNA in cicloheximide-treated cells was reduced by 3-fold. In contrast, down-regulation of SFPQ/PSF in a recombinant virus replicon system indicated that, while the accumulation of viral mRNA is reduced by 5-fold, vRNA levels are slightly increased. In vitro transcription of recombinant RNPs generated in SFPQ/PSF-silenced cells indicated a 4-5-fold reduction in polyadenylation but no alteration in cap snatching. These results indicate that SFPQ/PSF is a host factor essential for influenza virus transcription that increases the efficiency of viral mRNA polyadenylation and open the possibility to develop new antivirals targeting the accumulation of primary transcripts, a very early step during infection.

The influenza virus RNA synthesis machine: advances in its structure and function.

The influenza A viruses are the causative agents of respiratory disease that occurs as yearly epidemics and occasional pandemics. These viruses are endemic in wild avian species and can sometimes break the species barrier to infect and generate new virus lineages in humans. The influenza A virus genome consists of eight single-stranded, negative-polarity RNAs that form ribonucleoprotein complexes by association to the RNA polymerase and the nucleoprotein. In this review we focus on the structure of this RNA-synthesis machines and the included RNA polymerase, and on the mechanisms by which they express their genetic information as mRNAs and generate progeny ribonucleoproteins that will become incorporated into new infectious virions. New structural, biochemical and genetic data are rapidly accumulating in this very active area of research. We discuss these results and attempt to integrate the information into structural and functional models that may help the design of new experiments and further our knowledge on virus RNA replication and gene expression. This interplay between structural and functional data will eventually provide new targets for controlled attenuation or antiviral therapy.

Genetic trans-complementation establishes a new model for influenza virus RNA transcription and replication.

The influenza A viruses genome comprises eight single-stranded RNA segments of negative polarity. Each one is included in a ribonucleoprotein particle (vRNP) containing the polymerase complex and a number of nucleoprotein (NP) monomers. Viral RNA replication proceeds by formation of a complementary RNP of positive polarity (cRNP) that serves as intermediate to generate many progeny vRNPs. Transcription initiation takes place by a cap-snatching mechanism whereby the polymerase steals a cellular capped oligonucleotide and uses it as primer to copy the vRNP template. Transcription termination occurs prematurely at the polyadenylation signal, which the polymerase copies repeatedly to generate a 3'-terminal polyA. Here we studied the mechanisms of the viral RNA replication and transcription. We used efficient systems for recombinant RNP transcription/replication in vivo and well-defined polymerase mutants deficient in either RNA replication or transcription to address the roles of the polymerase complex present in the template RNP and newly synthesised polymerase complexes during replication and transcription. The results of trans-complementation experiments showed that soluble polymerase complexes can synthesise progeny RNA in trans and become incorporated into progeny vRNPs, but only transcription in cis could be detected. These results are compatible with a new model for virus RNA replication, whereby a template RNP would be replicated in trans by a soluble polymerase complex and a polymerase complex distinct from the replicative enzyme would direct the encapsidation of progeny vRNA. In contrast, transcription of the vRNP would occur in cis and the resident polymerase complex would be responsible for mRNA synthesis and polyadenylation.

The host-dependent interaction of alpha-importins with influenza PB2 polymerase subunit is required for virus RNA replication.

The influenza virus polymerase is formed by the PB1, PB2 and PA subunits and is required for virus transcription and replication in the nucleus of infected cells. As PB2 is a relevant host-range determinant we expressed a TAP-tagged PB2 in human cells and isolated intracellular complexes. Alpha-importin was identified as a PB2-associated factor by proteomic analyses. To study the relevance of this interaction for virus replication we mutated the PB2 NLS and analysed the phenotype of mutant subunits, polymerase complexes and RNPs. While mutant PB2 proteins showed reduced nuclear accumulation, they formed polymerase complexes normally when co expressed with PB1 and PA. However, mutant RNPs generated with a viral CAT replicon showed up to hundred-fold reduced CAT accumulation. Rescue of nuclear localisation of mutant PB2 by insertion of an additional SV40 TAg-derived NLS did not revert the mutant phenotype of RNPs. Furthermore, determination of recombinant RNP accumulation in vivo indicated that PB2 NLS mutations drastically reduced virus RNA replication. These results indicate that, above and beyond its role in nuclear accumulation, PB2 interaction with alpha-importins is required for virus RNA replication. To ascertain whether PB2-alpha-importin binding could contribute to the adaptation of H5N1 avian viruses to man, their association in vivo was determined. Human alpha importin isoforms associated efficiently to PB2 protein of an H3N2 human virus but bound to diminished and variable extents to PB2 from H5N1 avian or human strains, suggesting that the function of alpha importin during RNA replication is important for the adaptation of avian viruses to the human host.

Analysis of the interaction of influenza virus polymerase complex with human cell factors.

The influenza virus polymerase is formed by the PB1, PB2 and PA subunits and is required for virus transcription and replication in the nucleus of infected cells. Here we present the characterisation of the complexes formed intracellularly by the influenza polymerase in human cells. The virus polymerase was expressed by cotransfection of the polymerase subunits cDNAs, one of which fused to the tandem-affinity purification (TAP) tag. The intracellular complexes were purified by the TAP approach, which involves IgG-Sepharose and calmodulin-agarose chromatography, under very mild conditions. The purified complexes contained the heterotrimeric polymerase and a series of associated proteins that were not apparent in purifications of untagged polymerase used as a control. Several influenza polymerase-associated proteins were identified by MALDI-MS and their presence in purified polymerase-containing complexes were verified by Western blot. Their relevance for influenza infection was established by colocalisation with virus ribonucleoproteins in human infected cells. Most of the associated human factors were nuclear proteins involved in cellular RNA synthesis, modification and nucleo-cytoplasmic export, but some were cytosolic proteins involved in translation and transport. The interactions recognised in this proteomic approach suggest that the influenza polymerase might be involved in steps of the infection cycle other than RNA replication and transcription.

Oligomerization of the influenza virus polymerase complex in vivo.

The influenza virus polymerase is a heterotrimer formed by the PB1, PB2 and PA subunits and is responsible for virus transcription and replication. We have expressed the virus polymerase complex by co-transfection of the subunit cDNAs, one of which was tandem affinity purification (TAP)-tagged, into human cells. The intracellular polymerase complexes were purified by the TAP approach, involving two affinity chromatography steps, IgG-Sepharose and calmodulin-agarose. Gel-filtration analysis indicated that, although most of the purified polymerase behaved as a heterotrimer, a significant proportion of the purified material migrated as polymerase dimers, trimers and higher oligomers. Co-purification of polymerase complexes alternatively tagged in the same subunit confirmed that the polymerase complex might form oligomers intracellularly. The implications of this observation for virus infection are discussed.

Three-dimensional model for the isolated recombinant influenza virus polymerase heterotrimer.

The genome of influenza A virus is organized into eight ribonucleoprotein complexes (RNPs), each containing one RNA polymerase complex. This RNA polymerase has also been found non-associated to RNPs and is possibly involved in distinct functions in the infection cycle. We have expressed the virus RNA polymerase complex by co-tranfection of the PB1, PB2 and PA genes in mammalian cells and the heterotrimer was purified by the TAP tag procedure. Its 3D structure was determined by electron microscopy and single-particle image processing. The model obtained resembles the structure previously reported for the polymerase complex associated to viral RNPs but appears to be in a more open conformation. Detailed model comparison indicated that specific areas of the complex show important conformational changes as compared to the structure for the RNP-associated polymerase, particularly in regions known to interact with the adjacent NP monomers in the RNP. Also, the PB2 subunit seems to undergo a substantial displacement as a result of the association of the polymerase to RNPs. The structural model presented suggests that a core conformation of the polymerase in solution exists but the interaction with other partners, such as proteins or RNA, will trigger distinct conformational changes to activate new functional properties.