Temperature-induced reorganisation of Schistocephalus solidus (Cestoda) proteome during the transition to the warm-blooded host

ABSTRACT The protein composition of the cestode Schistocephalus solidus was measured in an experiment simulating the trophic transmission of the parasite from a cold-blooded to a warm-blooded host. The first hour of host colonisation was studied in a model experiment, in which sticklebacks Gasterosteus aculeatus infected with S. solidus were heated at 40°C for 1 h. As a result, a decrease in the content of one tegument protein was detected in the plerocercoids of S. solidus. Sexual maturation of the parasites was initiated in an experiment where S. solidus larvae were taken from fish and cultured in vitro at 40°C for 48 h. Temperature-independent changes in the parasite proteome were investigated by incubating plerocercoids at 22°C for 48 h in culture medium. Analysis of the proteome allowed us to distinguish the temperature-induced genes of S. solidus, as well as to specify the molecular markers of the plerocercoid and adult worms. The main conclusion of the study is that the key enzymes of long-term metabolic changes (glycogen consumption, protein production, etc.) in parasites during colonisation of a warm-blooded host are induced by temperature.

Tegument Protein pp150 Sequence-Specific Peptide Blocks Cytomegalovirus Infection

Human cytomegalovirus (HCMV) tegument protein pp150 is essential for the completion of the final steps in virion maturation. Earlier studies indicated that three pp150nt (N-terminal one-third of pp150) conformers cluster on each triplex (Tri1, Tri2A and Tri2B), and extend towards small capsid proteins atop nearby major capsid proteins, forming a net-like layer of tegument densities that enmesh and stabilize HCMV capsids. Based on this atomic detail, we designed several peptides targeting pp150nt. Our data show significant reduction in virus growth upon treatment with one of these peptides (pep-CR2) with an IC50 of 1.33 μM and no significant impact on cell viability. Based on 3D modeling, pep-CR2 specifically interferes with the pp150–capsid binding interface. Cells pre-treated with pep-CR2 and infected with HCMV sequester pp150 in the nucleus, indicating a mechanistic disruption of pp150 loading onto capsids and subsequent nuclear egress. Furthermore, pep-CR2 effectively inhibits mouse cytomegalovirus (MCMV) infection in cell culture, paving the way for future animal testing. Combined, these results indicate that CR2 of pp150 is amenable to targeting by a peptide inhibitor, and can be developed into an effective antiviral.

Structure of human cytomegalovirus virion reveals host tRNA binding to capsid-associated tegument protein pp150

Under the Baltimore nucleic acid-based virus classification scheme, the herpesvirus human cytomegalovirus (HCMV) is a Class I virus, meaning that it contains a double-stranded DNA genome—and no RNA. Here, we report sub-particle cryoEM reconstructions of HCMV virions at 2.9 Å resolution revealing structures resembling non-coding transfer RNAs (tRNAs) associated with the virion’s capsid-bound tegument protein, pp150. Through deep sequencing, we show that these RNA sequences match human tRNAs, and we built atomic models using the most abundant tRNA species. Based on our models, tRNA recruitment is mediated by the electrostatic interactions between tRNA phosphate groups and the helix-loop-helix motif of HCMV pp150. The specificity of these interactions may explain the absence of such tRNA densities in murine cytomegalovirus and other human herpesviruses.

VP8, the Major Tegument Protein of Bovine Herpesvirus-1, Is Partially Packaged during Early Tegument Formation in a VP22-Dependent Manner

Bovine herpesvirus-1 (BoHV-1) is a major cause of rhinotracheitis and vulvovaginitis in cattle. VP8, the major tegument protein of BoHV-1, is essential for viral replication in the host. VP8 is phosphorylated by the viral kinase US3, mediating its translocation to the cytoplasm. VP8 remains nuclear when not phosphorylated. Interestingly, VP8 has a significant presence in mature BoHV-1YmVP8, in which the VP8 phosphorylation sites are mutated. This suggests that VP8 might be packaged during primary envelopment of BoHV-1. This was investigated by mass spectrometry and Western blotting, which showed VP8, as well as VP22, to be constituents of the primary enveloped virions. VP8 and VP22 were shown to interact via co-immunoprecipitation experiments, in both BoHV-1-infected and VP8-transfected cells. VP8 and VP22 also co-localised with one another and with nuclear lamin-associated protein 2 in BoHV-1-infected cells, suggesting an interaction between VP8 and VP22 in the perinuclear region. In cells infected with VP22-deleted BoHV-1 (BoHV-1ΔUL49), VP8 was absent from the primary enveloped virions, implying that VP22 might be critical for the early packaging of VP8. In conclusion, a novel VP22-dependent mechanism for packaging of VP8 was identified, which may be responsible for a significant amount of VP8 in the viral particle.

Disruption of the Interaction between ORF33 and the Conserved Carboxyl-Terminus of ORF45 Abolishes Progeny Virion Production of Kaposi Sarcoma-Associated Herpesvirus

The Open Reading Frame 45 (ORF45) of Kaposi sarcoma-associated herpesvirus (KSHV) is a gammaherpesvirus-specific, immediate-early, tegument protein required for efficient viral replication and virion production. We have previously shown that ORF45 interacts with the conserved herpesviral protein ORF33 through the highly conserved C-terminal 19 amino acids (C19) of ORF45. Because the deletion of C19 abolished ORF33 accumulation and viral production, we reasoned that this interaction could be critical for viral production and explored as an antiviral target for gammaherpesviruses. In work described in this article, we characterize this interaction in further detail, first by revealing that this interaction is conserved among gammaherpesviruses, then by identifying residues in C19 critical for its interaction with and stabilization of ORF33. More importantly, we show that disruption of the interaction, either by mutating key residues (W403A or W405A) in C19 or by using competing cell penetration peptide TAT-C19, dramatically reduce the yield of KSHV progeny viruses. Our results not only reveal critical roles of this interaction to viral production but also provide a proof of concept for targeting the ORF33-ORF45 interaction as a novel antiviral strategy against KSHV and other gammaherpesviruses.

OPTN is a host intrinsic restriction factor against neuroinvasive HSV-1 infection

Fast-replicating neurotropic herpesviruses exemplified by herpes simplex virus-1 (HSV-1) naturally infect the central nervous system (CNS). However, most individuals intrinsically suppress the virus during a primary infection and preclude it from significantly damaging the CNS. Optineurin (OPTN) is a conserved autophagy receptor with little understanding of its role in neurotropic viral infections. We show that OPTN selectively targets HSV-1 tegument protein, VP16, and the fusion glycoprotein, gB, to degradation by autophagy. OPTN-deficient mice challenged with HSV-1 show significant cognitive decline and susceptibility to lethal CNS infection. OPTN deficiency unveils severe consequences for recruitment of adaptive immunity and suppression of neuronal necroptosis. Ocular HSV-1 infection is lethal without OPTN and is rescued using a necroptosis inhibitor. These results place OPTN at the crux of neuronal survival from potentially lethal CNS viral infections.

Human Cytomegalovirus Essential Tegument Protein pp150 is Amenable to Targeting by Sequence Specific Peptides and ELP-conjugates

Human cytomegalovirus (HCMV) tegument protein pp150 is essential for the completion of final steps in virion maturation. Earlier studies indicated that three pp150nt (N terminal one-third of pp150) conformers cluster on each triplex (Tri1, Tri2A and Tri2B) and extend towards small capsid proteins atop nearby major capsid proteins forming a net-like layer of tegument densities that enmesh and stabilize HCMV capsids. Based on this atomic detail, we designed several peptides targeting pp150nt. Our data show significant reduction in virus growth upon treatment with one of these peptides (pep-CR2) with an IC50 of 1.33 μM. Based on 3D modeling, pep-CR2 specifically interferes with the pp150-capsid binding interface. Cells pre-treated with pep-CR2 and infected with HCMV sequester pp150 in the nucleus indicating a mechanistic disruption of pp150 loading onto capsids and subsequent nuclear egress. To enhance the in-vivo inhibitory potential and bioavailability of pep-CR2, we conjugated it with a carrier molecule (elastin like polypeptide (ELP)). The ELP-pep-CR2 conjugate was expressed in E.coli and purified. Upon treatment with ELP-pep-CR2, HCMV showed significant titer reductions with no significant impact on cell viability. These results indicate that CR2 of pp150 is amenable to targeting by a peptide inhibitor and can be developed into an effective antiviral.

Suppression of JAK-STAT signaling by Epstein-Barr virus tegument protein BGLF2 through recruitment of SHP1 phosphatase and promotion of STAT2 degradation

Some lytic proteins encoded by Epstein-Barr virus (EBV) suppress host interferon (IFN) signaling to facilitate viral replication. In this study we sought to identify and characterize EBV proteins antagonizing IFN signaling. The induction of IFN-stimulated genes (ISGs) by IFN-β was effectively suppressed by EBV. A functional screen was therefore performed to identify IFN-antagonizing proteins encoded by EBV. EBV tegument protein BGLF2 was identified as a potent suppressor of JAK-STAT signaling. This activity was found to be independent of its stimulatory effect on p38 and JNK pathways. Association of BGLF2 with STAT2 resulted in more pronounced K48-linked polyubiquitination and proteasomal degradation of the latter. Mechanistically, BGLF2 promoted the recruitment of SHP1 phosphatase to STAT1 to inhibit its tyrosine phosphorylation. In addition, BGLF2 associated with cullin 1 E3 ubiquitin ligase to facilitate its recruitment to STAT2. Consequently, BGLF2 suppressed ISG induction by IFN-β. Furthermore, BGLF2 also suppressed type II and type III IFN signaling, although the suppressive effect on type II IFN response was milder. When pre-treated with IFN-β, host cells became less susceptible to primary infection of EBV. This phenotype was reversed when expression of BGLF2 was enforced. Finally, genetic disruption of BGLF2 in EBV led to more pronounced induction of ISGs. Taken together, our study unveils the roles of BGLF2 not only in the subversion of innate IFN response but also in lytic infection and reactivation of EBV. Importance Epstein-Barr virus (EBV) is an oncogenic virus associated with the development of lymphoid and epithelial malignancies. EBV has to subvert interferon-mediated host antiviral response to replicate and cause diseases. It is therefore of great interest to identify and characterize interferon-antagonizing proteins produced by EBV. In this study we perform a screen to search for EBV proteins that suppress the action of interferons. We further show that BGLF2 protein of EBV is particularly strong in this suppression. This is achieved by inhibiting two key proteins STAT1 and STAT2 that mediate the antiviral activity of interferons. BGLF2 recruits a host enzyme to remove the phosphate group from STAT1 thereby inactivating its activity. BGLF2 also redirects STAT2 for degradation. A recombinant virus in which BGLF2 gene has been disrupted can activate host interferon response more robustly. Our findings reveal a novel mechanism by which EBV BGLF2 protein suppresses interferon signaling.

CRISPR/Cas9-constructed pseudorabies virus mutants reveal the importance of UL13 in alphaherpesvirus escape from genome silencing

Latent and recurrent productive infection of long-living cells, such as neurons, enables alphaherpesviruses to persist in their host populations. Still, the viral factors involved in these events remain largely obscure. Using a complementation assay in compartmented primary peripheral nervous system (PNS) neuronal cultures, we previously reported that productive replication of axonally-delivered genomes is facilitated by PRV tegument proteins. Here, we sought to unravel the role of tegument protein UL13 in this escape from silencing. We first constructed four new PRV mutants in the virulent Becker strain using CRISPR/Cas9-mediated gene replacement: (i) PRV Becker defective for UL13 expression (PRV ΔUL13), (ii) PRV where UL13 is fused to eGFP (PRV UL13-eGFP) and two control viruses (iii and iv) PRV where VP16 is fused with mTurquoise at either the N-terminus (PRV mTurq-VP16) or C-terminus (PRV VP16-mTurq). Live cell imaging of PRV capsids showed efficient retrograde transport after axonal infection with PRV UL13-eGFP, although we did not detect dual-color particles. Surprisingly, immunofluorescence staining of particles in mid-axons indicated that UL13 might be co-transported with PRV capsids in PNS axons. Superinfecting nerve cell bodies with UV-inactivated PRV ΔUL13 failed to efficiently promote escape from genome silencing when compared to UV-PRV wild type and UV-PRV UL13-eGFP superinfection. However, UL13 does not act directly in the escape from genome silencing, as AAV-mediated UL13 expression in neuronal cell bodies was not sufficient to provoke escape from genome silencing. Based on this, we suggest that UL13 may contribute to initiation of productive infection through phosphorylation of other tegument proteins. Importance Alphaherpesviruses have mastered various strategies to persist in an immunocompetent host, including the induction of latency and reactivation in peripheral nervous system (PNS) ganglia. We recently discovered that the molecular mechanism underlying escape from latency by the alphaherpesvirus pseudorabies virus (PRV) relies on a structural viral tegument protein. This study aimed at unravelling the role of tegument protein UL13 in PRV escape from latency. First, we confirmed the use of CRISPR/Cas9-mediated gene replacement as a versatile tool to modify the PRV genome. Next, we used our new set of viral mutants and AAV vectors to conclude on the indirect role of UL13 in PRV escape from latency in primary neurons and on its spatial localization during retrograde capsid transport in axons. Based on these findings, we speculate that UL13 phosphorylates one or more tegument proteins, thereby priming these putative proteins to induce escape from genome silencing.

The autophagy-initiating protein kinase ULK1 phosphorylates human cytomegalovirus tegument protein pp28 and regulates efficient virus release

Autophagy is a catabolic process contributing to intrinsic cellular defense by degrading viral particles or proteins, however, several viruses hijack this pathway for their own benefit. The role of autophagy during human cytomegalovirus (HCMV) replication has not been definitely clarified yet. Utilizing siRNA-based screening, we observed that depletion of many autophagy-related proteins resulted in reduced virus release suggesting a requirement of autophagy-related factors for efficient HCMV replication. Additionally, we could show that the autophagy-initiating serine/threonine-protein kinase ULK1 as well as other constituents of the ULK1 complex were upregulated at early times of infection and stayed upregulated throughout the replication cycle. We demonstrate that an indirect interference with ULK1 through inhibition of the upstream regulator AMPK impaired virus release. Furthermore, this result was verified by direct abrogation of ULK1 kinase activity utilizing the ULK1-specific kinase inhibitors SBI-0206965 and ULK-101. Analysis of viral protein expression in the presence of ULK-101 revealed a connection between the cellular kinase ULK1 and the viral tegument protein pp28 (pUL99) and we identified pp28 as a novel viral substrate of ULK1 by in vitro kinase assays. In the absence of ULK1 kinase activity, large pp28- and pp65-positive structures could be detected in the cytoplasm at late time points of infection. Transmission electron microscopy demonstrated that these structures represent large perinuclear protein accumulations presumably representing aggresomes. Our results indicate that HCMV manipulates ULK1 and further components of the autophagic machinery to ensure efficient release of viral particles.IMPORTANCE The catabolic program of autophagy represents a powerful immune defense against viruses that is, however, counteracted by antagonizing viral factors. Understanding the exact interplay between autophagy and HCMV infection is of major importance since autophagy-related proteins emerged as promising targets for pharmacologic intervention. Our study provides evidence for a proviral role of several autophagy-related proteins suggesting that HCMV has developed strategies to usurp components of the autophagic machinery for its own benefit. In particular, we observed a strong upregulation of the autophagy-initiating protein kinase ULK1 and further components of the ULK1 complex during HCMV replication. In addition, both siRNA-mediated depletion of ULK1 and interference with ULK1 protein kinase activity by two chemically different inhibitors resulted in impaired viral particle release. Thus, we propose that ULK1 kinase activity is required for efficient HCMV replication and thus represents a promising novel target for future antiviral drug development.