RESULTS:
1 - 4 of 4 for "Steve Goodbourn"
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.
The cellular localization of avian influenza virus PB1-F2 protein alters the magnitude of IFN2 promoter and NFκB-dependent promoter antagonism in chicken cells
The accessory protein PB1-F2 of influenza A virus (IAV) functions in a chicken host to prolong infectious virus shedding and thus the transmission window. Here we show that this delay in virus clearance by PB1-F2 in chickens is accompanied by reduced transcript levels of type 1 interferon (IFN)-induced genes and NFκB-activated pro-inflammation cytokines. In vitro two avian influenza isolate-derived PB1-F2 proteins H9N2 UDL01 and H5N1 5092 exhibited the same antagonism of the IFN and pro-inflammation induction pathways seen in vivo but to different extents. The two PB1-F2 proteins had different cellular localization in chicken cells with H5N1 5092 being predominantly mitochondrial-associated and H9N2 UDL being cytoplasmic but not mitochondrial-localized. We hypothesized that PB1-F2 localization might influence the functionality of the protein during infection and that the protein sequence could alter cellular localization. We demonstrated that the sequence of the C-terminus of PB1-F2 determined cytoplasmic localization in chicken cells and this was linked with protein instability. Mitochondrial localization of PB1-F2 resulted in reduced antagonism of an NFκB-dependent promoter. In parallel mitochondrial localization of PB1-F2 increased the potency of chicken IFN 2 induction antagonism. We suggest that mitochondrial localization of PB1-F2 restricts interaction with cytoplasmic-located IKKβ reducing NFκB-responsive promoter antagonism but enhances antagonism of the IFN2 promoter through interaction with the mitochondrial adaptor MAVS. Our study highlights the differential mechanisms by which IAV PB1-F2 protein can dampen the avian host innate signalling response.
A single amino acid substitution in the V protein of Nipah virus alters its ability to block interferon signalling in cells from different species
The V protein of the paramyxovirus Nipah virus (NiV) has been shown to antagonize the interferon (IFN) response in human cells via sequestration of STAT1 and STAT2. This study describes a mutant of the NiV V protein referred to as V(AAHL) that is unable to antagonize IFN signalling and demonstrates that a single amino acid substitution is responsible for its inactivity. The molecular basis for this was identified as a failure to interact with STAT1 and STAT2. It was also shown that NiV V but not V(AAHL) was functional as an IFN antagonist in human monkey rabbit dog horse pig and bat cells which suggests that the ability of NiV to block IFN signalling is not a major constraint that prevents this virus from crossing species barriers.