Comprehensive proteomic analysis of HCoV-OC43 virions and virus-modulated extracellular vesicles.

Unlabelled: Viruses are obligate parasites that depend on the cellular machinery for their propagation. Several viruses also incorporate cellular proteins that facilitate viral spread. Defining these cellular proteins is critical to decipher viral life cycles and delineate novel therapeutic strategies.

RNA helicase DDX3X modulates herpes simplex virus 1 nuclear egress.

DDX3X is a mammalian RNA helicase that regulates RNA metabolism, cancers, innate immunity and several RNA viruses. We discovered that herpes simplex virus 1, a nuclear DNA replicating virus, redirects DDX3X to the nuclear envelope where it surprisingly modulates the exit of newly assembled viral particles. DDX3X depletion also leads to an accumulation of virions in intranuclear herniations.

Optimizing human coronavirus OC43 growth and titration.

Coronaviruses have been at the forefront of the news for the last 2 years. Unfortunately, SARS-CoV-2, the etiologic agent for the COVID-19 pandemic, must be manipulated in biosecurity level 3 settings, which significantly limits research. Meanwhile, several less pathogenic human coronaviruses (HCoV) exist and can be studied in much more common biosafety level 2 laboratories.

SUN2 Modulates the Propagation of HSV-1.

Herpesviruses assemble new viral particles in the nucleus. These nucleocapsids bud through the inner nuclear membrane to produce enveloped viral particles in the perinuclear space before fusing with the outer nuclear membrane to reach the cytoplasm. This unusual route is necessary since viral capsids are too large to pass through nuclear pores.

Proteomics of Herpes Simplex Virus Type 1 Nuclear Capsids.

Herpes simplex virus replicates in the nucleus, where new capsids are assembled. It produces procapsids devoid of nucleic acid but containing the preVP22a scaffold protein. These thermo-unstable particles then mature into A-, B- or C-nuclear icosahedral capsids, depending on their ability to shed the proteolytically processed scaffold and incorporation of the viral genome.

The XPO6 Exportin Mediates Herpes Simplex Virus 1 gM Nuclear Release Late in Infection.

The glycoprotein M of herpes simplex virus 1 (HSV-1) is dynamically relocated from nuclear membranes to the -Golgi network (TGN) during infection, but molecular partners that promote this relocalization are unknown. Furthermore, while the presence of the virus is essential for this phenomenon, it is not clear if this is facilitated by viral or host proteins. Past attempts to characterize glycoprotein M (gM) interacting partners identified the viral protein gN by coimmunoprecipitation and the host protein E-Syt1 through a proteomics approach.

[Intracellular transport of Alphaherpesvirinae].

The Alphaherpesvirinae sub-family includes viruses primarily associated with cold sores, genital herpes, chicken pox and shingles in humans, but are responsible for several other pathologies and additionally infect many animals. These viruses are large entities that travel through various cellular compartments during their life cycle. As for the transport of cellular cargoes, this involves several budding and fusion steps as well as transport of viral particles along the cytoskeleton.

Analysis and Sorting of Individual HSV-1 Particles by Flow Virometry.

Flow cytometry has been instrumental in characterizing normal and infected cells. However, until recently, it was not possible to use such an approach to analyze small entities such as bacteria, let alone viruses, owing to the 0.5 μm resolution of most instruments.

Characterization of Extracellular HSV-1 Virions by Proteomics.

The analysis of HSV-1 mature extracellular virions by proteomics requires highly enriched samples to limit false-positives and favor the detection of true components. The protocol described below involves the removal of highly contaminating serum proteins and purification of the virions by a series of differential and density centrifugation steps. In addition, L-particles, which are viral particles devoid of a genome and capsid but present in the extracellular milieu, are depleted on Ficoll 400 gradients.

Cellular Protein Kinase D Modulators Play a Role during Multiple Steps of Herpes Simplex Virus 1 Egress.

The assembly of new herpes simplex virus 1 (HSV-1) particles takes place in the nucleus. These particles then travel across the two nuclear membranes and acquire a final envelope from a cellular compartment. The contribution of the cell to the release of the virus is, however, little known.

Flow Virometry: a Powerful Tool To Functionally Characterize Viruses.

For several decades, flow cytometry has been a common approach to analyze cells and sort them to near-purity. It enables one to probe inner cellular molecules, surface receptors, or infected cells. However, the analysis of smaller entities such as viruses and exocytic vesicles has been more difficult but is becoming mainstream.

Extended Synaptotagmin 1 Interacts with Herpes Simplex Virus 1 Glycoprotein M and Negatively Modulates Virus-Induced Membrane Fusion.

Enveloped viruses typically encode their own fusion machinery to enter cells. Herpesviruses are unusual, as they fuse with a number of cellular compartments throughout their life cycles. As uncontrolled fusion of the host membranes should be avoided in these events, tight regulation of the viral fusion machinery is critical.

Quantitative Evaluation of Protein Heterogeneity within Herpes Simplex Virus 1 Particles.

Several virulence genes have been identified thus far in the herpes simplex virus 1 genome. It is also generally accepted that protein heterogeneity among virions further impacts viral fitness. However, linking this variability directly with infectivity has been challenging at the individual viral particle level.

Rab7A regulates tau secretion.

The axonal microtubule-associated protein TAU, involved in Alzheimer's disease (AD), can be found in the extracellular space where it could be taken up by neurons, an event that is believed to contribute to the propagation of tau pathology in the brain. Since the small GTPase Rab7A is involved in the trafficking of endosomes, autophagosomes, and lysosomes, and RAB7A gene expression and protein levels are up-regulated in AD patients, we tested the hypothesis that Rab7A was involved in tau secretion. We previously reported that both primary cortical neurons and HeLa cells over-expressing human TAU can release tau.

The ATP-Dependent RNA Helicase DDX3X Modulates Herpes Simplex Virus 1 Gene Expression.

The human protein DDX3X is a DEAD box ATP-dependent RNA helicase that regulates transcription, mRNA maturation, and mRNA export and translation. DDX3X concomitantly modulates the replication of several RNA viruses and promotes innate immunity. We previously showed that herpes simplex virus 1 (HSV-1), a human DNA virus, incorporates DDX3X into its mature particles and that DDX3X is required for optimal HSV-1 infectivity.

Subcellular trafficking and functional importance of herpes simplex virus type 1 glycoprotein M domains.

Herpes simplex virus type 1 (HSV-1) glycoprotein M (gM/UL10) is a 473 aa type III transmembrane protein that resides in various membrane compartments. HSV-1 gM contains several putative trafficking motifs, but their functional relevance remains to be elucidated. We show here that transiently expressed gM 19–343 was sufficient for transport to the trans-Golgi network (TGN), whilst gM 133–473, where the first two transmembrane domains were deleted, and gM 1–342, which lacked the final residue of the last transmembrane domain, were retained in the endoplasmic reticulum (ER), indicating that all transmembrane domains are required for proper folding and ER exit.

Herpes simplex virus 1 gN partners with gM to modulate the viral fusion machinery.

Unlabelled: Herpes simplex virus 1 (HSV-1) capsids are assembled in the nucleus, where they incorporate the viral genome. They then transit through the two nuclear membranes and are wrapped by a host-derived envelope. In the process, several HSV-1 proteins are targeted to the nuclear membranes, but their roles in viral nuclear egress are unclear.

Characterization of extracellular HSV-1 virions by proteomics.

The analysis of herpes simplex virus type 1 mature extracellular virions by proteomics requires highly enriched samples to limit false positives and favor the detection of true components. The protocol described below involves the removal of highly contaminating serum proteins and purification of the virions by a series of differential and density centrifugation steps. In addition, L-particles, which are viral particles devoid of genome and capsid but present in the extracellular milieu, are depleted on Ficoll 400 gradients.

Analysis of the early steps of herpes simplex virus 1 capsid tegumentation.

Herpes simplex virus type 1 particles are multilayered structures with a DNA genome surrounded by a capsid, tegument, and envelope. While the protein content of mature virions is known, the sequence of addition of the tegument and the intracellular compartments where this occurs are intensely debated. To probe this process during the initial stages of egress, we used two approaches: an in vitro nuclear egress assay, which reconstitutes the exit of nuclear capsids to the cytoplasm, and a classical nuclear capsid sedimentation assay.

Inhibition of the host translation shutoff response by herpes simplex virus 1 triggers nuclear envelope-derived autophagy.

Macroautophagy is a cellular pathway that degrades intracellular pathogens and contributes to antigen presentation. Herpes simplex virus 1 (HSV-1) infection triggers both macroautophagy and an additional form of autophagy that uses the nuclear envelope as a source of membrane. The present study constitutes the first in-depth analysis of nuclear envelope-derived autophagy (NEDA).

Analysis of virion-incorporated host proteins required for herpes simplex virus type 1 infection through a RNA interference screen.

Viruses are strictly dependent on cells to propagate and many incorporate host proteins in their viral particles, but the significance of this incorporation is poorly understood. Recently, we performed the first comprehensive characterization of the mature herpes simplex virus type 1 (HSV-1) in which up to 49 distinct cellular proteins were identified by mass spectrometry. In the present study, we sought to identify if these cellular factors are relevant for the HSV-1 life cycle.

Analysis of herpes simplex virus type I nuclear particles by flow cytometry.

Flow cytometry has been instrumental to characterize cell populations and examine their inner molecules and processes. In most instances, whole cells are analyzed, and hence, particle size is not an issue. Viruses are 2-3 orders of magnitude smaller than cells so flow cytometry has typically been used to study viral markers within whole infected cells.

Herpesviruses exploit several host compartments for envelopment.

Enveloped viruses acquire their host-derived membrane at a variety of intracellular locations. Herpesviruses are complex entities that undergo several budding and fusion events during an infection. All members of this large family are believed to share a similar life cycle.

Deciphering novel host-herpesvirus interactions by virion proteomics.

Over the years, a vast array of information concerning the interactions of viruses with their hosts has been collected. However, recent advances in proteomics and other system biology techniques suggest these interactions are far more complex than anticipated. One particularly interesting and novel aspect is the analysis of cellular proteins incorporated into mature virions.

Biochemical analysis of infected cell polypeptide (ICP)0, ICP4, UL7 and UL23 incorporated into extracellular herpes simplex virus type 1 virions.

Herpes simplex virus type 1 (HSV-1) capsids assemble in the nucleus but acquire their teguments from various cellular compartments. Unfortunately, little is known about their exact arrangement and when they coat the newly produced capsids. The complexity of the virions is further highlighted by our recent proteomics analysis that detected the presence of several novel or controversial components in extracellular HSV-1 virions.