RESULTS:
1 - 6 of 6 for "David Hughes"
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.
Discovering antiviral restriction factors and pathways using genetic screens
Viral infections activate the powerful interferon (IFN) response that induces the expression of several hundred IFN stimulated genes (ISGs). The principal role of this extensive response is to create an unfavourable environment for virus replication and to limit spread; however untangling the biological consequences of this large response is complicated. In addition to a seemingly high degree of redundancy several ISGs are usually required in combination to limit infection as individual ISGs often have low to moderate antiviral activity. Furthermore what ISG or combination of ISGs are antiviral for a given virus is usually not known. For these reasons and since the function(s) of many ISGs remains unexplored genome-wide approaches are well placed to investigate what aspects of this response result in an appropriate virus-specific phenotype. This review discusses the advances screening approaches have provided for the study of host defence mechanisms including clustered regularly interspaced short palindromic repeats/CRISPR associated protein 9 (CRISPR/Cas9) ISG expression libraries and RNA interference (RNAi) technologies.
Understanding virus resistance due to ISG15-loss-of-function
Viral infections induce profound cellular responses resulting the expression of hundreds of IFN-stimulated genes (ISGs). Some ISGs have specific antiviral activity while others regulate the cellular response. For most viruses the specific antiviral ISG(s) is not known which has potential consequences for the quest for new therapeutics. The ubiquitin-like protein ISG15 is a major regulator of antiviral response and inherited ISG15-deficiency leads to autoinflammatory interferonopathies where patients exhibit elevated ISG expression in the absence of infection. Using CRISPR/Cas9 knockout technology we have recapitulated these effects in cultured cells confirming ‘free’ ISG15’s role as a central regulator of type-I IFN antiviral response. We also show that during an antiviral response ISG15-deficiency leads to significant physiological defects (inhibition of translation and proliferation) and resistance to parainfluenza viruses. We asked if virus resistance was due to the direct antiviral activity of ISGs or whether cells were non-permissive due to physiological defects. We took advantage of the knowledge that IFIT1 is the principle antiviral ISG for parainfluenza virus 5 (PIV5). Knockdown of IFIT1 restored PIV5 infection in ISG15-deficient cells confirming that resistance was due to the antiviral response and not due to physiological state related to ISG15-deficiency. We also compared infections with related viruses where IFIT1 has known intermediate antiviral activity (PIV2) and low activity (PIV3); restoration of replication with these viruses reflected their sensitivity to IFIT1 restriction. Based on the observations in IFIT1-knockdown cells we propose a novel platform for the identification of antiviral ISGs based on recovery of virus infection.
Characterization of a novel wood mouse virus related to murid herpesvirus 4
Two novel gammaherpesviruses were isolated one from a field vole (Microtus agrestis) and the other from wood mice (Apodemus sylvaticus). The genome of the latter designated wood mouse herpesvirus (WMHV) was completely sequenced. WMHV had the same genome structure and predicted gene content as murid herpesvirus 4 (MuHV4; murine gammaherpesvirus 68). Overall nucleotide sequence identity between WMHV and MuHV4 was 85 % and most of the 10 kb region at the left end of the unique region was particularly highly conserved especially the viral tRNA-like sequences and the coding regions of genes M1 and M4. The partial sequence (71 913 bp) of another gammaherpesvirus Brest herpesvirus (BRHV) which was isolated ostensibly from a white-toothed shrew (Crocidura russula) was also determined. The BRHV sequence was 99.2 % identical to the corresponding portion of the WMHV genome. Thus WMHV and BRHV appeared to be strains of a new virus species. Biological characterization of WMHV indicated that it grew with similar kinetics to MuHV4 in cell culture. The pathogenesis of WMHV in wood mice was also extremely similar to that of MuHV4 except for the absence of inducible bronchus-associated lymphoid tissue at day 14 post-infection and a higher load of latently infected cells at 21 days post-infection.
Evidence for the presence of a low-level, persistent baculovirus infection of Mamestra brassicae insects.
A laboratory culture of Mamestra brassicae insects (MbLC) harbours a latent or occult baculovirus that resembles M. brassicae multiple nucleocapsid nucleopolyhedrovirus (MbMNPV). Although conventional extraction techniques have failed to detect the presence of virus in MbLC control virusfree insects (MbWS) died of an MbMNPV-like infection after being fed MbLC fat-body cells. This suggested that the MbLC cells harboured infectious MbMNPV albeit at low levels. We have also demonstrated that fat-body cells from MbLC but not from MbWS contain mRNA specific for the polyhedrin gene and transcriptional factors that are capable of activating baculovirus late and very late gene promoters linked to a reporter gene encoding chloramphenicol acetyltransferase. Our data provide indirect evidence that the latent MbMNPV in the MbLC insects is maintained as a persistent infection with the expression of viral genes at a low level.