Mirko Cortese

Dengue Virus Perturbs Mitochondrial Morphodynamics to Dampen Innate Immune Responses.

Summary With no antiviral drugs or widely available vaccines, Dengue virus (DENV) constitutes a public health concern. DENV replicates at ER-derived cytoplasmic structures that include substructures called convoluted membranes (CMs); however, the purpose of these membrane alterations remains unclear. We determine that DENV nonstructural protein (NS)4B, a promising drug target with unknown function, associates with mitochondrial proteins and alters mitochondria morphology to promote infection. During infection, NS4B induces elongation of mitochondria, which physically contact CMs. This restructuring compromises the integrity of mitochondria-associated membranes, sites of ER-mitochondria interface critical for innate immune signaling. The spatio-temporal parameters of CM biogenesis and mitochondria elongation are linked to loss of activation of the fission factor Dynamin-Related Protein-1. Mitochondria elongation promotes DENV replication and alleviates RIG-I-dependent activation of interferon responses. As Zika virus infection induces similar mitochondria elongation, this perturbation may protect DENV and related viruses from innate immunity and create a favorable replicative environment.

Dengue Virus Non-structural Protein 1 Modulates Infectious Particle Production via Interaction with the Structural Proteins

Non-structural protein 1 (NS1) is one of the most enigmatic proteins of the Dengue virus (DENV), playing distinct functions in immune evasion, pathogenesis and viral replication. The recently reported crystal structure of DENV NS1 revealed its peculiar three-dimensional fold; however, detailed information on NS1 function at different steps of the viral replication cycle is still missing. By using the recently reported crystal structure, as well as amino acid sequence conservation, as a guide for a comprehensive site-directed mutagenesis study, we discovered that in addition to being essential for RNA replication, DENV NS1 is also critically required for the production of infectious virus particles. Taking advantage of a trans-complementation approach based on fully functional epitope-tagged NS1 variants, we identified previously unreported interactions between NS1 and the structural proteins Envelope (E) and precursor Membrane (prM). Interestingly, coimmunoprecipitation revealed an additional association with capsid, arguing that NS1 interacts via the structural glycoproteins with DENV particles. Results obtained with mutations residing either in the NS1 Wing domain or in the β-ladder domain suggest that NS1 might have two distinct functions in the assembly of DENV particles. By using a trans-complementation approach with a C-terminally KDEL-tagged ER-resident NS1, we demonstrate that the secretion of NS1 is dispensable for both RNA replication and infectious particle production. In conclusion, our results provide an extensive genetic map of NS1 determinants essential for viral RNA replication and identify a novel role of NS1 in virion production that is mediated via interaction with the structural proteins. These studies extend the list of NS1 functions and argue for a central role in coordinating replication and assembly/release of infectious DENV particles.

Recombinant human cytomegalovirus (HCMV) RL13 binds human immunoglobulin G Fc.

The human cytomegalovirus (HCMV) protein RL13 has recently been described to be present in all primary isolates but rapidly mutated in culture adapted viruses. Although these data suggest a crucial role for this gene product in HCMV primary infection, no function has so far been assigned to this protein. Working with RL13 expressed in isolation in transfected human epithelial cells, we demonstrated that recombinant RL13 from the clinical HCMV isolates TR and Merlin have selective human immunoglobulin (Ig)-binding properties towards IgG1 and IgG2 subtypes. An additional Fc binding protein, RL12, was also identified as an IgG1 and IgG2 binding protein but not further characterized. The glycoprotein RL13 trafficked to the plasma membrane where it bound and internalized exogenous IgG or its constant fragment (Fcγ). Analysis of RL13 ectodomain mutants suggested that the RL13 Ig-like domain is responsible for the Fc binding activity. Ligand-dependent internalization relied on a YxxL endocytic motif located in the C-terminal tail of RL13. Additionally, we showed that the tyrosine residue could be replaced by phenylalanine but not by alanine, indicating that the internalization signal was independent from phosphorylation events. In sum, RL13 binds human IgG and may contribute to HCMV immune evasion in the infected host, but this function does not readily explain the instability of the RL13 gene during viral propagation in cultured cells.

Correlative light and electron microscopy methods for the study of virus-cell interactions.

Electron microscopy (EM) is an invaluable tool to study the interactions of viruses with cells, and the ultrastructural changes induced in host cells by virus infection. Light microscopy (LM) is a complementary tool with the potential to locate rare events, label specific components, and obtain dynamic information. The combination of LM and EM in correlative light and electron microscopy (CLEM) is particularly powerful. It can be used to complement a static EM image with dynamic data from live imaging, identify the ultrastructure observed in LM, or, conversely, provide molecular specificity data for a known ultrastructure. Here, we describe methods and strategies for CLEM, discuss their advantages and limitations, and review applications of CLEM to study virus–host interactions.

A Combined Genetic-Proteomic Approach Identifies Residues within Dengue Virus NS4B Critical for Interaction with NS3 and Viral Replication

ABSTRACT Dengue virus (DENV) infection causes the most prevalent arthropod-borne viral disease worldwide. Approved vaccines are not available, and targets suitable for the development of antiviral drugs are lacking. One possible drug target is nonstructural protein 4B (NS4B), because it is absolutely required for virus replication; however, its exact role in the DENV replication cycle is largely unknown. With the aim of mapping NS4B determinants critical for DENV replication, we performed a reverse genetic screening of 33 NS4B mutants in the context of an infectious DENV genome. While the majority of these mutations were lethal, for several of them, we were able to select for second-site pseudoreversions, most often residing in NS4B and restoring replication competence. To identify all viral NS4B interaction partners, we engineered a fully viable DENV genome encoding an affinity-tagged NS4B. Mass spectrometry-based analysis of the NS4B complex isolated from infected cells identified the NS3 protease/helicase as a major interaction partner of NS4B. By combining the genetic complementation map of NS4B with a replication-independent expression system, we identified the NS4B cytosolic loop—more precisely, amino acid residue Q134—as a critical determinant for NS4B-NS3 interaction. An alanine substitution at this site completely abrogated the interaction and DENV RNA replication, and both were restored by pseudoreversions A69S and A137V. This strict correlation between the degree of NS4B-NS3 interaction and DENV replication provides strong evidence that this viral protein complex plays a pivotal role during the DENV replication cycle, hence representing a promising target for novel antiviral strategies. IMPORTANCE With no approved therapy or vaccine against dengue virus infection, the viral nonstructural protein 4B (NS4B) represents a possible drug target, because it is indispensable for virus replication. However, little is known about its precise structure and function. Here, we established the first comprehensive genetic interaction map of NS4B, identifying amino acid residues that are essential for virus replication, as well as second-site mutations compensating for their defects. Additionally, we determined the NS4B viral interactome in infected cells and identified the NS3 protease/helicase as a major interaction partner of NS4B. We mapped residues in the cytosolic loop of NS4B as critical determinants for interaction with NS3, as well as RNA replication. The strong correlation between NS3-NS4B interaction and RNA replication provides strong evidence that this complex plays a pivotal role in the viral replication cycle, hence representing a promising antiviral drug target.

Lessons from Reverse Vaccinology for viral vaccine design.

Although almost 15 years have passed since the birthdate of Reverse Vaccinology (RV), there are very limited applications of this approach to viral vaccines discovery. Undeniably, RV presents a series of advantages as it can virtually identify all potential antigens coded by a genome, irrespective of their abundance, phase of expression and immunogenicity. Additionally, it can be applied to all pathogens, including those that cannot be grown in vitro. In this review we summarize the few examples of RV application to viruses, in particular the Herpesviridae, and report the advantage and limitations of this approach. Next we focus on the novel approaches and additional technologies to vaccine development including structure based approach (Structural Vaccinology [SV]), synthetic biology and some examples of their application in the development of viral vaccines.

Rewiring cellular networks by members of the Flaviviridae family

Members of the Flaviviridae virus family comprise a large group of enveloped viruses with a single-strand RNA genome of positive polarity. Several genera belong to this family, including the Hepacivirus genus, of which hepatitis C virus (HCV) is the prototype member, and the Flavivirus genus, which contains both dengue virus and Zika virus. Viruses of these genera differ in many respects, such as the mode of transmission or the course of infection, which is either predominantly persistent in the case of HCV or acutely self-limiting in the case of flaviviruses. Although the fundamental replication strategy of Flaviviridae members is similar, during the past few years, important differences have been discovered, including the way in which these viruses exploit cellular resources to facilitate viral propagation. These differences might be responsible, at least in part, for the various biological properties of these viruses, thus offering the possibility to learn from comparisons. In this Review, we discuss the current understanding of how Flaviviridae viruses manipulate and usurp cellular pathways in infected cells. Specifically, we focus on comparing strategies employed by flaviviruses with those employed by hepaciviruses, and we discuss the importance of these interactions in the context of viral replication and antiviral therapies.

Membrane alterations induced by nonstructural proteins of human norovirus

Human noroviruses (huNoV) are the most frequent cause of non-bacterial acute gastroenteritis worldwide, particularly genogroup II genotype 4 (GII.4) variants. The viral nonstructural (NS) proteins encoded by the ORF1 polyprotein induce vesical clusters harboring the viral replication sites. Little is known so far about the ultrastructure of these replication organelles or the contribution of individual NS proteins to their biogenesis. We compared the ultrastructural changes induced by expression of norovirus ORF1 polyproteins with those induced upon infection with murine norovirus (MNV). Characteristic membrane alterations induced by ORF1 expression resembled those found in MNV infected cells, consisting of vesicle accumulations likely built from the endoplasmic reticulum (ER) which included single membrane vesicles (SMVs), double membrane vesicles (DMVs) and multi membrane vesicles (MMVs). In-depth analysis using electron tomography suggested that MMVs originate through the enwrapping of SMVs with tubular structures similar to mechanisms reported for picornaviruses. Expression of GII.4 NS1-2, NS3 and NS4 fused to GFP revealed distinct membrane alterations when analyzed by correlative light and electron microscopy. Expression of NS1-2 induced proliferation of smooth ER membranes forming long tubular structures that were affected by mutations in the active center of the putative NS1-2 hydrolase domain. NS3 was associated with ER membranes around lipid droplets (LDs) and induced the formation of convoluted membranes, which were even more pronounced in case of NS4. Interestingly, NS4 was the only GII.4 protein capable of inducing SMV and DMV formation when expressed individually. Our work provides the first ultrastructural analysis of norovirus GII.4 induced vesicle clusters and suggests that their morphology and biogenesis is most similar to picornaviruses. We further identified NS4 as a key factor in the formation of membrane alterations of huNoV and provide models of the putative membrane topologies of NS1-2, NS3 and NS4 to guide future studies.

The Human Cytomegalovirus UL116 Gene Encodes an Envelope Glycoprotein Forming a Complex with gH Independently from gL

ABSTRACT Human cytomegalovirus (HCMV) is a major cause of morbidity and mortality in transplant patients and is the leading viral cause of birth defects after congenital infection. HCMV infection relies on the recognition of cell-specific receptors by one of the viral envelope glycoprotein complexes. Either the gH/gL/gO or the gH/gL/UL128/UL130/UL131A (Pentamer) complex has been found to fulfill this role, accounting for HCMV entry into almost all cell types. We have studied the UL116 gene product, a putative open reading frame identified by in silico analysis and predicted to code for a secreted protein. Virus infection experiments in mammalian cells demonstrated that UL116 is expressed late in the HCMV replication cycle and is a heavily glycosylated protein that first localizes to the cellular site of virus assembly and then inserts into the virion envelope. Transient-transfection studies revealed that UL116 is efficiently transported to the plasma membrane when coexpressed with gH and that gL competes with UL116 for gH binding. Further evidence for gH/UL116 complex formation was obtained by coimmunoprecipitation experiments on both transfected and infected cells and biochemical characterization of the purified complex. In summary, our results show that the product of the UL116 gene is an HCMV envelope glycoprotein that forms a novel gH-based complex alternative to gH/gL. Remarkably, the gH/UL116 complex is the first herpesvirus gH-based gL-less complex. IMPORTANCE HCMV infection can cause severe disease in immunocompromised adults and infants infected in utero. The dissection of the HCMV entry machinery is important to understand the mechanism of viral infection and to identify new vaccine antigens. The gH/gL/gO and gH/gL/UL128/UL130/UL131 (Pentamer) complexes play a key role in HCMV cell entry and tropism. Both complexes are formed by an invariant gH/gL scaffold on which the other subunits assemble. Here, we show that the UL116 gene product is expressed in infected cells and forms a heterodimer with gH. The gH/UL116 complex is carried on the infectious virions, although in smaller amounts than gH/gL complexes. No gH/UL116/gL ternary complex formed in transfected cells, suggesting that the gH/UL116 complex is independent from gL. This new gH-based gL-free complex represents a potential target for a protective HCMV vaccine and opens new perspectives on the comprehension of the HCMV cell entry mechanism and tropism.

Human cytomegalovirus pUL10 interacts with leukocytes and impairs TCR-mediated T-cell activation.

Human cytomegalovirus (HCMV) is known to exert suppressive effects on the host immune system through expression of various viral genes, thus directly and indirectly affecting antiviral immunity of the infected individuals. We report here that HCMV UL10 encodes a protein (pUL10) with immunosuppressive properties. UL10 has been classified as a member of the HCMV RL11 gene family. Although pUL10 is known to be dispensable for viral replication in cultured cells, its amino‐acid sequence is well conserved among different HCMV isolates, suggesting that the protein has a crucial role in viral survival in the host environment. We show that pUL10 is cleaved from the cell surface of fibroblasts as well as epithelial cells and interacts with a cellular receptor ubiquitously expressed on the surface of human leukocytes, demonstrated by ex vivo cell‐based assays and flow cytometric analyses on both lymphoid cell lines and primary blood cells. Furthermore, preincubation of peripheral blood mononuclear cells with purified pUL10 ectodomain results in significantly impaired proliferation and substantially reduced pro‐inflammatory cytokine production, in particular in CD4+ T cells upon in vitro T‐cell stimulation. The inhibitory effect of pUL10 is also observed on antigen receptor‐mediated intracellular tyrosine phosphorylation in a T‐cell line. Based on these observations, we suggest that pUL10 is a newly identified immunomodulatory protein encoded by HCMV. Further elucidation of interactions between pUL10 and the host immune system during HCMV may contribute to finding ways towards new therapies for HCMV infection.