Inhibitor of growth protein 3 epigenetically silences endogenous retroviral elements and prevents innate immune activation

Endogenous retroviruses (ERVs) are subject to transcriptional repression in adult tissues, in part to prevent autoimmune responses. However, little is known about the epigenetic silencing of ERV expression. Here, we describe a new role for inhibitor of growth family member 3 (ING3), to add to an emerging group of ERV transcriptional regulators. Our results show that ING3 binds to several ERV promoters (for instance MER21C) and establishes an EZH2-mediated H3K27 trimethylation modification. Loss of ING3 leads to decreases of H3K27 trimethylation enrichment at ERVs, induction of MDA5-MAVS-interferon signaling, and functional inhibition of several virus infections. These data demonstrate an important new function of ING3 in ERV silencing and contributing to innate immune regulation in somatic cells

SETD3 is an actin histidine methyltransferase that prevents primary dystocia.

For more than 50 years, the methylation of mammalian actin at histidine 73 has been known to occur1. Despite the pervasiveness of His73 methylation, which we find is conserved in several model animals and plants, its function remains unclear and the enzyme that generates this modification is unknown. Here we identify SET domain protein 3 (SETD3) as the physiological actin His73 methyltransferase. Structural studies reveal that an extensive network of interactions clamps the actin peptide onto the surface of SETD3 to orient His73 correctly within the catalytic pocket and to facilitate methyl transfer. His73 methylation reduces the nucleotide-exchange rate on actin monomers and modestly accelerates the assembly of actin filaments. Mice that lack SETD3 show complete loss of actin His73 methylation in several tissues, and quantitative proteomics analysis shows that actin His73 methylation is the only detectable physiological substrate of SETD3. SETD3-deficient female mice have severely decreased litter sizes owing to primary maternal dystocia that is refractory to ecbolic induction agents. Furthermore, depletion of SETD3 impairs signal-induced contraction in primary human uterine smooth muscle cells. Together, our results identify a mammalian histidine methyltransferase and uncover a pivotal role for SETD3 and actin His73 methylation in the regulation of smooth muscle contractility. Our data also support the broader hypothesis that protein histidine methylation acts as a common regulatory mechanism

BST2/Tetherin Inhibition of Alphavirus Exit

Alphaviruses such as chikungunya virus (CHIKV) and Semliki Forest virus (SFV) are small enveloped RNA viruses that bud from the plasma membrane. Tetherin/BST2 is an interferon-induced host membrane protein that inhibits the release of many enveloped viruses via direct tethering of budded particles to the cell surface. Alphaviruses have highly organized structures and exclude host membrane proteins from the site of budding, suggesting that their release might be insensitive to tetherin inhibition. Here, we demonstrated that exogenously-expressed tetherin efficiently inhibited the release of SFV and CHIKV particles from host cells without affecting virus entry and infection. Alphavirus release was also inhibited by the endogenous levels of tetherin in HeLa cells. While rubella virus (RuV) and dengue virus (DENV) have structural similarities to alphaviruses, tetherin inhibited the release of RuV but not DENV. We found that two recently identified tetherin isoforms differing in length at the N-terminus exhibited distinct capabilities in restricting alphavirus release. SFV exit was efficiently inhibited by the long isoform but not the short isoform of tetherin, while both isoforms inhibited vesicular stomatitis virus exit. Thus, in spite of the organized structure of the virus particle, tetherin specifically blocks alphavirus release and shows an interesting isoform requirement

Genome-Wide RNAi Screen Identifies Novel Host Proteins Required for Alphavirus Entry

<div><p>The enveloped alphaviruses include important and emerging human pathogens such as Chikungunya virus and Eastern equine encephalitis virus. Alphaviruses enter cells by clathrin-mediated endocytosis, and exit by budding from the plasma membrane. While there has been considerable progress in defining the structure and function of the viral proteins, relatively little is known about the host factors involved in alphavirus infection. We used a genome-wide siRNA screen to identify host factors that promote or inhibit alphavirus infection in human cells. Fuzzy homologue (FUZ), a protein with reported roles in planar cell polarity and cilia biogenesis, was required for the clathrin-dependent internalization of both alphaviruses and the classical endocytic ligand transferrin. The tetraspanin membrane protein TSPAN9 was critical for the efficient fusion of low pH-triggered virus with the endosome membrane. FUZ and TSPAN9 were broadly required for infection by the alphaviruses Sindbis virus, Semliki Forest virus, and Chikungunya virus, but were not required by the structurally-related flavivirus Dengue virus. Our results highlight the unanticipated functions of FUZ and TSPAN9 in distinct steps of alphavirus entry and suggest novel host proteins that may serve as targets for antiviral therapy.</p></div

A Small-Molecule Oligosaccharyltransferase Inhibitor with Pan-flaviviral Activity

The mosquito-borne flaviviruses include important human pathogens such as dengue, Zika, West Nile, and yellow fever viruses, which pose a serious threat for global health. Recent genetic screens identified endoplasmic reticulum (ER)-membrane multiprotein complexes, including the oligosaccharyltransferase (OST) complex, as critical flavivirus host factors. Here, we show that a chemical modulator of the OST complex termed NGI-1 has promising antiviral activity against flavivirus infections. We demonstrate that NGI-1 blocks viral RNA replication and that antiviral activity does not depend on inhibition of the N-glycosylation function of the OST. Viral mutants adapted to replicate in cells deficient of the OST complex showed resistance to NGI-1 treatment, reinforcing the on-target activity of NGI-1. Lastly, we show that NGI-1 also has strong antiviral activity in primary and disease-relevant cell types. This study provides an example for advancing from the identification of genetic determinants of infection to a host-directed antiviral compound with broad activity against flaviviruses

Sindbis virus genome-wide RNAi screen.

<p>A. Strategy for initial and follow-up screens of Sindbis host factors. Initial screen was performed using pooled siRNAs and SINV infection at low multiplicity for 24 h. Selected siRNAs were tested individually under the same conditions; validation refers to those host factors in which ≥2/3 siRNAs inhibited or promoted SINV infection. Pooled siRNAs were also tested for effects on cell viability and for inhibition of single-cycle SINV infection. B. Ingenuity Pathway analysis of 55 proteins identified by the screen as promoting SINV-Luc infection. The top 14 overrepresented categories of molecular and cellular functions are shown, with the significance (p-values) and number of proteins indicated for each category in parentheses.</p

FUZ depletion inhibits alphavirus endocytosis.

<p>U-2 OS cells were transfected as in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003835#ppat-1003835-g002" target="_blank">Fig. 2 A</a> and tested as follows: A. SFV endocytosis. Cells with pre-bound SFV were incubated at 37°C for 30 min to permit endocytosis, and then fixed and stained with rabbit antibody to the envelope proteins before and after cell permeabilization, conditions that detect virus remaining on the cell surface (red/yellow) vs. internalized virus (green), respectively. B. Transferrin uptake. Cells were pre-bound with fluorescent transferrin on ice, incubated for 30 min at 37°C to permit endocytosis, and washed at pH 2.5 to strip off non-internalized transferrin before fixation. The control was acid-washed prior to 37°C incubation. C. Fluid phase uptake. Cells were imaged after incubation with fluorescent dextran for 3 h at 37°C followed by a 2 h chase. Panels A–C show confocal extended focus images (bar = 10 µM). Bar graphs with each panel represent the mean +/− SEM of 3 independent experiments with data normalized to NT control (*p<0.05, **p<0.01, ***p<0.001). All three siRNAs to FUZ and TSPAN9 produced similar results (data not shown).</p

ARCN1 depletion inhibits alphavirus binding to host cells.

<p>U-2 OS cells were transfected as in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003835#ppat-1003835-g002" target="_blank">Fig. 2A</a> and tested as follows: A. Virus fusion-infection. SINV or SFV was pre-bound to cells on ice and treated at low pH to trigger virus fusion with the plasma membrane. Infected cells were quantitated by immunofluorescence. B. Cell surface binding. SINV or SFV was bound to cells on ice and detected by immunofluorescence. A–B each represent the mean +/− SEM of 3–4 independent experiments with data normalized to the NT control (*p<0.05, **p<0.01).</p

A fast-growing dengue virus mutant reveals a dual role of STING in response to infection

The four dengue viruses (DENVs) have evolved multiple mechanisms to ensure its survival. Among these mechanisms is the ability to regulate its replication rate, which may contribute to avoiding premature immune activation that limit infection dissemination: DENVs associated with dengue epidemics have shown slower replication rate than pre-epidemic strains. Correspondingly, wild-type DENVs replicate more slowly than their clinically attenuated derivatives. To understand how DENVs ‘make haste slowly’, we generated and screened for DENV2 mutants with accelerated replication that also induced high type-I interferon (IFN) expression in infected cells. We chanced upon a single NS2B-I114T amino acid substitution, in an otherwise highly conserved amino acid residue. Accelerated DENV2 replication damaged host DNA as mutant infection was dependent on host DNA damage repair factors, namely RAD21, EID3 and NEK5. DNA damage induced cGAS/STING signalling and activated early type-I IFN response that inhibited infection dissemination. Unexpectedly, STING activation also supported mutant DENV replication in infected cells through STING-induced autophagy. Our findings thus show that DENV NS2B has multi-faceted role in controlling DENV replication rate and immune evasion and suggest that the dual role of STING in supporting virus replication within infected cells but inhibiting infection dissemination could be particularly advantageous for live attenuated vaccine development

Tests of ARCN1, FUZ, and TSPAN9 with other host cells and viruses.

<p>A. HeLa cells were transfected as in (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003835#ppat-1003835-g002" target="_blank">Fig. 2A</a>), infected with SINV-GFP (MOI = 50) and infection quantitated at 24 h post-infection by fluorescence microscopy. B. U-2 OS cells were transfected as in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003835#ppat-1003835-g002" target="_blank">Fig. 2A</a> and infection by the indicated viruses was tested as detailed in the methods. C. Primary human umbilical vein endothelial cells were transfected with the indicated siRNAs, incubated for 60 h, infected with SFV or CHIKV, and infection quantitated at 8–12 h post-infection by fluorescence microscopy. Values in A–C were normalized to a non-targeting siRNA control (NT), and the data in each panel represent the mean +/− SEM of 3 independent experiments (*p<0.05, **p<0.01, ***p<0.001).</p