Temperature sensitive influenza A virus genome replication results from low thermal stability of polymerase-cRNA complexes

BACKGROUND: The RNA-dependent RNA polymerase of Influenza A virus is a determinant of viral pathogenicity and host range that is responsible for transcribing and replicating the negative sense segmented viral genome (vRNA). Transcription produces capped and polyadenylated mRNAs whereas genome replication involves the synthesis of an alternative plus-sense transcript (cRNA) with unmodified termini that is copied back to vRNA. Viral mRNA transcription predominates at early stages of viral infection, while later, negative sense genome replication is favoured. However, the "switch" that regulates the transition from transcription to replication is poorly understood. RESULTS: We show that temperature strongly affects the balance between plus and minus-sense RNA synthesis with high temperature causing a large decrease in vRNA accumulation, a moderate decrease in cRNA levels but (depending on genome segment) either increased or unchanged levels of mRNA. We found no evidence implicating cellular heat shock protein activity in this effect despite the known association of hsp70 and hsp90 with viral polymerase components. Temperature-shift experiments indicated that polymerase synthesised at 41°C maintained transcriptional activity even though genome replication failed. Reduced polymerase association with viral RNA was seen in vivo and in confirmation of this, in vitro binding assays showed that temperature increased the rate of dissociation of polymerase from both positive and negative sense promoters. However, the interaction of polymerase with the cRNA promoter was particularly heat labile, showing rapid dissociation even at 37°C. This suggested that vRNA synthesis fails at elevated temperatures because the polymerase does not bind the promoter. In support of this hypothesis, a mutant cRNA promoter with vRNA-like sequence elements supported vRNA synthesis at higher temperatures than the wild-type promoter. CONCLUSION: The differential stability of negative and positive sense polymerase-promoter complexes explains why high temperature favours transcription over replication and has implications for the control of viral RNA synthesis at physiological temperatures. Furthermore, given the different body temperatures of birds and man, these finding suggest molecular hypotheses for how polymerase function may affect host range

The Cytoplasmic Location of Chicken Mx Is Not the Determining Factor for Its Lack of Antiviral Activity

Chicken Mx belongs to the Mx family of interferon-induced dynamin-like GTPases, which in some species possess potent antiviral properties. Conflicting data exist for the antiviral capability of chicken Mx. Reports of anti-influenza activity of alleles encoding an Asn631 polymorphism have not been supported by subsequent studies. The normal cytoplasmic localisation of chicken Mx may influence its antiviral capacity. Here we report further studies to determine the antiviral potential of chicken Mx against Newcastle disease virus (NDV), an economically important cytoplasmic RNA virus of chickens, and Thogoto virus, an orthomyxovirus known to be exquisitely sensitive to the cytoplasmic MxA protein from humans. We also report the consequences of re-locating chicken Mx to the nucleus.Chicken Mx was tested in virus infection assays using NDV. Neither the Asn631 nor Ser631 Mx alleles (when transfected into 293T cells) showed inhibition of virus-directed gene expression when the cells were subsequently infected with NDV. Human MxA however did show significant inhibition of NDV-directed gene expression. Chicken Mx failed to inhibit a Thogoto virus (THOV) minireplicon system in which the cytoplasmic human MxA protein showed potent and specific inhibition. Relocalisation of chicken Mx to the nucleus was achieved by inserting the Simian Virus 40 large T antigen nuclear localisation sequence (SV40 NLS) at the N-terminus of chicken Mx. Nuclear re-localised chicken Mx did not inhibit influenza (A/PR/8/34) gene expression during virus infection in cell culture or influenza polymerase activity in A/PR/8/34 or A/Turkey/50-92/91 minireplicon systems.The chicken Mx protein (Asn631) lacks inhibitory effects against THOV and NDV, and is unable to suppress influenza replication when artificially re-localised to the cell nucleus. Thus, the natural cytoplasmic localisation of the chicken Mx protein does not account for its lack of antiviral activity

Mutations close to functional motif IV in HSV-1 UL5 helicase that confer resistance to HSV helicase-primase inhibitors, variously affect virus growth rate and pathogenicity

Herpes simplex virus (HSV) helicase-primase (HP) is the target for a novel class of antiviral compounds, the helicase-primase inhibitors (HPIs), e.g. BAY 57-1293. Although mutations in herpesviruses conferring resistance to nucleoside analogues are commonly associated with attenuation in vivo, to date, this is not necessarily true for HPIs. HPI-resistant HSV mutants selected in tissue culture are reported to be equally pathogenic compared to parental virus in animal models. Here we demonstrate that a slow-growing HSV-1 mutant, with the BAY 57-1293-resistance mutation Gly352Arg in UL5 helicase, is clearly less virulent than its wild-type parent in a murine zosteriform infection model. This contrasts with published results obtained for a mutant containing a different HPI-resistance substitution (Gly352Val) at the same location, since this mutant was reported to be fully pathogenic. We believe our report to be the first to describe an HPI-resistant HSV-1 mutant, that is markedly less virulent in vivo and slowly growing in tissue culture compared to the parental strain. Another BAY 57-1293-resistant UL5 mutant (Lys356Gln), which showed faster growth characteristics in cell culture, however, was at least equally virulent compared to the parent strain

Asparagine 631 Variants of the Chicken Mx Protein Do Not Inhibit Influenza Virus Replication in Primary Chicken Embryo Fibroblasts or In Vitro Surrogate Assays▿

Whether chicken Mx inhibits influenza virus replication is an important question with regard to strategies aimed at enhancing influenza resistance in domestic flocks. The Asn631 polymorphism of the chicken Mx protein found in the Shamo (SHK) chicken line was previously reported to be crucial for the antiviral activity of this highly polymorphic chicken gene. Our aims were to determine whether cells from commercial chicken lines containing Asn631 alleles were resistant to influenza virus infection and to investigate the effects that other polymorphisms might have on Mx function. Unexpectedly, we found that the Asn631 genotype had no impact on multicycle replication of influenza virus (A/WSN/33 [H1N1]) in primary chicken embryo fibroblast lines. Furthermore, expression of the Shamo (SHK) chicken Mx protein in transfected 293T cells did not inhibit viral gene expression (A/PR/8/34 [H1N1], A/Duck/England/62 [H4N6], and A/Duck/Singapore/97 [H5N3]). Lastly, in minireplicon systems (A/PR/8/34 and A/Turkey/England/50-92/91 [H5N1]), which were highly sensitive to inhibition by the murine Mx1 and human MxA proteins, respectively, Shamo chicken Mx also proved ineffective in the context of avian as well as mammalian cell backgrounds. Our findings demonstrate that Asn631 chicken Mx alleles do not inhibit influenza virus replication of the five strains tested here and efforts to increase the frequency of Asn631 alleles in commercial chicken populations are not warranted. Nevertheless, chicken Mx variants with anti-influenza activity might still exist. The flow cytometry and minireplicon assays described herein could be used as efficient functional screens to identify such active chicken Mx alleles

Chicken Mx proteins lack activity in a THOV minireplicon system.

<p>293T cells were transfected with THOV PB1, PB2, PA and NP, a plasmid encoding a luciferase THOV minireplicon and a SEAP expressing plasmid, together with pcDNA3 or a plasmid expressing the indicated Mx protein (human MxA, the MxA T103A mutant, wild type chicken Mx proteins SHK (Asn631) and 8.1 (Ser631) and their nuclear-localised counterparts SHK NLS and 8.1 NLS). 48 h post-transfection, luciferase activity was measured and is shown as relative light units (rlu). The mean (and SD) of 3 replicates is shown. * indicates significant difference (Students <i>t</i>-test) relative to pcDNA3 (p<0.05).</p

Nuclear localized chicken Mx lacks anti-influenza activity.

<p>Panel A: Effect on influenza A/PR/8/34 minireplicon system. 293T cells were co-transfected with plasmids expressing the PB1, PB2, PA and NP proteins of influenza A/PR/8/34, a plasmid encoding a luciferase minireplicon, and a SEAP-expressing plasmid, together with either pcDNA3 or a plasmid expressing the indicated Mx protein (murine Mx1, human MxA, wild type chicken Mx proteins SHK (Asn631) and 8.1 (Ser631) and their nuclear-localised counterparts SHK NLS and 8.1 NLS). 48 h post-transfection, luciferase activity was measured and is shown as SEAP-corrected relative light units (rlu). The mean (and SD) of 3 replicates is shown. * indicates p<0.05 (Students <i>t</i>-test) relative to pcDNA3. Panel B: Effect on influenza A/Turkey/England/50-92/91 minireplicon system. DF-1 cells were transfected with plasmids encoding A/Turkey/England/50-92/91 PB1, PB2, PA and NP, A/PR/8/34 NS1, an influenza minireplicon plasmid encoding luciferase, and a SEAP expressing plasmid together with pcDNA3 or a plasmid expressing the indicated Mx protein (murine Mx1, human MxA, wild type chicken Mx proteins SHK (Asn631) and 8.1 (Ser631) and their nuclear-localised counterparts SHK NLS and 8.1 NLS). 48 h post-transfection, luciferase activity was measured and is shown as relative light units (rlu). The mean (and SD) of 6 replicates is shown. * indicates p<0.05 (Students <i>t</i>-test) relative to pcDNA3. Panel C: Effect on influenza A/PR/8/34 gene expression. 293T cells were co-transfected with Mx-expressing plasmids (or pcDNA3) and pEGFP-C1 and infected after 48 h with influenza A/PR/8/34. 15 h post-infection, the cells were stained for influenza vRNP and analysed by flow cytometry. The percentage of antigen positive cells is expressed relative to that of the pcDNA3 control. The mean (and range) of 2 replicates is shown for GFP positive cells co-transfected with the indicated constructs.</p

Chicken Mx proteins do not inhibit NDV-directed gene expression.

<p>293T cells were co-transfected with a plasmid expressing the DsRed-express fluorescent protein and either pcDNA3 or a plasmid expressing the indicated Mx protein (human MxA, the MxA mutant T013A, murine Mx1, the Mx1 mutant K49A, wild type SHK (Asn631) or 8.1 (Ser631) chicken Mx). 48 h post-transfection, the cells were infected with NDV-GFP at an MOI which achieved approximately 60% infection (as determined by flow cytometry). 15 h post-infection, the cells were fixed and analysed by flow cytometry. Cells were then gated according to their expression of DsRed, and analysed for GFP fluorescence in the FL1-H channel. Panel A shows representative histograms for the GFP fluorescence in DsRed positive cells that were co-transfected with the indicated plasmids. Sub-panel (a) shows the background GFP fluorescence in uninfected, pcDNA3-transfected cells. The fluorescence threshold marker (M1) demarcates between GFP negative and positive cells. Panel B shows data derived from 6 replicates and bar heights show the % GFP positive cells expressed relative to that for the pcDNA3 control. The mean (and SD) are shown for DsRed positive cells co-transfected with the constructs as indicated. * indicates a significant difference (Students <i>t</i>-test) relative to pcDNA3 (p<0.05).</p

A single drug-resistance mutation in HSV-1 UL52 primase points to a difference between two helicase-primase inhibitors in their mode of interaction with the antiviral target

Objectives: To investigate the mechanism of action of the helicase–primase inhibitors (HPIs) BAY 57-1293 and BILS 22 BS by selection and characterization of drug-resistant herpes simplex virus (HSV)-1 mutants. Methods: HSV-1 mutants were selected using BAY 57-1293 in Vero cells. Resistance mutations identified in the UL5 helicase or UL52 primase genes were validated by marker transfer. Cross-resistance to the structurally distinct BILS 22 BS was measured by ID50 determinations. Results (i) A single mutation (UL52: A899T) confers 43-fold resistance to BAY 57-1293, but does not confer any resistance to BILS 22 BS. (ii) A double mutant (UL52: A899T and UL5: K356T) is 2500-fold resistant to BAY 57-1293, which is more than 17 times the sum of fold-resistance due to the individual mutations, UL52: A899T (43-fold) and UL5: K356T (100-fold). (iii) Virus containing the single helicase mutation and the double mutant with mutations in both helicase and primase showed equal resistance to BILS 22 BS (70-fold). Conclusions: By measuring the relative inhibitory concentrations required to overcome particular mutations in the helicase and primase proteins, evidence was obtained that BAY 57-1293 interacts with both components of the helicase–primase complex to achieve maximum potency, whereas for BILS 22BS, this may not be the case. Furthermore, our observations suggest that BAY 57-1293 interacts simultaneously with UL5 and UL52. Overall, the results suggest that these two potent HPIs interact differently with the helicase–primase complex