Varicella zoster virus (VZV) is the etiological agent of chickenpox and shingles, diseases characterized by epidermal skin blistering. Using a calcium-induced keratinocyte differentiation model we investigated the interaction between epidermal differentiation and VZV infection. RNA-seq analysis showed that VZV infection has a profound effect on differentiating keratinocytes, altering the normal process of epidermal gene expression to generate a signature that resembles patterns of gene expression seen in both heritable and acquired skin-blistering disorders. Further investigation by real-time PCR, protein analysis and electron microscopy revealed that VZV specifically reduced expression of specific suprabasal cytokeratins and desmosomal proteins, leading to disruption of epidermal structure and function. These changes were accompanied by an upregulation of kallikreins and serine proteases. Taken together VZV infection promotes blistering and desquamation of the epidermis, both of which are necessary to the viral spread and pathogenesis. At the same time, analysis of the viral transcriptome provided evidence that VZV gene expression was significantly increased following calcium treatment of keratinocytes. Using reporter viruses and immunohistochemistry we confirmed that VZV gene and protein expression in skin is linked with cellular differentiation. These studies highlight the intimate host-pathogen interaction following VZV infection of skin and provide insight into the mechanisms by which VZV remodels the epidermal environment to promote its own replication and spread.
The gammaherpesvirus alcelaphine herpesvirus 1 (AlHV-1) causes malignant catarrhal fever in susceptible ungulates but infects its natural host, wildebeest, without obvious clinical signs. In tissue culture, AlHV-1 is initially predominantly cell associated and virulent but on extended culture becomes cell-free and attenuated. We wanted to determine what changes in protein composition had taken place during the transition from virulent to attenuated virus in culture. Purified virus preparations were fractionated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and proteins were analyzed by liquid chromatography-electrospray ionization-tandem mass spectrometry. Peptides were identified in serial gel slices by using MASCOT software to interrogate virus-specific and nonredundant sequence databases. Twenty-three AlHV-1-encoded proteins and six cellular proteins were identified in the attenuated and virulent viruses. Two polypeptides were detected in only the virulent virus preparations, while one other protein was found in only the attenuated virus. Two of these virus-specific proteins were identified by a single peptide, suggesting that these may be low-abundance virion proteins rather than markers of attenuation or pathogenesis. The results suggest that attenuation of AlHV-1 is not the result of gross changes in the composition of the virus particle but probably due to altered viral gene expression in the infected cell.
Mechanisms by which certain RNA viruses, such as hepatitis C virus, establish persistent infections and cause chronic disease are of fundamental importance in viral pathogenesis. Mammalian positive-stranded RNA viruses establishing persistence typically possess genome-scale ordered RNA secondary structure (GORS) in their genomes. Murine norovirus (MNV) persists in immunocompetent mice and provides an experimental model to functionally characterize GORS. Substitution mutants were constructed with coding sequences in NS3/4- and NS6/7-coding regions replaced with sequences with identical coding and (di-)nucleotide composition but disrupted RNA secondary structure (F1, F2, F1/F2 mutants). Mutants replicated with similar kinetics to wild-type (WT) MNV3 in RAW264.7 cells and primary macrophages, exhibited similar (highly restricted) induction and susceptibility to interferon-coupled cellular responses and equal replication fitness by serial passaging of co-cultures. In vivo, both WT and F1/F2 mutant viruses persistently infected mice, although F1, F2 and F1/F2 mutant viruses were rapidly eliminated 1–7 days post-inoculation in competition experiments with WT. F1/F2 mutants recovered from tissues at 9 months showed higher synonymous substitution rates than WT and nucleotide substitutions that potentially restored of RNA secondary structure. GORS plays no role in basic replication of MNV but potentially contributes to viral fitness and persistence in vivo.
The Roslin Institute & Royal (Dick) School of Veterinary Studies, University of Edinburgh, Easter Bush Veterinary Campus, Roslin, Midlothian EH25 9RG, UK Herpesviruses encode microRNAs (miRNAs) that target both virus and host genes; however, their role in herpesvirus biology is understood poorly. We identified previously eight miRNAs encoded by ovine herpesvirus-2 (OvHV-2), the causative agent of malignant catarrhal fever (MCF), and have now investigated the role of these miRNAs in regulating expression of OvHV-2 genes that play important roles in virus biology. ORF20 (cell cycle inhibition), ORF50 (reactivation) and ORF73 (latency maintenance) each contain predicted targets for several OvHV-2 miRNAs. Cotransfection of miRNA mimics with luciferase reporter constructs containing the predicted targets showed the 59 UTRs of ORF20 and ORF73 contain functional targets for ovhv-miR-2 and ovhv2-miR-8, respectively, and the 39 UTR of ORF50 contains a functional target for ovhv2-miR-5. Transfection of BJ1035 cells (an OvHV-2-infected bovine T-cell line) with the relevant miRNA mimic resulted in a significant decrease in ORF50 and a smaller but non-significant decrease in ORF20. However, we were unable to demonstrate a decrease in ORF73. MCF is a disease of dysregulated lymphocyte proliferation; miRNA inhibition of ORF20 expression may play a role in this aberrant lymphocyte proliferation. The proteins encoded by ORF50 and ORF73 play opposing roles in latency. It has been hypothesized that miRNA-induced inhibition of virus genes acts to ensure that fluctuations in virus mRNA levels do not result in reactivation under conditions that are unfavourable for viral replication and our data supported this hypothesis.
11We produced a genome wide CRISPR knockout library, btCRISPRko.v1, targeting all protein coding genes in 12 the cattle genome and used it to identify host genes important for Bovine Herpes Virus Type 1 (BHV-1) 13 replication. By infecting library transduced MDBK cells with a GFP tagged BHV-1 virus and FACS sorting 14 them based on their GFP intensity, we identified a list of pro-viral and anti-viral candidate host genes that might 15 affect various aspects of the virus biology, such as cell entry, RNA transcription and viral protein trafficking. 16Among them were VPS51, VPS52 and VPS53 that encode for subunits of two membrane tethering complexes 17 EARP and GARP. Simultaneous loss of both complexes in MDBKs resulted in a significant reduction in the 18 production of infectious cell free BHV-1 virions, suggesting the vital roles they play during capsid re-19 envelopment with endocytosed membrane tubules prior to endosomal recycling mediated cellular egress. We 20 also observed potential capsid retention and aggregation in the nuclei of these cells, indicating that they might 21 also indirectly affect capsid egress from the nucleus. The btCRISPRko.v1 library generated here greatly 22 expanded our capability in BHV-1 related host gene discovery; we hope it will facilitate efforts intended to 23 study interactions between the host and other pathogens in cattle and also basic host cell biology. 24 25 26 27 28 29 30Bovine Herpes Virus Type 1(BHV-1) causes major economic loss through multiple disease manifestations such 31 as infectious bovine rhinotracheitis (IBR), infectious pustular vulvovaginitis, balanoposthitis and abortions in 32 cows 1 . The virus is endemic in Ireland and the UK, with 80% of herds seropositive for infections 2-4 . Following 33 successful clearance of acute infection, BHV-1 establishes lifelong latency in sensory neurons via neuronal 34 retrograde transport. Stressful life events such as a pregnancy, co-mingling and transporting during extreme 35 weather can trigger reactivation to induce clinical diseases. Vaccination can be used to control IBR; however, a 36 recent study showed that strains isolated from respiratory cases and aborted fetuses corresponded to modified 37 live vaccine strains 5 . Improved knowledge in host-pathogen interactions is needed to develop better disease 38 control and resistance strategies. 39Like other alpha-herpesvirus, BHV-1 replicates its dsDNA genome in the host nucleus but matures into 40 infectious particles in the cytoplasm. To initiate a lytic cycle of infection, envelope glycoprotein gC of the virus 41 interacts with cell surface Heparan Sulfate proteoglycans 6,7 , enabling close contact and binding between other 42 glycoproteins and cell surface proteins. The binding of gD to the poliovirus receptor (PVR) and nectin-1 8-10 and 43 interaction between gB or gH/gL with putative receptor paired immunoglobulin-like type 2 receptor α or 44 PILRα 11-14 brings the virus closer to the cellular membrane and catalyzes cell penetrance by membrane fusion or 45 endocytosi...
Herpesviruses encode miRNAs that target both virus and host genes; however their role in herpesvirus biology is still poorly understood. We previously identified thirty five miRNAs encoded by OvHV-2; the causative agent of malignant catarrhal fever (MCF) and are investigating the role of these miRNAs in regulating expression of OvHV-2 genes that play important roles in virus biology. Analysis, using RNAHybrid predicted that two OvHV-2 encoded miRNAs, ovhv2-miR-17-10 and ovhv2-miR-61-1, target transcripts coding for the OvHV-2 bZIP protein Ov2. In other herpesvirus bZIP proteins are known to play important roles in lytic virus replication. Here we show by Flow cytometry and western blotting that ovhv2-miR-17-10 and ovhv2-miR-61-1, reduce the expression of Ov2 protein. The predicted target sites for both miRNAs within the Ov2 gene were disrupted whilst retaining the Ov2 coding sequence. Mutation of the ovhv2-miR-61-1 target sequence restored Ov2 protein expression levels to control levels confirming the identity of its target site. However, it was not possible to determine the binding site of ovhv2-miR-17-10 possibly due to potential G:U pairing introduced during the mutation process. The targeting of Ov2 by two virus-encoded miRNAs suggests an important regulatory role for Ov2 in OvHV-2 replication or reactivation.
The gammaherpesvirus alcelaphine herpesvirus 1 (AlHV-1) causes fatal malignant catarrhal fever (MCF) in susceptible species including cattle, but infects its reservoir host, wildebeest, without causing disease. Pathology in cattle may be influenced by virus-host cell interactions mediated by the virus glycoproteins. Cloning and expression of a haemagglutinin-tagged version of the AlHV-1 glycoprotein B (gB) was used to demonstrate that the AlHV-1-specific monoclonal antibody 12B5 recognised gB and that gB was the main component of the gp115 complex of AlHV-1, a glycoprotein complex of five components identified on the surface of AlHV-1 by immunoprecipitation and radiolabelling. Analysis of AlHV-1 virus particles showed that the native form of gB was detected by mAb 12B5 as a band of about 70 kDa, whilst recombinant gB expressed by transfected HEK293T cells appeared to be subject to additional cleavage and incomplete post-translational processing. Antibody 12B5 recognised an epitope on the N-terminal furin-cleaved fragment of gB on AlHV-1 virus particles. It could be used to detect recombinant and virus-expressed gB on western blots and on the surface of infected cells by flow cytometry, whilst recombinant gB was detected on the surface of transfected cells by immunofluorescence. Recombinant gB has potential as an antigen for ELISA detection of MCF virus infection and as a candidate vaccine antigen.
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