RESULTS:
1 - 8 of 8 for "Richard E. Randall"
Single-cycle parainfluenza virus type 5 vectors for producing recombinant proteins, including a humanized anti-V5 tag antibody
Parainfluenza virus type 5 (PIV5) can cause either persistent or acute/lytic infections in a wide range of mammalian tissue culture cells. Here we have generated PIV5 fusion (F)-expressing helper cell lines that support the replication of F-deleted viruses. As proof of the principle that F-deleted single-cycle infectious viruses can be used as safe and efficient expression vectors we have cloned and expressed a humanized (Hu) version of the mouse anti-V5 tag antibody (clone SV5-Pk1). We show that multiple different cell lines can be infected and express high levels of the Hu anti-V5 antibody with Chinese hamster ovary cells expressing 20–50 mg l−1 after 5 days when cells were grown to a density of ~1×106 cells per millilitre at the time of infection. We suggest that PIV5-based vectors may be further developed to produce recombinant proteins both in vitro and in vivo.
Persistent paramyxovirus infections: in co-infections the parainfluenza virus type 5 persistent phenotype is dominant over the lytic phenotype
Parainfluenza virus type 5 (PIV5) can either have a persistent or a lytic phenotype in cultured cells. We have previously shown that the phenotype is determined by the phosphorylation status of the phosphoprotein (P). Single amino acid substitutions at critical residues including a serine-to-phenylalanine substitution at position 157 on P result in a switch between persistent and lytic phenotypes. Here using PIV5 vectors expressing either mCherry or GFP with persistent or lytic phenotypes we show that in co-infections the persistent phenotype is dominant. Thus in contrast to the cell death observed with cells infected solely with the lytic variant in co-infected cells persistence is immediately established and both lytic and persistent genotypes persist. Furthermore 10–20 % of virus released from dually infected cells contains both genotypes indicating that PIV5 particles can package more than one genome. Co-infected cells continue to maintain both genotypes/phenotypes during cell passage as do individual colonies of cells derived from a culture of persistently infected cells. A refinement of our model on how the dynamics of virus selection may occur in vivo is presented.
Human interactome of the influenza B virus NS1 protein
NS1 proteins of influenza A and B viruses share limited sequence homology yet both are potent manipulators of host cell processes particularly interferon (IFN) induction. Although many cellular partners are reported for A/NS1 only a few (e.g. PKR and ISG15) have been identified for B/NS1. Here affinity-purification and mass spectrometry were used to expand the known host interactome of B/NS1. We identified 22 human proteins as new putative targets for B/NS1 validating several including DHX9 ILF3 YBX1 and HNRNPC. Consistent with two RNA-binding domains in B/NS1 many of the identified factors bind RNA and some interact with B/NS1 in an RNA-dependent manner. Functional characterization of several B/NS1 interactors identified SNRNP200 as a potential positive regulator of host IFN responses while ILF3 exhibited dual roles in both IFN induction and influenza B virus replication. These data provide a resource for future investigations into the mechanisms underpinning host cell modulation by influenza B virus NS1.
In memoriam – Richard M. Elliott (1954–2015)
The multifunctional NS1 protein of influenza A viruses
The non-structural (NS1) protein of influenza A viruses is a non-essential virulence factor that has multiple accessory functions during viral infection. In recent years the major role ascribed to NS1 has been its inhibition of host immune responses especially the limitation of both interferon (IFN) production and the antiviral effects of IFN-induced proteins such as dsRNA-dependent protein kinase R (PKR) and 2'5'-oligoadenylate synthetase (OAS)/RNase L. However it is clear that NS1 also acts directly to modulate other important aspects of the virus replication cycle including viral RNA replication viral protein synthesis and general host-cell physiology. Here we review the current literature on this remarkably multifunctional viral protein. In the first part of this article we summarize the basic biochemistry of NS1 in particular its synthesis structure and intracellular localization. We then discuss the various roles NS1 has in regulating viral replication mechanisms host innate/adaptive immune responses and cellular signalling pathways. We focus on the NS1–RNA and NS1–protein interactions that are fundamental to these processes and highlight apparent strain-specific ways in which different NS1 proteins may act. In this regard the contributions of certain NS1 functions to the pathogenicity of human and animal influenza A viruses are also discussed. Finally we outline practical applications that future studies on NS1 may lead to including the rational design and manufacture of influenza vaccines the development of novel antiviral drugs and the use of oncolytic influenza A viruses as potential anti-cancer agents.
Interferons and viruses: an interplay between induction, signalling, antiviral responses and virus countermeasures
The interferon (IFN) system is an extremely powerful antiviral response that is capable of controlling most if not all virus infections in the absence of adaptive immunity. However viruses can still replicate and cause disease in vivo because they have some strategy for at least partially circumventing the IFN response. We reviewed this topic in 2000 [Goodbourn S. Didcock L. & Randall R. E. (2000). J Gen Virol 81 2341–2364] but since then a great deal has been discovered about the molecular mechanisms of the IFN response and how different viruses circumvent it. This information is of fundamental interest but may also have practical application in the design and manufacture of attenuated virus vaccines and the development of novel antiviral drugs. In the first part of this review we describe how viruses activate the IFN system how IFNs induce transcription of their target genes and the mechanism of action of IFN-induced proteins with antiviral action. In the second part we describe how viruses circumvent the IFN response. Here we reflect upon possible consequences for both the virus and host of the different strategies that viruses have evolved and discuss whether certain viruses have exploited the IFN response to modulate their life cycle (e.g. to establish and maintain persistent/latent infections) whether perturbation of the IFN response by persistent infections can lead to chronic disease and the importance of the IFN system as a species barrier to virus infections. Lastly we briefly describe applied aspects that arise from an increase in our knowledge in this area including vaccine design and manufacture the development of novel antiviral drugs and the use of IFN-sensitive oncolytic viruses in the treatment of cancer.
Improved growth of enteric adenovirus type 40 in a modified cell line that can no longer respond to interferon stimulation
Human enteric adenoviruses propagate poorly in conventional human cell lines used to grow other adenovirus serotypes. As human enteric adenoviruses have a defect in counteracting the cellular interferon (IFN) response in cell culture to aid in growth of the virus a 293-based cell line defective in its ability to respond to IFN was constructed. This cell line (293-SV5/V) constitutively expresses V-protein of the paramyxovirus Simian virus 5 which degrades the signal transducer and activator of transcription 1 (STAT1) and thereby prevents the STAT1-mediated IFN response. Analysis of human enteric adenovirus type 40 (HAdV-40)-infected 293-SV5/V cells compared with parental 293 cells shows that the recombinant line allows more rapid production of virus and results in higher titres. These results suggest that the defect in HAdV-40 in counteracting the IFN response can be overcome at least partially through the use of 293-SV5/V cell lines.
Construction of solid matrix-antibody-antigen complexes containing simian immunodeficiency virus p27 using tag-specific monoclonal antibody and tag-linked antigen
We have previously shown that immunization with solid matrix-antigen-antibody (SMAA) complexes induces both vigorous humoral and cell-mediated immune responses and have suggested that this method of vaccination may be developed for use in humans and potentially as a vaccine against AIDS. Here we demonstrate that a small oligopeptide can act as a tag for the construction of SMAA complexes using a tag-specific monoclonal antibody and tag-linked antigens. We show that a 14-amino acid oligopeptide present in the phospho (P) and V proteins of simian virus 5 (SV5) retains its antigenicity when attached to the C terminus of three ‘foreign’ proteins [p27 and gp110 of simian immunodeficiency virus (SIV) and glutathione S-transferase] such that these proteins can be incorporated into SMAA complexes using a monoclonal antibody (MAb) that was originally raised against the native SV5 P and V proteins. Mice were immunized with SMAA complexes containing recombinant p27-TAG and MAbs have been isolated that recognized native SIV p27. The significance of these results in terms of the development of SMAA complexes as human vaccines is discussed.