A Spatio-Temporal Analysis of Matrix Protein and Nucleocapsid Trafficking during Vesicular Stomatitis Virus Uncoating

To study VSV entry and the fate of incoming matrix (M) protein during virus uncoating we used recombinant viruses encoding M proteins with a C-terminal tetracysteine tag that could be fluorescently labeled using biarsenical (Lumio) compounds. We found that uncoating occurs early in the endocytic pathway and is inhibited by expression of dominant-negative (DN) Rab5, but is not inhibited by DN-Rab7 or DN-Rab11. Uncoating, as defined by the separation of nucleocapsids from M protein, occurred between 15 and 20 minutes post-entry and did not require microtubules or an intact actin cytoskeleton. Unexpectedly, the bulk of M protein remained associated with endosomal membranes after uncoating and was eventually trafficked to recycling endosomes. Another small, but significant fraction of M distributed to nuclear pore complexes, which was also not dependent on microtubules or polymerized actin. Quantification of fluorescence from high-resolution confocal micrographs indicated that after membrane fusion, M protein diffuses across the endosomal membrane with a concomitant increase in fluorescence from the Lumio label which occurred soon after the release of RNPs into the cytoplasm. These data support a new model for VSV uncoating in which RNPs are released from M which remains bound to the endosomal membrane rather than the dissociation of M protein from RNPs after release of the complex into the cytoplasm following membrane fusion

Decline in Titers of Anti-Idiotypic Antibodies Specific to Autoantibodies to GAD65 (GAD65Ab) Precedes Development of GAD65Ab and Type 1 Diabetes.

The humoral Idiotypic Network consisting of antibodies and their anti-idiotypic antibodies (anti-Id) can be temporarily upset by antigen exposure. In the healthy immune response the original equilibrium is eventually restored through counter-regulatory mechanisms. In certain autoimmune diseases however, autoantibody levels exceed those of their respective anti-Id, indicating a permanent disturbance in the respective humoral Idiotypic Network. We investigated anti-Id directed to a major Type 1 diabetes (T1D)-associated autoantibody (GAD65Ab) in two independent cohorts during progression to disease. Samples taken from participants of the Natural History Study showed significantly lower anti-Id levels in individuals that later progressed to T1D compared to non-progressors (anti-Id antibody index of 0.06 vs. 0.08, respectively, p = 0.02). We also observed a significant inverse correlation between anti-Id levels and age at sampling, but only in progressors (p = 0.014). Finally, anti-Id levels in progressors showed a significant decline during progression as compared to longitudinal anti-Id levels in non-progressors (median rate of change: -0.0004 vs. +0.0004, respectively, p = 0.003), suggesting a loss of anti-Id during progression. Our analysis of the Diabetes Prediction in Skåne cohort showed that early in life (age 2) individuals at risk have anti-Id levels indistinguishable from those in healthy controls, indicating that low anti-Id levels are not an innate characteristic of the immune response in individuals at risk. Notably, anti-Id levels declined significantly in individuals that later developed GAD65Ab suggesting that the decline in anti-Id levels precedes the emergence of GAD65Ab (median rate of change: -0.005) compared to matched controls (median rate of change: +0.001) (p = 0.0016). We conclude that while anti-Id are present early in life, their levels decrease prior to the appearance of GAD65Ab and to the development of T1D

Synchronized Retrovirus Fusion in Cells Expressing Alternative Receptor Isoforms Releases the Viral Core into Distinct Sub-cellular Compartments

Disparate enveloped viruses initiate infection by fusing with endosomes. However, the highly diverse and dynamic nature of endosomes impairs mechanistic studies of fusion and identification of sub-cellular sites supporting the nucleocapsid release. We took advantage of the extreme stability of avian retrovirus-receptor complexes at neutral pH and of acid-dependence of virus-endosome fusion to isolate the latter step from preceding asynchronous internalization/trafficking steps. Viruses were trapped within endosomes in the presence of NH4Cl. Removal of NH4Cl resulted in a quick and uniform acidification of all subcellular compartments, thereby initiating synchronous viral fusion. Single virus imaging demonstrated that fusion was initiated within seconds after acidification and often culminated in the release of the viral core from an endosome. Comparative studies of cells expressing either the transmembrane or GPI-anchored receptor isoform revealed that the transmembrane receptor delivered the virus to more fusion-permissive compartments. Thus the identity of endosomal compartments, in addition to their acidity, appears to modulate viral fusion. A more striking manifestation of the virus delivery to distinct compartments in the presence of NH4Cl was the viral core release into the cytosol of cells expressing the transmembrane receptor and into endosomes of cells expressing the GPI-anchored isoform. In the latter cells, the newly released cores exhibited restricted mobility and were exposed to a more acidic environment than the cytoplasm. These cores appear to enter into the cytosol after an additional slow temperature-dependent step. We conclude that the NH4Cl block traps the virus within intralumenal vesicles of late endosomes in cells expressing the GPI-anchored receptor. Viruses surrounded by more than one endosomal membrane release their core into the cytoplasm in two steps – fusion with an intralumenal vesicle followed by a yet unknown temperature-dependent step that liberates the core from late endosomes

Labeling of Multiple HIV-1 Proteins with the Biarsenical-Tetracysteine System

Due to its small size and versatility, the biarsenical-tetracysteine system is an attractive way to label viral proteins for live cell imaging. This study describes the genetic labeling of the human immunodeficiency virus type 1 (HIV-1) structural proteins (matrix, capsid and nucleocapsid), enzymes (protease, reverse transcriptase, RNAse H and integrase) and envelope glycoprotein 120 with a tetracysteine tag in the context of a full-length virus. We measure the impact of these modifications on the natural virus infection and, most importantly, present the first infectious HIV-1 construct containing a fluorescently-labeled nucleocapsid protein. Furthermore, due to the high background levels normally associated with the labeling of tetracysteine-tagged proteins we have also optimized a metabolic labeling system that produces infectious virus containing the natural envelope glycoproteins and specifically labeled tetracysteine-tagged proteins that can easily be detected after virus infection of T-lymphocytes. This approach can be adapted to other viral systems for the visualization of the interplay between virus and host cell during infection

Recombinant Vesicular Stomatitis Virus Vaccine Vectors Expressing Filovirus Glycoproteins Lack Neurovirulence in Nonhuman Primates

The filoviruses, Marburg virus and Ebola virus, cause severe hemorrhagic fever with high mortality in humans and nonhuman primates. Among the most promising filovirus vaccines under development is a system based on recombinant vesicular stomatitis virus (rVSV) that expresses an individual filovirus glycoprotein (GP) in place of the VSV glycoprotein (G). The main concern with all replication-competent vaccines, including the rVSV filovirus GP vectors, is their safety. To address this concern, we performed a neurovirulence study using 21 cynomolgus macaques where the vaccines were administered intrathalamically. Seven animals received a rVSV vector expressing the Zaire ebolavirus (ZEBOV) GP; seven animals received a rVSV vector expressing the Lake Victoria marburgvirus (MARV) GP; three animals received rVSV-wild type (wt) vector, and four animals received vehicle control. Two of three animals given rVSV-wt showed severe neurological symptoms whereas animals receiving vehicle control, rVSV-ZEBOV-GP, or rVSV-MARV-GP did not develop these symptoms. Histological analysis revealed major lesions in neural tissues of all three rVSV-wt animals; however, no significant lesions were observed in any animals from the filovirus vaccine or vehicle control groups. These data strongly suggest that rVSV filovirus GP vaccine vectors lack the neurovirulence properties associated with the rVSV-wt parent vector and support their further development as a vaccine platform for human use

Non-neutralizing antibodies elicited by recombinant Lassa-Rabies vaccine are critical for protection against Lassa fever

Lassa fever (LF), caused by Lassa virus (LASV), is a viral hemorrhagic fever for which no approved vaccine or potent antiviral treatment is available. LF is a WHO priority disease and, together with rabies, a major health burden in West Africa. Here we present the development and characterization of an inactivated recombinant LASV and rabies vaccine candidate (LASSARAB) that expresses a codon-optimized LASV glycoprotein (coGPC) and is adjuvanted by a TLR-4 agonist (GLA-SE). LASSARAB elicits lasting humoral response against LASV and RABV in both mouse and guinea pig models, and it protects both guinea pigs and mice against LF. We also demonstrate a previously unexplored role for non-neutralizing LASV GPC-specific antibodies as a major mechanism of protection by LASSARAB against LF through antibody-dependent cellular functions. Overall, these findings demonstrate an effective inactivated LF vaccine and elucidate a novel humoral correlate of protection for LF.NIH grants R01 AI105204 to M.J.S., by the Jefferson Vaccine Center, and by the Fundação para a Ciência e Tecnologia (FCT) scholarship PD/BD/105847/2014 (to T.A.-M.). This work was also funded in part through the NIAID Division of Intramural Research and the NIAID Division of Clinical Research, Battelle Memorial Institute’s prime contract with the U.S. National Institute of Allergy and Infectious Diseases (NIAID) under Contract No. HHSN272200700016Iinfo:eu-repo/semantics/publishedVersio

IFITM3 Inhibits Influenza A Virus Infection by Preventing Cytosolic Entry

To replicate, viruses must gain access to the host cell's resources. Interferon (IFN) regulates the actions of a large complement of interferon effector genes (IEGs) that prevent viral replication. The interferon inducible transmembrane protein family members, IFITM1, 2 and 3, are IEGs required for inhibition of influenza A virus, dengue virus, and West Nile virus replication in vitro. Here we report that IFN prevents emergence of viral genomes from the endosomal pathway, and that IFITM3 is both necessary and sufficient for this function. Notably, viral pseudoparticles were inhibited from transferring their contents into the host cell cytosol by IFN, and IFITM3 was required and sufficient for this action. We further demonstrate that IFN expands Rab7 and LAMP1-containing structures, and that IFITM3 overexpression is sufficient for this phenotype. Moreover, IFITM3 partially resides in late endosomal and lysosomal structures, placing it in the path of invading viruses. Collectively our data are consistent with the prediction that viruses that fuse in the late endosomes or lysosomes are vulnerable to IFITM3's actions, while viruses that enter at the cell surface or in the early endosomes may avoid inhibition. Multiple viruses enter host cells through the late endocytic pathway, and many of these invaders are attenuated by IFN. Therefore these findings are likely to have significance for the intrinsic immune system's neutralization of a diverse array of threats

Molecular Determinants and Dynamics of Hepatitis C Virus Secretion

The current model of hepatitis C virus (HCV) production involves the assembly of virions on or near the surface of lipid droplets, envelopment at the ER in association with components of VLDL synthesis, and egress via the secretory pathway. However, the cellular requirements for and a mechanistic understanding of HCV secretion are incomplete at best. We combined an RNA interference (RNAi) analysis of host factors for infectious HCV secretion with the development of live cell imaging of HCV core trafficking to gain a detailed understanding of HCV egress. RNAi studies identified multiple components of the secretory pathway, including ER to Golgi trafficking, lipid and protein kinases that regulate budding from the trans-Golgi network (TGN), VAMP1 vesicles and adaptor proteins, and the recycling endosome. Our results support a model wherein HCV is infectious upon envelopment at the ER and exits the cell via the secretory pathway. We next constructed infectious HCV with a tetracysteine (TC) tag insertion in core (TC-core) to monitor the dynamics of HCV core trafficking in association with its cellular cofactors. In order to isolate core protein movements associated with infectious HCV secretion, only trafficking events that required the essential HCV assembly factor NS2 were quantified. TC-core traffics to the cell periphery along microtubules and this movement can be inhibited by nocodazole. Sub-populations of TC-core localize to the Golgi and co-traffic with components of the recycling endosome. Silencing of the recycling endosome component Rab11a results in the accumulation of HCV core at the Golgi. The majority of dynamic core traffics in association with apolipoprotein E (ApoE) and VAMP1 vesicles. This study identifies many new host cofactors of HCV egress, while presenting dynamic studies of HCV core trafficking in infected cells