The non-classical major histocompatibility complex II protein SLA-DM is crucial for African swine fever virus replication.

African swine fever virus (ASFV) is a lethal animal pathogen that enters its host cells through endocytosis. So far, host factors specifically required for ASFV replication have been barely identified. In this study a genome-wide CRISPR/Cas9 knockout screen in porcine cells indicated that the genes RFXANK, RFXAP, SLA-DMA, SLA-DMB, and CIITA are important for productive ASFV infection. The proteins encoded by these genes belong to the major histocompatibility complex II (MHC II), or swine leucocyte antigen complex II (SLA II). RFXAP and CIITA are MHC II-specific transcription factors, whereas SLA-DMA/B are subunits of the non-classical MHC II molecule SLA-DM. Targeted knockout of either of these genes led to severe replication defects of different ASFV isolates, reflected by substantially reduced plating efficiency, cell-to-cell spread, progeny virus titers and viral DNA replication. Transgene-based reconstitution of SLA-DMA/B fully restored the replication capacity demonstrating that SLA-DM, which resides in late endosomes, plays a crucial role during early steps of ASFV infection.

N6-methyladenosine is required for efficient RNA synthesis of Ebola virus and other haemorrhagic fever viruses.

N6-methyladenosine (m6A) is one of the most abundant modifications of cellular RNA, where it serves various functions. m6A methylation of many viral RNA species has also been described; however, little is known about the m6A epitranscriptome of haemorrhagic fever-causing viruses like Ebola virus (EBOV). Here, we analysed the importance of the methyltransferase METTL3 for the life cycle of this virus. We found that METTL3 interacts with the EBOV nucleoprotein and the transcriptional activator VP30 to support viral RNA synthesis, and that METTL3 is recruited into EBOV inclusions bodies, where viral RNA synthesis occurs. Analysis of the m6A methylation pattern of EBOV mRNAs showed that they are methylated by METTL3. Further studies revealed that METTL3 interaction with the viral nucleoprotein, as well as its importance for RNA synthesis and protein expression, is also observed for other haemorrhagic fever viruses such as Junín virus (JUNV) and Crimean-Congo haemorrhagic fever virus (CCHFV). The negative effects on viral RNA synthesis due to loss of m6A methylation are independent of innate immune sensing, as METTL3 knockout did not affect type I interferon induction in response to viral RNA synthesis or infection. Our results suggest a novel function for m6A that is conserved among diverse haemorrhagic fever-causing viruses (i.e. EBOV, JUNV and CCHFV), making METTL3 a promising target for broadly-acting antivirals.

Clinical, neuropathological, and immunological short- and long-term feature of a mouse model mimicking human herpes virus encephalitis.

Herpes simplex encephalitis (HSE) is one of the most serious diseases of the nervous system in humans. However, its pathogenesis is still only poorly understood. Although several mouse models of predominantly herpes simplex virus 1 (HSV-1) infections mimic different crucial aspects of HSE, central questions remain unanswered. They comprise the specific temporofrontal tropism, viral spread within the central nervous system (CNS), as well as potential molecular and immunological barriers that drive virus into latency while only rarely resulting in severe HSE. We have recently proposed an alternative mouse model by using a pseudorabies virus (PrV) mutant that more faithfully represents the striking features of human HSE: temporofrontal meningoencephalitis with few severely, but generally only moderately to subclinically affected mice as well as characteristic behavioral abnormalities. Here, we characterized this animal model using 6- to 8-week-old female CD-1 mice in more detail. Long-term investigation over 6 months consistently revealed a biphasic course of infection accompanied by recurring clinical signs including behavioral alterations and mainly mild meningoencephalitis restricted to the temporal and frontal lobes. By histopathological and immunological analyses, we followed the kinetics and spatial distribution of inflammatory lesions as well as the underlying cytokine expression in the CNS over 21 days within the acute phase of infection. Affecting the temporal lobes, the inflammatory infiltrate was composed of lymphocytes and macrophages showing a predominantly lymphocytic shift 15 days after infection. A strong increase was observed in cytokines CXCL10, CCL2, CCL5, and CXCL1 recruiting inflammatory cells to the CNS. Unlike the majority of infected mice, strongly affected animals demonstrated extensive temporal lobe edema, which is typically present in severe human HSE cases. In summary, these results support the validity of our animal model for in-depth investigation of HSE pathogenesis.

A Genome-Wide CRISPR/Cas9 Screen Reveals the Requirement of Host Sphingomyelin Synthase 1 for Infection with Pseudorabies Virus Mutant gD-Pass.

Herpesviruses are large DNA viruses, which encode up to 300 different proteins including enzymes enabling efficient replication. Nevertheless, they depend on a multitude of host cell proteins for successful propagation. To uncover cellular host factors important for replication of pseudorabies virus (PrV), an alphaherpesvirus of swine, we performed an unbiased genome-wide CRISPR/Cas9 forward screen. To this end, a porcine CRISPR-knockout sgRNA library (SsCRISPRko.v1) targeting 20,598 genes was generated and used to transduce porcine kidney cells. Cells were then infected with either wildtype PrV (PrV-Ka) or a PrV mutant (PrV-gD-Pass) lacking the receptor-binding protein gD, which regained infectivity after serial passaging in cell culture. While no cells survived infection with PrV-Ka, resistant cell colonies were observed after infection with PrV-gD-Pass. In these cells, sphingomyelin synthase 1 (SMS1) was identified as the top hit candidate. Infection efficiency was reduced by up to 90% for PrV-gD-Pass in rabbit RK13-sgms1KO cells compared to wildtype cells accompanied by lower viral progeny titers. Exogenous expression of SMS1 partly reverted the entry defect of PrV-gD-Pass. In contrast, infectivity of PrV-Ka was reduced by 50% on the knockout cells, which could not be restored by exogenous expression of SMS1. These data suggest that SMS1 plays a pivotal role for PrV infection, when the gD-mediated entry pathway is blocked.

Role of Vesicle-Associated Membrane Protein-Associated Proteins (VAP) A and VAPB in Nuclear Egress of the Alphaherpesvirus Pseudorabies Virus.

The molecular mechanism affecting translocation of newly synthesized herpesvirus nucleocapsids from the nucleus into the cytoplasm is still not fully understood. The viral nuclear egress complex (NEC) mediates budding at and scission from the inner nuclear membrane, but the NEC is not sufficient for efficient fusion of the primary virion envelope with the outer nuclear membrane. Since no other viral protein was found to be essential for this process, it was suggested that a cellular machinery is recruited by viral proteins. However, knowledge on fusion mechanisms involving the nuclear membranes is rare. Recently, vesicle-associated membrane protein-associated protein B (VAPB) was shown to play a role in nuclear egress of herpes simplex virus 1 (HSV-1). To test this for the related alphaherpesvirus pseudorabies virus (PrV), we mutated genes encoding VAPB and VAPA by CRISPR/Cas9-based genome editing in our standard rabbit kidney cells (RK13), either individually or in combination. Single as well as double knockout cells were tested for virus propagation and for defects in nuclear egress. However, no deficiency in virus replication nor any effect on nuclear egress was obvious suggesting that VAPB and VAPA do not play a significant role in this process during PrV infection in RK13 cells.

Diazadispiroalkane Derivatives Are New Viral Entry Inhibitors.

Herpesviruses are widespread and can cause serious illness. Many currently available antiviral drugs have limited effects, result in rapid development of resistance, and often exhibit dose-dependent toxicity. Especially for human cytomegalovirus (HCMV), new well-tolerated compounds with novel mechanisms of action are urgently needed. In this study, we characterized the antiviral activity of two new diazadispiroalkane derivatives, 11826091 and 11826236. These two small molecules exhibited strong activity against low-passage-number HCMV. Pretreatment of cell-free virus with these compounds greatly reduced infection. Time-of-addition assays where 11826091 or 11826236 was added to cells before infection, before and during infection, or during or after infection demonstrated an inhibitory effect on early steps of infection. Interestingly, 11826236 had an effect by addition to cells after infection. Results from entry assays showed the major effect to be on attachment. Only 11826236 had a minimal effect on penetration comparable to heparin. Further, no effect on virus infection was found for cell lines with a defect in heparan sulfate expression or lacking all surface glycosaminoglycans, indicating that these small molecules bind to heparan sulfate on the cell surface. To test this further, we extended our analyses to pseudorabies virus (PrV), a member of the Alphaherpesvirinae, which is known to use cell surface heparan sulfate for initial attachment via nonessential glycoprotein C (gC). While infection with PrV wild type was strongly impaired by 11826091 or 11826236, as with heparin, a mutant lacking gC was unaffected by either treatment, demonstrating that primary attachment to heparan sulfate via gC is targeted by these small molecules.

BH3-only sensors Bad, Noxa and Puma are Key Regulators of Tacaribe virus-induced Apoptosis.

Pathogenicity often differs dramatically among even closely related arenavirus species. For instance, Junín virus (JUNV), the causative agent of Argentine hemorrhagic fever (AHF), is closely related to Tacaribe virus (TCRV), which is normally avirulent in humans. While little is known about how host cell pathways are regulated in response to arenavirus infection, or how this contributes to virulence, these two viruses have been found to differ markedly in their ability to induce apoptosis. However, details of the mechanism(s) governing the apoptotic response to arenavirus infections are unknown. Here we confirm that TCRV-induced apoptosis is mitochondria-regulated, with associated canonical hallmarks of the intrinsic apoptotic pathway, and go on to identify the pro- and anti-apoptotic Bcl-2 factors responsible for regulating this process. In particular, levels of the pro-apoptotic BH3-only proteins Noxa and Puma, as well as their canonical transcription factor p53, were strongly increased. Interestingly, TCRV infection also led to the accumulation of the inactive phosphorylated form of another pro-apoptotic BH3-only protein, Bad (i.e. as phospho-Bad). Knockout of Noxa or Puma suppressed apoptosis in response to TCRV infection, whereas silencing of Bad increased apoptosis, confirming that these factors are key regulators of apoptosis induction in response to TCRV infection. Further, we found that while the highly pathogenic JUNV does not induce caspase activation, it still activated upstream pro-apoptotic factors, consistent with current models suggesting that JUNV evades apoptosis by interfering with caspase activation through a nucleoprotein-mediated decoy function. This new mechanistic insight into the role that individual BH3-only proteins and their regulation play in controlling apoptotic fate in arenavirus-infected cells provides an important experimental framework for future studies aimed at dissecting differences in the apoptotic responses between arenaviruses, their connection to other cell signaling events and ultimately the relationship of these processes to pathogenesis.

Generation and characterization of monoclonal antibodies specific for the Pseudorabies Virus nuclear egress complex.

During herpesvirus replication, newly synthesized nucleocapsids exit the nucleus by a vesicle-mediated transport, which requires the nuclear egress complex (NEC), composed of the conserved viral proteins designated as pUL31 and pUL34 in the alphaherpesviruses pseudorabies virus (PrV) and herpes simplex viruses. Oligomerization of the heterodimeric NEC at the inner nuclear membrane (INM) results in membrane bending and budding of virus particles into the perinuclear space. The INM-derived primary envelope then fuses with the outer nuclear membrane to release nucleocapsids into the cytoplasm. The two NEC components are necessary and sufficient for induction of vesicle budding and scission as shown after co-expression in eukaryotic cells or in synthetic membranes. However, where and when the NEC is formed, how membrane curvature is mediated and how it is regulated, remains unclear. While monospecific antisera raised against the different components of the PrV NEC aided in the characterization and intracellular localization of the individual proteins, no NEC specific tools have been described yet for any herpesvirus. To gain more insight into vesicle budding and scission, we aimed at generating NEC specific monoclonal antibodies (mAbs). To this end, mice were immunized with bacterially expressed soluble PrV NEC, which was previously used for structure determination. Besides pUL31- and pUL34-specific mAbs, we also identified mAbs, which reacted only in the presence of both proteins indicating specificity for the complex. Confocal microscopy with those NEC-specific mAbs revealed small puncta (approx. 0.064 µm2) along the nuclear rim in PrV wild type infected cells. In contrast, ca. 5-fold larger speckles (approx. 0.35 µm2) were detectable in cells infected with a PrV mutant lacking the viral protein kinase pUS3, which is known to accumulate primary enveloped virions in the PNS within large invaginations of the INM, or in cells co-expressing pUL31 and pUL34. Kinetic experiments showed that while the individual proteins were detectable already between 2-4 hours after infection, the NEC-specific mAbs produced significant staining only after 4-6 hours in accordance with timing of nuclear egress. Taken together, the data indicate that these mAbs specifically label the PrV NEC.

An improved animal model for herpesvirus encephalitis in humans.

Herpesviral encephalitis caused by Herpes Simplex Virus 1 (HSV-1) is one of the most devastating diseases in humans. Patients present with fever, mental status changes or seizures and when untreated, sequelae can be fatal. Herpes Simplex Encephalitis (HSE) is characterized by mainly unilateral necrotizing inflammation effacing the frontal and mesiotemporal lobes with rare involvement of the brainstem. HSV-1 is hypothesized to invade the CNS via the trigeminal or olfactory nerve, but viral tropism and the exact route of infection remain unclear. Several mouse models for HSE have been developed, but they mimic natural infection only inadequately. The porcine alphaherpesvirus Pseudorabies virus (PrV) is closely related to HSV-1 and Varicella Zoster Virus (VZV). While pigs can control productive infection, it is lethal in other susceptible animals associated with severe pruritus leading to automutilation. Here, we describe the first mutant PrV establishing productive infection in mice that the animals are able to control. After intranasal inoculation with a PrV mutant lacking tegument protein pUL21 and pUS3 kinase activity (PrV-ΔUL21/US3Δkin), nearly all mice survived despite extensive infection of the central nervous system. Neuroinvasion mainly occurred along the trigeminal pathway. Whereas trigeminal first and second order neurons and autonomic ganglia were positive early after intranasal infection, PrV-specific antigen was mainly detectable in the frontal, mesiotemporal and parietal lobes at later times, accompanied by a long lasting lymphohistiocytic meningoencephalitis. Despite this extensive infection, mice showed only mild to moderate clinical signs, developed alopecic skin lesions, or remained asymptomatic. Interestingly, most mice exhibited abnormalities in behavior and activity levels including slow movements, akinesia and stargazing. In summary, clinical signs, distribution of viral antigen and inflammatory pattern show striking analogies to human encephalitis caused by HSV-1 or VZV not observed in other animal models of disease.

Function of Torsin AAA+ ATPases in Pseudorabies Virus Nuclear Egress.

Newly assembled herpesvirus nucleocapsids traverse the intact nuclear envelope by a vesicle-mediated nucleo-cytoplasmic transport for final virion maturation in the cytoplasm. For this, they bud at the inner nuclear membrane resulting in primary enveloped particles in the perinuclear space (PNS) followed by fusion of the primary envelope with the outer nuclear membrane (ONM). While the conserved viral nuclear egress complex orchestrates the first steps, effectors of fusion of the primary virion envelope with the ONM are still mostly enigmatic but might include cellular proteins like SUN2 or ESCRT-III components. Here, we analyzed the influence of the only known AAA+ ATPases located in the endoplasmic reticulum and the PNS, the Torsins (Tor), on nuclear egress of the alphaherpesvirus pseudorabies virus. For this overexpression of wild type and mutant proteins as well as CRISPR/Cas9 genome editing was applied. Neither single overexpression nor gene knockout (KO) of TorA or TorB had a significant impact. However, TorA/B double KO cells showed decreased viral titers at early time points of infection and an accumulation of primary virions in the PNS pointing to a delay in capsid release during nuclear egress.

Mutational Functional Analysis of the Pseudorabies Virus Nuclear Egress Complex-Nucleocapsid Interaction.

Herpesvirus nucleocapsids leave the nucleus by a vesicle-mediated translocation mediated by the viral nuclear egress complex (NEC). The NEC is composed of two conserved viral proteins, designated pUL34 and pUL31 in the alphaherpesvirus pseudorabies virus (PrV). It is required for efficient nuclear egress and is sufficient for vesicle formation and scission from the inner nuclear membrane (INM). Structure-based mutagenesis identified a lysine at position 242 (K242) in pUL31, located in the most membrane distal part of the NEC, to be crucial for efficient nucleocapsid incorporation into budding vesicles. Replacing the lysine by alanine (K242A) resulted in accumulations of empty vesicles in the perinuclear space, despite the presence of excess nucleocapsids in the nucleus. However, it remained unclear whether the defect in capsid incorporation was due to interference with a direct, electrostatic interaction between the capsid and the NEC or structural restrictions. To test this, we replaced K242 with several amino acids, thereby modifying the charge, size, and side chain orientation. In addition, virus recombinants expressing pUL31-K242A were passaged and screened for second-site mutations. Compensatory mutations at different locations in pUL31 or pUL34 were identified, pointing to an inherent flexibility of the NEC. In summary, our data suggest that the amino acid at position 242 does not directly interact with the nucleocapsid but that rearrangements in the NEC coat are required for efficient nucleocapsid envelopment at the INM.IMPORTANCE Herpesviruses encode an exceptional vesicle formation and scission machinery, which operates at the inner nuclear membrane, translocating the viral nucleocapsid from the nucleus into the perinuclear space. The conserved herpesviral nuclear egress complex (NEC) orchestrates this process. High-resolution imaging approaches as well as the recently solved crystal structures of the NEC provided deep insight into the molecular details of vesicle formation and scission. Nevertheless, the molecular mechanism of nucleocapsid incorporation remained unclear. In accordance with structure-based predictions, a basic amino acid could be pinpointed in the most membrane-distal domain of the NEC (pUL31-K242), indicating that capsid incorporation might depend on a direct electrostatic interaction. Our follow-up study, described here, however, shows that the positive charge is not relevant but that the overall structure matters.