Gerald M. McInerney

Foot-and-mouth disease virus 3C protease induces cleavage of translation initiation factors eIF4A and eIF4G within infected cells.

ABSTRACT Infection of cells by foot-and-mouth disease virus (FMDV) results in the rapid inhibition of host cell protein synthesis. This process is accompanied by the early cleavage of the translation initiation factor eIF4G, a component of the cap-binding complex eIF4F. This cleavage is mediated by the leader (L) protease. Subsequently, as the virus proteins accumulate, secondary cleavages of eIF4G occur. Furthermore, eIF4A (46 kDa), a second component of eIF4F, is also cleaved in these later stages of the infection cycle. The 33-kDa cleavage product of eIF4A has lost a fragment from its N terminus. Transient-expression assays demonstrated that eIF4A was not cleaved in the presence of FMDV L or with the poliovirus 2A protease (which also mediates eIF4G cleavage) but was cleaved when the FMDV 3C protease was expressed. The FMDV 3C protease was also shown in such assays to induce cleavage of eIF4G, resulting in the production of cleavage products different from those generated by the L protease. Consistent with these results, within cells infected with a mutant FMDV lacking the L protease or within cells containing an FMDV replicon lacking L-P1 coding sequences it was again shown that eIF4A and eIF4G were cleaved.

Semliki Forest Virus Nonstructural Protein 2 Is Involved in Suppression of the Type I Interferon Response

ABSTRACT The type I interferons (IFNs) are potent mediators of antiviral immunity, and many viruses have developed means to block their expression or their effects. Semliki Forest virus (SFV) infection induces rapid and profound silencing of host cell gene expression, a process believed to be important for the inhibition of the IFN response. In SFV-infected cells, a large proportion of the nonstructural protein nsp2 is found in the nucleus, but a role for this localization has not been described. In this work we demonstrate that a viral mutant, SFV4-RDR, in which the nuclear localization sequence of nsp2 has been rendered inactive, induces a significantly more robust IFN response in infected cells. This mutant virus replicates at a rate similar to that of the parental SFV4 strain and also shuts off host cell gene expression to similar levels, indicating that the general cellular shutoff is not responsible for the inhibition of IFN expression. Further, the rate of virus-induced nuclear translocation of early IFN transcription factors was not found to differ between the wild-type and mutant viruses, indicating that the effect of nsp2 is at a later stage. These results provide novel information about the mode of action of this viral IFN antagonist.

Age-dependent TLR3 expression of the intestinal epithelium contributes to rotavirus susceptibility.

Rotavirus is a major cause of diarrhea worldwide and exhibits a pronounced small intestinal epithelial cell (IEC) tropism. Both human infants and neonatal mice are highly susceptible, whereas adult individuals remain asymptomatic and shed only low numbers of viral particles. Here we investigated age-dependent mechanisms of the intestinal epithelial innate immune response to rotavirus infection in an oral mouse infection model. Expression of the innate immune receptor for viral dsRNA, Toll-like receptor (Tlr) 3 was low in the epithelium of suckling mice but strongly increased during the postnatal period inversely correlating with rotavirus susceptibility, viral shedding and histological damage. Adult mice deficient in Tlr3 (Tlr3−/−) or the adaptor molecule Trif (TrifLps2/Lps2) exerted significantly higher viral shedding and decreased epithelial expression of proinflammatory and antiviral genes as compared to wild-type animals. In contrast, neonatal mice deficient in Tlr3 or Trif did not display impaired cell stimulation or enhanced rotavirus susceptibility. Using chimeric mice, a major contribution of the non-hematopoietic cell compartment in the Trif-mediated antiviral host response was detected in adult animals. Finally, a significant age-dependent increase of TLR3 expression was also detected in human small intestinal biopsies. Thus, upregulation of epithelial TLR3 expression during infancy might contribute to the age-dependent susceptibility to rotavirus infection.

A novel quantitative flow cytometry-based assay for autophagy.

Autophagy is a cellular degradation process with an increasingly recognised importance in many biological pathways such as nutrient sensing, stress responses and development. We present a straightforward assay for autophagy which combines the sensitivity of the EGFP-LC3 reporter protein with the throughput capacity and quantitative power of flow cytometry. Because saponin extraction is specific for the non-autophagosome associated EGFP-LC3-I form of the protein, flow cytometry can be used to measure total fluorescence of saponin extracted HOS-EGFP-LC3 cells as a measure of the levels of autophagosome associated EGFP-LC3-II. Combined with inhibitors of degradation, we have adapted this assay to differentiate between constitutive and induced autophagy and to quantify the changes in flux of the system. Moreover, using direct antibody staining for the endogenous LC3 protein, we have extended this assay to the detection of autophagosome formation in non-transfected cells.

Sequestration of G3BP coupled with efficient translation inhibits stress granules in Semliki Forest virus infection.

Semliki Forest virus nsP3 sequesters G3BP to inhibit stress granule formation on viral mRNAs. Furthermore, the efficient translation of viral mRNAs containing a translation enhancer element assists disruption of SGs in infected cells. This work thus describes a novel mechanism for SG disruption.

G3BP–Caprin1–USP10 complexes mediate stress granule condensation and associate with 40S subunits

Stress granule condensation (SGC) of translationally arrested mRNAs requires G3BP, and G3BP-mediated SGC is inhibited by serine 149 phosphorylation, regulated by mutually exclusive binding of Caprin1 and USP10, and requires its RGG region for SGC and for interactions with 40S ribosomal subunits.

MAVS, cGAS, and endogenous retroviruses in T-independent B cell responses

Multivalent molecules with repetitive structures including bacterial capsular polysaccharides and viral capsids elicit antibody responses through B cell receptor (BCR) crosslinking in the absence of T cell help. We report that immunization with these T cell–independent type 2 (TI-2) antigens causes up-regulation of endogenous retrovirus (ERV) RNAs in antigen-specific mouse B cells. These RNAs are detected via a mitochondrial antiviral signaling protein (MAVS)–dependent RNA sensing pathway or reverse-transcribed and detected via the cGAS-cGAMP-STING pathway, triggering a second, sustained wave of signaling that promotes specific immunoglobulin M production. Deficiency of both MAVS and cGAS, or treatment of MAVS-deficient mice with reverse transcriptase inhibitors, dramatically inhibits TI-2 antibody responses. These findings suggest that ERV and two innate sensing pathways that detect them are integral components of the TI-2 B cell signaling apparatus. Endogenous retroviruses materially contribute to humoral immunity in mice. [Also see Perspective by Grasset and Cerutti] Endogenous retroviruses trigger B cells Scattered across our genome are endogenous retroviruses (ERVs), ancient “footprints” of previous viral infections. Scientists do not fully understand their functions, but Zeng et al. now report a role for ERVs in mobilizing a particular type of B cell–driven immune response in mice (T cell–independent, TID), which is usually mounted in response to viral capids or bacterial polysaccharides (see the Perspective by Grasset and Cerutti). Immunizing mice with a model TID antigen elicited an increase in ERV RNA and DNA in the cytoplasm of B cells. Innate immune receptors that recognize cytoplasmic nucleotides then triggered signaling cascades that resulted in the production of immunoglobulin M. Science, this issue p. 1486; see also p. 1454

A Differential Role for Macropinocytosis in Mediating Entry of the Two Forms of Vaccinia Virus into Dendritic Cells

Vaccinia virus (VACV) is being developed as a recombinant viral vaccine vector for several key pathogens. Dendritic cells (DCs) are specialised antigen presenting cells that are crucial for the initiation of primary immune responses; however, the mechanisms of uptake of VACV by these cells are unclear. Therefore we examined the binding and entry of both the intracellular mature virus (MV) and extracellular enveloped virus (EV) forms of VACV into vesicular compartments of monocyte-derived DCs. Using a panel of inhibitors, flow cytometry and confocal microscopy we have shown that neither MV nor EV binds to the highly expressed C-type lectin receptors on DCs that are responsible for capturing many other viruses. We also found that both forms of VACV enter DCs via a clathrin-, caveolin-, flotillin- and dynamin-independent pathway that is dependent on actin, intracellular calcium and host-cell cholesterol. Both MV and EV entry were inhibited by the macropinocytosis inhibitors rottlerin and dimethyl amiloride and depended on phosphotidylinositol-3-kinase (PI(3)K), and both colocalised with dextran but not transferrin. VACV was not delivered to the classical endolysosomal pathway, failing to colocalise with EEA1 or Lamp2. Finally, expression of early viral genes was not affected by bafilomycin A, indicating that the virus does not depend on low pH to deliver cores to the cytoplasm. From these collective results we conclude that VACV enters DCs via macropinocytosis. However, MV was consistently less sensitive to inhibition and is likely to utilise at least one other entry pathway. Definition and future manipulation of these pathways may assist in enhancing the activity of recombinant vaccinia vectors through effects on antigen presentation.

Early Alpha/Beta Interferon Production by Myeloid Dendritic Cells in Response to UV-Inactivated Virus Requires Viral Entry and Interferon Regulatory Factor 3 but Not MyD88

ABSTRACT Alpha/beta interferons (IFN-α/β) are key mediators of innate immunity and important modulators of adaptive immunity. The mechanisms by which IFN-α/β are induced are becoming increasingly well understood. Recent studies showed that Toll-like receptors 7 and 8 expressed by plasmacytoid dendritic cells (pDCs) mediate the endosomal recognition of incoming viral RNA genomes, a process which requires myeloid differentiation factor 88 (MyD88). Here we investigate the requirements for virus-induced IFN-α/β production in cultures of bone marrow-derived murine myeloid DCs (mDCs). Using recombinant Semliki Forest virus blocked at different steps in the viral life cycle, we show that replication-defective virus induced IFN-α/β in mDCs while fusion-defective virus did not induce IFN-α/β. The response to replication-defective virus was largely intact in MyD88−/− mDC cultures but was severely reduced in mDC cultures from mice lacking IFN regulatory factor 3. Our observations suggest that mDCs respond to incoming virus via a pathway that differs from the fusion-independent, MyD88-mediated endosomal pathway described for the induction of IFN-α/β in pDCs. We propose that events during or downstream of viral fusion, but prior to replication, can activate IFN-α/β in mDCs. Thus, mDCs may contribute to the antiviral response activated by the immune system at early time points after infection.

A forward genetic screen reveals roles for Nfkbid, Zeb1, and Ruvbl2 in humoral immunity

Using chemical germ-line mutagenesis, we screened mice for defects in the humoral immune response to a type II T-independent immunogen and an experimental alphavirus vector. A total of 26 mutations that impair humoral immunity were recovered, and 19 of these mutations have been positionally cloned. Among the phenovariants were bumble, cellophane, and Worker ascribed to mutations in Nfkbid, Zeb1, and Ruvbl2, respectively. We show that IκBNS, the nuclear IκB-like protein encoded by Nfkbid, is required for the development of marginal zone and peritoneal B-1 B cells and additionally required for extrafollicular antibody responses to T-independent and -dependent immunogens. Zeb1 is also required for marginal zone and peritoneal B-1 B-cell development as well as T-cell development, germinal center formation, and memory B-cell responses. Finally, Ruvbl2 is required for T-cell development and maximal T-dependent antibody responses. Collectively, the mutations that we identified give us insight into the points at which disruption of an antibody response can occur. All of the mutations identified to date directly affect lymphocyte development or function; none have an exclusive effect on cells of the innate immune system.