Stem cell-derived porcine macrophages as a new platform for studying host-pathogen interactions.

BACKGROUND: Infectious diseases of farmed and wild animals pose a recurrent threat to food security and human health. The macrophage, a key component of the innate immune system, is the first line of defence against many infectious agents and plays a major role in shaping the adaptive immune response. However, this phagocyte is a target and host for many pathogens. Understanding the molecular basis of interactions between macrophages and pathogens is therefore crucial for the development of effective strategies to combat important infectious diseases. RESULTS: We explored how porcine pluripotent stem cells (PSCs) can provide a limitless in vitro supply of genetically and experimentally tractable macrophages. Porcine PSC-derived macrophages (PSCdMs) exhibited molecular and functional characteristics of ex vivo primary macrophages and were productively infected by pig pathogens, including porcine reproductive and respiratory syndrome virus (PRRSV) and African swine fever virus (ASFV), two of the most economically important and devastating viruses in pig farming. Moreover, porcine PSCdMs were readily amenable to genetic modification by CRISPR/Cas9 gene editing applied either in parental stem cells or directly in the macrophages by lentiviral vector transduction. CONCLUSIONS: We show that porcine PSCdMs exhibit key macrophage characteristics, including infection by a range of commercially relevant pig pathogens. In addition, genetic engineering of PSCs and PSCdMs affords new opportunities for functional analysis of macrophage biology in an important livestock species. PSCs and differentiated derivatives should therefore represent a useful and ethical experimental platform to investigate the genetic and molecular basis of host-pathogen interactions in pigs, and also have wider applications in livestock.

Does the Zinc Finger Antiviral Protein (ZAP) Shape the Evolution of Herpesvirus Genomes?

An evolutionary arms race occurs between viruses and hosts. Hosts have developed an array of antiviral mechanisms aimed at inhibiting replication and spread of viruses, reducing their fitness, and ultimately minimising pathogenic effects. In turn, viruses have evolved sophisticated counter-measures that mediate evasion of host defence mechanisms. A key aspect of host defences is the ability to differentiate between self and non-self. Previous studies have demonstrated significant suppression of CpG and UpA dinucleotide frequencies in the coding regions of RNA and small DNA viruses. Artificially increasing these dinucleotide frequencies results in a substantial attenuation of virus replication, suggesting dinucleotide bias could facilitate recognition of non-self RNA. The interferon-inducible gene, zinc finger antiviral protein (ZAP) is the host factor responsible for sensing CpG dinucleotides in viral RNA and restricting RNA viruses through direct binding and degradation of the target RNA. Herpesviruses are large DNA viruses that comprise three subfamilies, alpha, beta and gamma, which display divergent CpG dinucleotide patterns within their genomes. ZAP has recently been shown to act as a host restriction factor against human cytomegalovirus (HCMV), a beta-herpesvirus, which in turn evades ZAP detection by suppressing CpG levels in the major immediate-early transcript IE1, one of the first genes expressed by the virus. While suppression of CpG dinucleotides allows evasion of ZAP targeting, synonymous changes in nucleotide composition that cause genome biases, such as low GC content, can cause inefficient gene expression, especially in unspliced transcripts. To maintain compact genomes, the majority of herpesvirus transcripts are unspliced. Here we discuss how the conflicting pressures of ZAP evasion, the need to maintain compact genomes through the use of unspliced transcripts and maintaining efficient gene expression may have shaped the evolution of herpesvirus genomes, leading to characteristic CpG dinucleotide patterns.

Hematopoietic cell-mediated dissemination of murine cytomegalovirus is regulated by NK cells and immune evasion.

Cytomegalovirus (CMV) causes clinically important diseases in immune compromised and immune immature individuals. Based largely on work in the mouse model of murine (M)CMV, there is a consensus that myeloid cells are important for disseminating CMV from the site of infection. In theory, such dissemination should expose CMV to cell-mediated immunity and thus necessitate evasion of T cells and NK cells. However, this hypothesis remains untested. We constructed a recombinant MCMV encoding target sites for the hematopoietic specific miRNA miR-142-3p in the essential viral gene IE3. This virus disseminated poorly to the salivary gland following intranasal or footpad infections but not following intraperitoneal infection in C57BL/6 mice, demonstrating that dissemination by hematopoietic cells is essential for specific routes of infection. Remarkably, depletion of NK cells or T cells restored dissemination of this virus in C57BL/6 mice after intranasal infection, while dissemination occurred normally in BALB/c mice, which lack strong NK cell control of MCMV. These data show that cell-mediated immunity is responsible for restricting MCMV to hematopoietic cell-mediated dissemination. Infected hematopoietic cells avoided cell-mediated immunity via three immune evasion genes that modulate class I MHC and NKG2D ligands (m04, m06 and m152). MCMV lacking these 3 genes spread poorly to the salivary gland unless NK cells were depleted, but also failed to replicate persistently in either the nasal mucosa or salivary gland unless CD8+ T cells were depleted. Surprisingly, CD8+ T cells primed after intranasal infection required CD4+ T cell help to expand and become functional. Together, our data suggest that MCMV can use both hematopoietic cell-dependent and -independent means of dissemination after intranasal infection and that cell mediated immune responses restrict dissemination to infected hematopoietic cells, which are protected from NK cells during dissemination by viral immune evasion. In contrast, viral replication within mucosal tissues depends on evasion of T cells.

The host ubiquitin-dependent segregase VCP/p97 is required for the onset of human cytomegalovirus replication.

The human cytomegalovirus major immediate early proteins IE1 and IE2 are critical drivers of virus replication and are considered pivotal in determining the balance between productive and latent infection. IE1 and IE2 are derived from the same primary transcript by alternative splicing and regulation of their expression likely involves a complex interplay between cellular and viral factors. Here we show that knockdown of the host ubiquitin-dependent segregase VCP/p97, results in loss of IE2 expression, subsequent suppression of early and late gene expression and, ultimately, failure in virus replication. RNAseq analysis showed increased levels of IE1 splicing, with a corresponding decrease in IE2 splicing following VCP knockdown. Global analysis of viral transcription showed the expression of a subset of viral genes is not reduced despite the loss of IE2 expression, including UL112/113. Furthermore, Immunofluorescence studies demonstrated that VCP strongly colocalised with the viral replication compartments in the nucleus. Finally, we show that NMS-873, a small molecule inhibitor of VCP, is a potent HCMV antiviral with potential as a novel host targeting therapeutic for HCMV infection.

Expression of ovine herpesvirus -2 encoded microRNAs in an immortalised bovine - cell line.

Ovine herpesvirus-2 (OvHV-2) infects most sheep, where it establishes an asymptomatic, latent infection. Infection of susceptible hosts e.g. cattle and deer results in malignant catarrhal fever, a fatal lymphoproliferative disease characterised by uncontrolled lymphocyte proliferation and non MHC restricted cytotoxicity. The same cell populations are infected in both cattle and sheep but only in cattle does virus infection cause dysregulation of cell function leading to disease. The mechanism by which OvHV-2 induces this uncontrolled proliferation is unknown. A number of herpesviruses have been shown to encode microRNAs (miRNAs) that have roles in control of both viral and cellular gene expression. We hypothesised that OvHV-2 encodes miRNAs and that these play a role in pathogenesis. Analysis of massively parallel sequencing data from an OvHV-2 persistently-infected bovine lymphoid cell line (BJ1035) identified forty-five possible virus-encoded miRNAs. We previously confirmed the expression of eight OvHV-2 miRNAs by northern hybridization. In this study we used RT-PCR to confirm the expression of an additional twenty-seven OvHV-2-encoded miRNAs. All thirty-five OvHV-2 miRNAs are expressed from the same virus genome strand and the majority (30) are encoded in an approximately 9 kb region that contains no predicted virus open reading frames. Future identification of the cellular and virus targets of these miRNAs will inform our understanding of MCF pathogenesis.

Ovine herpesvirus-2-encoded microRNAs target virus genes involved in virus latency.

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. Co-transfection of miRNA mimics with luciferase reporter constructs containing the predicted targets showed the 5' UTRs of ORF20 and ORF73 contain functional targets for ovhv-miR-2 and ovhv2-miR-8, respectively, and the 3' 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.

Cytomegalovirus-based cancer vaccines expressing TRP2 induce rejection of melanoma in mice.

Cytomegalovirus (CMV) induces strong and long-lasting immune responses, which make it an attractive candidate for a cancer vaccine vector. In this study, we tested whether a tumor antigen expressed in CMV can induce a strong anti-tumor effect. We expressed an unmodified melanoma antigen, mouse tyrosinase-related protein 2 (TRP2), in mouse cytomegalovirus (MCMV). Prophylactic vaccination of the mice with a single dose of MCMV-TRP2 induced rejection of B16 melanoma challenge; therapeutic vaccination with MCMV-TRP2 prolonged the survival of the mice challenged with B16 cells. Additionally, vaccination with MCMV-TRP2 five months before tumor challenge still induced tumor rejection, which indicated that the vaccine induced long-term protection. Furthermore, MCMV-TRP2 protected mice against B16 melanoma challenge regardless of the pre-existing CMV infection. We found that vaccination with MCMV-TRP2 induced long-lasting TRP2 specific antibodies but not CD8 T cells. In addition, depletion of CD4 and CD8 T cells did not compromise the antitumor effect by MCMV-TRP2; while in B cell deficient (µMT) mice, the vaccine lost its antitumor effect. These results indicate that antibodies, not T cells, are important in mediating the antitumor effect during the effector phase by the vaccine. We also made a spread deficient MCMV-TRP2 lacking the essential glycoprotein gL, which showed a similar antitumor effect. In conclusion, our study indicates that tumor antigen (TRP2) expressed in MCMV induces a strong and long-lasting anti-melanoma effect through an antibody-dependent mechanism. Our findings demonstrate that CMV might be a promising vector for the development of cancer vaccines.

Competition for antigen at the level of the APC is a major determinant of immunodominance during memory inflation in murine cytomegalovirus infection.

The unique ability of CMV to drive the expansion of virus-specific T cell populations during the course of a lifelong, persistent infection has generated interest in the virus as a potential vaccine strategy. When designing CMV-based vaccine vectors to direct immune responses against HIV or tumor Ags, it becomes important to understand how and why certain CMV-specific populations are chosen to inflate over time. To investigate this, we designed recombinant murine CMVs (MCMVs) encoding a SIINFEKL-enhanced GFP fusion protein under the control of endogenous immediate early promoters. When mice were infected with these viruses, T cells specific for the SIINFEKL epitope inflated and profoundly dominated T cells specific for nonrecombinant (i.e., MCMV-derived) Ags. Moreover, when the virus encoded SIINFEKL, T cells specific for nonrecombinant Ags displayed a phenotype indicative of less frequent exposure to Ag. The immunodominance of SIINFEKL-specific T cells could not be altered by decreasing the number of SIINFEKL-specific cells available to respond, or by increasing the number of cells specific for endogenous MCMV Ags. In contrast, coinfection with viruses expressing and lacking SIINFEKL enabled coinflation of T cells specific for both SIINFEKL and nonrecombinant Ags. Because coinfection allows presentation of SIINFEKL and MCMV-derived Ags by different cells within the same animal, these data reveal that competition for, or availability of, Ag at the level of the APC determines the composition of the inflationary response to MCMV. SIINFEKL's strong affinity for H-2K(b), as well as its early and abundant expression, may provide this epitope's competitive advantage.

Competition between T cells maintains clonal dominance during memory inflation induced by MCMV.

Both human cytomegalovirus (HCMV) and murine cytomegalovirus (MCMV) establish persistent infections that induce the accumulation of virus-specific T cells over time in a process called memory inflation. It has been proposed that T cells expressing T-cell receptors (TCRs) with high affinity for HCMV-derived peptides are preferentially selected after acute HCMV infection. To test this in the murine model, small numbers of OT-I transgenic T cells, which express a TCR with high affinity for the SIINFEKL peptide, were transferred into congenic mice and recipients were challenged with recombinant MCMV expressing SIINFEKL. OT-I T cells were selectively enriched during the first 3 weeks of infection. Similarly, in the absence of OT-I T cells, the functional avidity of SIINFEKL-specific T cells increased from early to late times postinfection. However, even when exceedingly small numbers of OT-I T cells were transferred, their inflation limited the inflation of host-derived T cells specific for SIINFEKL. Importantly, subtle minor histocompatibility differences led to late rejection of the transferred OT-I T cells in some mice, which allowed host-derived T cells to inflate substantially. Thus, T cells with a high functional avidity are selected shortly after MCMV infection and continuously sustain their clonal dominance in a competitive manner.

Histone modifications associated with herpes simplex virus type 1 genomes during quiescence and following ICP0-mediated de-repression.

In the current study, it was shown that repressed virus genomes in quiescently infected MRC5 cells adopt a repressed histone-associated structure marked by the enrichment of deacetylated histones at a wide variety of herpes simplex virus type 1 (HSV-1) promoters. In addition, it was shown that genome de-repression, mediated by HSV-2 superinfection or delivery of ICP0 using a recombinant adenovirus vector, resulted in the enrichment of acetylated histones on HSV DNA. These data indicate that ICP0-mediated genome de-repression is intimately linked to enrichment of acetylated histones at virus promoters. The fold change in association of pan-acetylated histone H3 following Ad.TRE.ICP0-mediated de-repression consistently revealed promoter-specific variation, with the highest fold changes (>50-fold) being observed at the latency-associated transcript promoter and enhancer regions. Chromatin immunoprecipitation analyses using an antibody specific to the C terminus of histone H3 as a surrogate measure of nucleosome occupancy revealed little variability in the total loading of histone H3 at the various HSV promoters. This observation suggests that acetylation of histone H3 in response to ICP0 expression is not uniformly targeted across the HSV-1 genome during ICP0-mediated de-repression.