Epstein-Barr Virus BBLF1 Mediates Secretory Vesicle Transport to Facilitate Mature Virion Release.

Enveloped viruses undergo a complex multistep process of assembly, maturation, and release into the extracellular space utilizing host secretory machinery. Several studies of the herpesvirus subfamily have shown that secretory vesicles derived from the trans-Golgi network (TGN) or endosomes transport virions into the extracellular space. However, the regulatory mechanism underlying the release of Epstein-Barr virus, a human oncovirus, remains unclear. We demonstrate that disruption of BBLF1, a tegument component, suppressed viral release and resulted in the accumulation of viral particles on the inner side of the vesicular membrane. Organelle separation revealed the accumulation of infectious viruses in fractions containing vesicles derived from the TGN and late endosomes. Deficiency of an acidic amino acid cluster in BBLF1 reduced viral secretion. Moreover, truncational deletion of the C-terminal region of BBLF1 increased infectious virus production. These findings suggest that BBLF1 regulates the viral release pathway and reveal a new aspect of tegument protein function. IMPORTANCE Several viruses have been linked to the development of cancer in humans. Epstein-Barr virus (EBV), the first identified human oncovirus, causes a wide range of cancers. Accumulating literature has demonstrated the role of viral reactivation in tumorigenesis. Elucidating the functions of viral lytic genes induced by reactivation, and the mechanisms of lytic infection, is essential to understanding pathogenesis. Progeny viral particles synthesized during lytic infection are released outside the cell after the assembly, maturation, and release steps, leading to further infection. Through functional analysis using BBLF1-knockout viruses, we demonstrated that BBLF1 promotes viral release. The acidic amino acid cluster in BBLF1 was also important for viral release. Conversely, mutants lacking the C terminus exhibited more efficient virus production, suggesting that BBLF1 is involved in the fine-tuning of progeny release during the EBV life cycle.

A Role for Human Skin Mast Cells in Dengue Virus Infection and Systemic Spread.

Dengue virus (DENV) is a mosquito-borne flavivirus that causes serious global human disease and mortality. Skin immune cells are an important component of initial DENV infection and systemic spread. Here, we show that mast cells are a target of DENV in human skin and that DENV infection of skin mast cells induces degranulation and alters cytokine and growth factor expression profiles. Importantly, to our knowledge, we also demonstrate for the first time that DENV localizes within secretory granules in infected skin mast cells. In addition, DENV within extracellular granules was infectious in vitro and in vivo, trafficking through lymph to draining lymph nodes in mice. We demonstrate an important role for human skin mast cells in DENV infection and identify a novel mechanism for systemic spread of DENV infection from the initial peripheral mosquito injection site.

Autophagy-associated dengue vesicles promote viral transmission avoiding antibody neutralization.

One of the major defense mechanisms against virus spread in vivo is the blocking of viral infectibility by neutralizing antibodies. We describe here the identification of infectious autophagy-associated dengue vesicles released from infected cells. These vesicles contain viral proteins E, NS1, prM/M, and viral RNA, as well as host lipid droplets and LC3-II, an autophagy marker. The viral RNA can be protected within the autophagic organelles since anti-dengue neutralizing antibodies do not have an effect on the vesicle-mediated transmission that is able to initiate a new round of infection in target cells. Importantly, such infectious vesicles were also detected in a patient serum. Our study suggests that autophagy machinery plays a new role in dengue virus transmission. This discovery explains the inefficiency of neutralizing antibody upon dengue infection as a potential immune evasion mechanism in vivo.

Intercellular Transmission of Viral Populations with Vesicles.

A common paradigm holds that during cell-to-cell transmission, viruses behave as lone soldiers. Recently, we discovered not only that enteroviruses are transmitted via vesicles as populations of viral particles but also that this type of transmission enhances their infection efficiency (Y. H. Chen et al., Cell 160: 619-630, 2015). This mechanism could be advantageous for the overall fitness of the viral population, promoting genetic interplay by enabling viral quasispecies to collectively infect a susceptible host cell. Here, we discuss these findings in the context of viral pathogenesis and also propose that this novel type of vesicular transmission is widespread among different virus families and includes populations of both viral particles and naked viral genomes.

Correlative scanning-transmission electron microscopy reveals that a chimeric flavivirus is released as individual particles in secretory vesicles.

The intracellular morphogenesis of flaviviruses has been well described, but flavivirus release from the host cell remains poorly documented. We took advantage of the optimized production of an attenuated chimeric yellow fever/dengue virus for vaccine purposes to study this phenomenon by microscopic approaches. Scanning electron microscopy (SEM) showed the release of numerous viral particles at the cell surface through a short-lived process. For transmission electron microscopy (TEM) studies of the intracellular ultrastructure of the small number of cells releasing viral particles at a given time, we developed a new correlative microscopy method: CSEMTEM (for correlative scanning electron microscopy - transmission electron microscopy). CSEMTEM analysis suggested that chimeric flavivirus particles were released as individual particles, in small exocytosis vesicles, via a regulated secretory pathway. Our morphological findings provide new insight into interactions between flaviviruses and cells and demonstrate that CSEMTEM is a useful new method, complementary to SEM observations of biological events by intracellular TEM investigations.

Providence virus (family: Carmotetraviridae) replicates vRNA in association with the Golgi apparatus and secretory vesicles.

Providence virus (PrV) is the sole member of the family Carmotetraviridae (formerly Tetraviridae) sharing the characteristic T=4 capsid architecture with other tetravirus families. Despite significant structural similarities, PrV differs from other tetraviruses in terms of genome organization, non-structural protein sequence and regulation of gene expression. In addition, it is the only tetravirus that infects tissue culture cells. Previous studies showed that in persistently infected Helicoverpa zea MG8 cells, the PrV replicase associates with detergent-resistant membranes in punctate cytosolic structures, which is similar to the distribution of an alpha-like tetravirus replicase (Helicoverpa armigera stunt virus). Here, we demonstrate that the site of PrV vRNA replication coincides with the presence of PrV p40/p104 proteins in infected cells and that these replication proteins associate with the Golgi apparatus and secretory vesicles in transfected cells.

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.

Herpes simplex virus utilizes the large secretory vesicle pathway for anterograde transport of tegument and envelope proteins and for viral exocytosis from growth cones of human fetal axons.

Axonal transport of herpes simplex virus (HSV-1) is essential for viral infection and spread in the peripheral nervous system of the host. Therefore, the virus probably utilizes existing active transport and targeting mechanisms in neurons for virus assembly and spread from neurons to skin. In the present study, we used transmission immunoelectron microscopy to investigate the nature and origin of vesicles involved in the anterograde axonal transport of HSV-1 tegument and envelope proteins and of vesicles surrounding partially and fully enveloped capsids in growth cones. This study aimed to elucidate the mechanism of virus assembly and exit from axons of human fetal dorsal root ganglia neurons. We demonstrated that viral tegument and envelope proteins can travel in axons independently of viral capsids and were transported to the axon terminus in two types of transport vesicles, tubulovesicular membrane structures and large dense-cored vesicles. These vesicles and membrane carriers were derived from the trans-Golgi network (TGN) and contained key proteins, such as Rab3A, SNAP-25, GAP-43, and kinesin-1, involved in the secretory and exocytic pathways in axons. These proteins were also observed on fully and partially enveloped capsids in growth cones and on extracellular virions. Our findings provide further evidence to the subassembly model of separate transport in axons of unenveloped capsids from envelope and tegument proteins with final virus assembly occurring at the axon terminus. We postulate that HSV-1 capsids invaginate tegument- and envelope-bearing TGN-derived vesicles and utilize the large secretory vesicle pathway of exocytosis for exit from axons.

HIV type 1 can act as an APC upon acquisition from the host cell of peptide-loaded HLA-DR and CD86 molecules.

It is well documented that a wide range of host-derived cell surface constituents is inserted within HIV type 1 (HIV-1) and located on the exterior of the virion. Although no virus-associated protein of host origin has been shown to be absolutely required for virus replication, studies have revealed that many of these proteins are functional and can affect several steps of the virus life cycle. In this study, we found that HIV-1 acquires peptide-loaded class II MHC (MHC-II) and the costimulatory CD86 molecules from the host cell. Moreover, we present evidence that virions bearing such peptide-loaded MHC-II and CD86 proteins can lead to activation of the transcription factors NF-kappa B and NF-AT in an Ag-specific human T cell line. A linear correlation was found between activation of NF-kappa B and the amount of peptide-loaded MHC-II molecules inserted within HIV-1. Finally, transcription of unintegrated and integrated HIV-1 DNA was promoted upon exposure of peptide-specific human T cells to viruses bearing both peptide-loaded MHC-II and CD86 proteins. These data suggest that HIV-1 can operate as an APC depending on the nature of virus-anchored host cell membrane components. It can be proposed that HIV-1 can manipulate one of its primary targets through the process of incorporation of host-derived proteins.

Cell surface expression of the HIV-1 envelope glycoproteins is directed from intracellular CTLA-4-containing regulated secretory granules.

The envelope glycoprotein (Env) of HIV-1 is incorporated into virions that bud from the cell surface of infected T cells. With immunofluorescence microscopy and subcellular membrane fractionation techniques, the intracellular fate of Env in the secretory pathway of HIV-1-infected T cells was evaluated. Rather than trafficking constitutively from the Golgi to the cell surface, Env is directed to intracellular CTLA-4-containing granules, whose recruitment to the cell surface is regulated. The use of the regulated pathway for intracellular Env storage before virion assembly holds implications for the staging of Env exposure at the cell surface of infected cells and of coordinating HIV virion assembly.