- Volume 98, Issue 3, 2017

Histone deacetylase 6 (HDAC6) is a unique cytoplasmic deacetylase that regulates various important biological processes by preventing protein aggregation and deacetylating different non-histone substrates including tubulin, heat shock protein 90, cortactin, retinoic acid inducible gene I and β-catenin. Growing evidence has indicated a dual role for HDAC6 in viral infection and pathogenesis: HDAC6 may represent a host defence mechanism against viral infection by modulating microtubule acetylation, triggering antiviral immune response and stimulating protective autophagy, or it may be hijacked by the virus to enhance proinflammatory response. In this review, we will highlight current data illustrating the complexity and importance of HDAC6 in viral pathogenesis. We will summarize the structure and functional specificity of HDAC6, and its deacetylase- and ubiquitin-dependent activity in key cellular events in response to virus infection. We will also discuss how HDAC6 exerts its direct or indirect histone modification ability in viral lytic–latency switch.

Recent studies in human immunodeficiency virus (HIV) have garnered interest for the role of CC chemokine receptor 6 (CCR6) and its known ligands, CC chemokine ligand 20 (CCL20) and human β-defensins, in viral entry, dissemination and antiviral immunity. Several studies have suggested that CCR6 may also act as a weak co-receptor of HIV entry, in addition to the canonical CXC chemokine receptor 4 (CXCR4) and CCR5. However, the pathogenic significance has yet to be demonstrated as the observations for preferential infection of CD4+CCR6+ over CD4+CCR6− T cells appear to be independent of CCR6 expression. This indicates means for preferential infection other than CCR6 co-receptor use. Attention has also turned to the inadvertent role of the CCR6/CCL20 axis in attracting key immune cells, including TH17 cells and dendritic cells, to sites of infection and propagating the virus to other sites of the body. This review article will summarize the latest evidence that the CCR6/CCL20 chemokine axis is playing an important role in HIV pathogenesis and immunity. Further work with in vivo studies is needed to establish the biological and, hence, therapeutic significance of these findings.

The broad range of cellular functions governed by ion channels represents an attractive target for viral manipulation. Indeed, modulation of host cell ion channel activity by viral proteins is being increasingly identified as an important virus–host interaction. Recent examples have demonstrated that virion entry, virus egress and the maintenance of a cellular environment conducive to virus persistence are, in part, dependent on virus manipulation of ion channel activity. Most excitingly, evidence has emerged that targeting ion channels pharmacologically can impede virus life cycles. Here, we discuss current examples of virus–ion channel interactions and the potential of targeting ion channel function as a new, pharmacologically safe and broad-ranging anti-viral therapeutic strategy.

Dicistroviridae is a family of small non-enveloped viruses with monopartite, linear, positive-sense RNA genomes of approximately 8–10 kb. Viruses of all classified species infect arthropod hosts, with some having devastating economic consequences, such as acute bee paralysis virus in domesticated honeybees and taura syndrome virus in shrimp farming. Conversely, the host specificity and other desirable traits exhibited by several members of this group make them potential natural enemies for intentional use against arthropod pests, such as triatoma virus against triatomine bugs that vector Chagas disease. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) Report on the taxonomy of the Dicistroviridae which is available at www.ictv.global/report/dicistroviridae.

Substitutions in the PA N-terminus (PAN) of influenza A viruses are associated with viral pathogenicity. During our previous study, which identified PAN-V63I and -A37S/I61T/V63I/V100A substitutions as virulence determinants, we observed a severe decrease in virus growth and transcription/replication capacity posed by PAN-A37S/V100A substitution. To further delineate the significance of substitutions at these positions, we generated mutant H7N7 viruses bearing the substitutions PAN-A37S, -A37S/I61T, -A37S/V63I, -V100A, -I61T/V100A and -V63I/V100A by reverse genetics. Our results showed that all mutant viruses except PAN-V100A showed a significantly reduced growth capability in infected cells. At the same time, the PAN-A37S, -A37S/I61T and -A37S/V63I mutant viruses displayed decreased viral transcription and replication by diminishing virus RNA synthesis activity. Biochemical assays indicated that the substitutions PAN-A37S, -A37S/I61T and -A37S/V63I suppressed the polymerase and endonuclease activities when compared with those of the wild-type. Together, our results demonstrated that the PAN-A37S, -A37S/I61T and -A37S/V63I substitutions contributed to a decreased pathogenicity of avian H7N7 influenza A virus.

Hepatitis C virus (HCV) readily establishes chronic infection, which is characterized by failure of virus-specific CD8+ T cells. HCV uses epitope mutation and T-cell exhaustion to escape from the host immune response. Previously, we engineered high-affinity T-cell receptors (HATs) targeting human immunodeficiency virus escape mutants. In this study, the affinity of a T-cell receptor specific for the HLA-A2-restricted HCV immunodominant epitope NS3 1406–1415 (KLVALGINAV) was improved from a KD of 6.6 µM to 40 pM. These HATs could also target HCV NS3 naturally occurring variants, including an escape variant vrt1 (KLVVLGINAV), with high affinities. The HATs can be used as high-affinity targeting molecules at the centre of the immune synapse for the HLA-restricted NS3 antigen. By fusing the HAT with a T-cell activation molecule, an anti-CD3 single-chain variable fragment, we constructed a molecule called high-affinity T-cell activation core (HATac), which can redirect functional CTLs possessing any specificity to recognize and kill cells presenting HCV NS3 antigens. This capability was verified with T2 cells loaded with prototype or variant peptides and HepG2 cells expressing the truncated NS3 prototype or variant proteins. The results indicate that HATac targeting the HLA-restricted NS3 antigen may provide a useful tool for circumventing immune escape mutants and T-cell exhaustion caused by HCV infection.

The foot-and-mouth disease virus (FMDV) capsid precursor, P1-2A, is cleaved by FMDV 3C protease to yield VP0, VP3, VP1 and 2A. Cleavage of the VP1/2A junction is the slowest. Serotype O FMDVs with uncleaved VP1-2A (having a K210E substitution in VP1; at position P2 in cleavage site) have been described previously and acquired a second site substitution (VP1 E83K) during virus rescue. Furthermore, introduction of the VP1 E83K substitution alone generated a second site change at the VP1/2A junction (2A L2P, position P2′ in cleavage site). These virus adaptations have now been analysed using next-generation sequencing to determine sub-consensus level changes in the virus; this revealed other variants within the E83K mutant virus population that changed residue VP1 K210. The construction of serotype A viruses with a blocked VP1/2A cleavage site (containing K210E) has now been achieved. A collection of alternative amino acid substitutions was made at this site, and the properties of the mutant viruses were determined. Only the presence of a positively charged residue at position P2 in the cleavage site permitted efficient cleavage of the VP1/2A junction, consistent with analyses of diverse FMDV genome sequences. Interestingly, in contrast to the serotype O virus results, no second site mutations occurred within the VP1 coding region of serotype A viruses with the blocked VP1/2A cleavage site. However, some of these viruses acquired changes in the 2C protein that is involved in enterovirus morphogenesis. These results have implications for the testing of potential antiviral agents targeting the FMDV 3C protease.

Recent studies have shown that the currently circulating rubella viruses are mostly members of two genotypes, 1E and 2B. Also, genetically distinct viruses of genotype 1G have been found in East and West Africa. This study used a Mantel test to objectively include both genetic diversity and geographic location in the definition of lineages, and identified statistically justified lineages (n=13) and sub-lineages (n=9) of viruses within genotypes 1G, 1E and 2B. Genotype 2B viruses were widely distributed, while viruses of genotype 1E as well as 1G and 1J were much more geographically restricted. This analysis showed that more precise groupings for rubella viruses are possible, which should improve the ability to track rubella viruses worldwide. A year-by-year analysis revealed gaps in surveillance that need to be resolved in order to support the surveillance needed for enhanced control and elimination goals for rubella.

Preventing virally induced liver disease begins with an understanding of the host factors that define susceptibility to infection. Hepatitis C virus (HCV) is a global health issue, with an estimated 170 million infected individuals at risk of developing liver disease including fibrosis and hepatocellular carcinoma. The liver is the major reservoir supporting HCV replication and this hepatocellular tropism is defined by HCV engagement of cellular entry receptors. Hepatocytes are polarized in vivo and this barrier function limits HCV entry. We previously reported that activated macrophages promote HCV entry into polarized hepatocytes via a TNF-α-dependent process; however, the underlying mechanism was not defined. In this study, we show that several TNF superfamily members, including TNF-α, TNF-β, TWEAK and LIGHT, promote HCV entry via NF-κB-mediated activation of myosin light chain kinase (MLCK) and disruption of tight junctions. These observations support a model where HCV hijacks an inflammatory immune response to stimulate infection and uncovers a role for NF-κB-MLCK signalling in maintaining hepatocellular tight junctions.

Every year, tick-borne encephalitis virus (TBEV) causes severe central nervous system infection in 10 000 to 15 000 people in Europe and Asia. TBEV is maintained in the environment by an enzootic cycle that requires a tick vector and a vertebrate host, and the adaptation of TBEV to vertebrate and invertebrate environments is essential for TBEV persistence in nature. This adaptation is facilitated by the error-prone nature of the virus’s RNA-dependent RNA polymerase, which generates genetically distinct virus variants called quasispecies. TBEV shows a focal geographical distribution pattern where each focus represents a TBEV hotspot. Here, we sequenced and characterized two TBEV genomes, JP-296 and JP-554, from questing Ixodes ricinus ticks at a TBEV focus in central Sweden. Phylogenetic analysis showed geographical clustering among the newly sequenced strains and three previously sequenced Scandinavian strains, Toro-2003, Saringe-2009 and Mandal-2009, which originated from the same ancestor. Among these five Scandinavian TBEV strains, only Mandal-2009 showed a large deletion within the 3′ non-coding region (NCR), similar to the highly virulent TBEV strain Hypr. Deep sequencing of JP-296, JP-554 and Mandal-2009 revealed significantly high quasispecies diversity for JP-296 and JP-554, with intact 3′NCRs, compared to the low diversity in Mandal-2009, with a truncated 3′NCR. Single-nucleotide polymorphism analysis showed that 40 % of the single-nucleotide polymorphisms were common between quasispecies populations of JP-296 and JP-554, indicating a putative mechanism for how TBEV persists and is maintained within its natural foci.

Enterovirus 71 (EVA71), a virus of the genus Enterovirus in the family Picornaviridae, is one of the main causative agents of hand, foot and mouth disease in infected infants and young children. In this study, we report that cells with EVA71 infection exhibit increased levels of cytochrome c release and caspase-3 activation. EVA71 infection induces the conformational activation of pro-apoptotic protein Bax and the subsequent formation of oligomers of Bax in mitochondria. Inhibitors that block caspase-8 activation cannot inhibit apoptosis induced by EVA71 infection. Importantly, cells with Bax but not Bak or caspase-8 knockdown show resistance to apoptosis induced by EVA71 infection. Mitochondria isolated from EVA71-infected cells display clear Bax-binding ability and the subsequent release of cytochrome c. Therefore, these results indicate that EVA71 infection directly impacts the mitochondrial apoptotic pathway by modulating the recruitment and activation of Bax.

Epstein–Barr virus-induced gene 2 (EBI2) is an important chemotactic receptor that is involved in proper B-cell T-cell interactions. Epstein–Barr virus (EBV) has been shown to upregulate this gene upon infection of cell lines, but the timing and mechanism of this upregulation, as well as its importance to EBV infection, remain unknown. This work investigated EBV’s manipulation of EBI2 expression of primary naive B cells. EBV infection induces EBI2 expression resulting in elevated levels of EBI2 after 24 h until 7 days post-infection, followed by a dramatic decline (P=0.027). Increased EBI2 expression was not found in non-specifically stimulated B cells or when irradiated virus was used. The EBV lytic gene BRRF1 exhibited a similar expression pattern to EBI2 (R2 =0.4622). BRRF1-deficient EBV could not induce EBI2. However, B cells transduced with BRRF1 showed elevated expression of EBI2 (P=0.042), a result that was not seen with transduction of a different EBV lytic transfection factor, BRLF1. Based on these results, we conclude that EBI2 expression is directly influenced by EBV infection and that BRRF1 is necessary and sufficient for EBI2 upregulation during infection.

CX3CR1 is an important chemokine receptor expressed on the surface of microglia and blood leukocytes, including monocytes. Signalling through this receptor influences the immune activity of microglia and monocyte trafficking into the central nervous system (CNS) in several neurological diseases. During experimental herpes simplex virus 1 (HSV-1) encephalitis (HSE), CX3CR1 deficiency has been reported to exacerbate the outcome of the disease. However, the precise contribution of CX3CR1 expressed in resident cells of the CNS or peripheral monocytes in protection against HSE remains unclear. To dissect the role of CX3CR1 during HSE, we reconstituted irradiated C57BL/6 WT and CX3CR1−/− mice with CX3CR1−/− (CX3CR1−/−→WT) and WT (WT→CX3CR1−/−) bone marrow cells, respectively. Our results showed that following intranasal infection with 1.2×106 p.f.u. of HSV-1, mortality rates were significantly higher in CX3CR1−/− (61.7 %) and WT→CX3CR1−/− (66.2 %) compared to WT (16.6 %; P=0.012 and P=0.016, respectively) and CX3CR1−/−→WT animals (20 %; P=0.013 and P=0.011, respectively). Higher mortality rates in CX3CR1−/− and WT→CX3CR1−/− mice were associated with increased infectious viral titres and wider HSV dissemination in brains, as well as an overproduction of inflammatory cytokines and chemokines including IL-1β, IL-6, IFN-γ, C-C motif ligand 2 and C-C motif ligand 5. Furthermore, CX3CR1 deficiency in resident cells of the CNS resulted in excessive and sustained Ly6Chi inflammatory monocyte and neutrophil infiltration into the brain. These data suggest that CX3CR1 deficiency in resident cells of the CNS affects mouse survival, HSV-1 replication control and cerebral inflammatory response whereas its deficiency in the haematopoietic system does not appear to influence the outcome of HSE.

Equine herpesvirus 5 (EHV5) is a commonly detected gammaherpesvirus, which, along with the closely related EHV2, constitute the only two known percaviruses that infect horses. Apart from detection in horse populations worldwide and the recent publication of the whole genome, there is little known about the biology and pathogenesis of this virus, with many assumptions made by parallels with EHV2. The long-term survival of gammaherpesviruses within infected hosts involves the establishment and maintenance of latency in selected cell and tissues types, particularly lymphocytes. A latent gammaherpesvirus infection is characterized by a limited number of genes expressing in a particular cell or tissue type. In this study, we have used in vitro co-culturing to detect EHV5 in equine PBMCs and characterize the predominant cellular site for the establishment and maintenance of a latent infection. These experiments were conducted by isolating PBMCs from 10 horses and sorting subpopulations into two T lymphocyte (CD4 and CD8), B lymphocyte and macrophage enriched or depleted fractions. These lymphocyte and macrophage fractions were examined for the presence of latent EHV5 by in vitro co-culturing with equine foetal kidney cells. The lymphocyte fraction enriched with B lymphocytes had a significantly increased (P=0.005) number of plaques formed during co-culturing, whereas the B lymphocyte depleted fraction had a significant reduction in the number of plaques formed after co-culturing. Taken together, these results demonstrate that equine gammaherpesviruses establish latency in the equine PBMCs, with the predominant site for maintenance of latent virus being B lymphocytes.

Protein phosphorylation has important regulatory functions in cell homeostasis and is tightly regulated by kinases and phosphatases. The tegument of human cytomegalovirus (CMV) contains not only several proteins reported to be extensively phosphorylated but also cellular protein phosphatases (PP1 and PP2A). To investigate this apparent inconsistency, we evaluated the phosphorylation status of the tegument proteins pUL32 and pp65 by enzymatic dephosphorylation and MS. Enzymatic dephosphorylation with bacterial λ phosphatase, but not with PP1, shifted the pUL32-specific signal on reducing SDS-PAGE from ~150 to ~148 kDa, a mass still much larger than the ~118 kDa obtained from our diffusion studies and from the calculated protein mass of ~113 kDa. Remarkably, inhibition of phosphatases through treatment with the phosphatase inhibitors calyculin A and okadaic acid resulted in a shift to ~190 or ~180 kDa, respectively, indicating that a considerable number of potential phosphorylated residues on pUL32 are not phosphorylated under normal conditions. MS revealed a general state of hypophosphorylation of CMV phosphoproteins with only 17 phosphorylated residues detected on pUL32 and 19 on pp65, respectively. Moreover, bioinformatics analysis shows that the C-terminal two-thirds of pUL32 are intrinsically disordered and that most phosphorylations map to this region. In conclusion, we show that important CMV tegument proteins are indeed phosphorylated, though to a lesser extent than previously reported, and the difference in mobility on SDS-PAGE and calculated mass of pUL32 may not be attributed to phosphorylation but more likely due to the partially intrinsically disordered nature of pUL32.

Duck enteritis virus (DEV) is an acute, septic, sexually transmitted disease that occurs in ducks, geese and other poultry. Autophagy is an evolutionarily ancient pathway that is important in many viral infections. Despite extensive study, the interplay between DEV and autophagy of host cells is not clearly understood. In this study, we found that DEV infection triggers autophagy in duck embryo fibroblast (DEF) cells, as demonstrated by the appearance of autophagosome-like double- or single-membrane vesicles in the cytoplasm of host cells and the number of GFP-LC3 dots. In addition, increased conversion of the autophagy marker protein LC3-I and LC3-II and decreased p62/SQSTM1 indicated complete autophagy flux. Heat-inactivated DEV infection did not induce autophagy, suggesting that the trigger of autophagy in DEF cells depended on DEV replication. When autophagy was pharmacologically inhibited by LY294002 or wortmannin, DEV replication decreased. The DEV offspring yield decreased when small interference RNA was used to interfere with autophagy related to the genes Beclin-1 and ATG5. In contrast, after treating DEF cells with rapamycin, an inducer of autophagy, DEV replication increased. These results indicated that DEV infection induced autophagy in DEF cells and autophagy facilitated DEV replication.

We have previously established that mucosal uptake of recombinant fowlpox virus (rFPV) vaccines is far superior to other vector-based vaccines. Specifically, intranasal priming with rFPV vaccines can recruit unique antigen-presenting cells, which induce excellent mucosal and systemic HIV-specific CD8+ T-cell immunity. In this study, we have for the first time investigated the in vivo dissemination, safety and expression kinetics of rFPV post intranasal delivery using recombinant viruses expressing green fluorescent protein or mCherry. Both confocal microscopy of tissue sections using green fluorescent protein and in vivo Imaging System (IVIS) spectrum live animal and whole organ imaging studies using mCherry revealed that (i) the peak antigen expression occurs 12 to 24 h post vaccination and no active viral gene expression is detected 96 h post vaccination. (ii) The virus only infects the initial vaccination site (lung and nasal cavity) and does not disseminate to distal sites such as the spleen or gut. (iii) More importantly, rFPV does not cross the olfactory receptor neuron pathway. Collectively, our findings indicate that rFPV vector-based vaccines have all the hallmarks of a safe and effective mucosal delivery vector, suitable for clinical evaluation.