Viral Exit Research Articles

Electron micrographs of human and avian influenza viruses with high and low pathogenicity

Background: An outbreak of highly pathogenic avian influenza (HPAI) H5N1 virus in Thailand was first reported in 2004. To date, electron micrographs demonstrating the morphology of HPAI H5N1 virus particle are quite limited. Objective: To demonstrate the morphology of HPAI H5N1 virus particles, avian influenza viruses with low pathogenicity, seasonal influenza viruses, and H5N1 structural components in infected cells. The M amino acid residues that might affect the viral morphology were also analyzed. Methods: Electron micrographs of negatively-stained virus particles and positively-stained thin sections of the HPAI H5N1 virus infected cells were visualized under a transmission electron microscope. M amino acid sequences of the study viruses were retrieved from the GenBank database and aligned with those of reference strains with known morphology and residues that are unique for the morphological type of the virus particles. Results: Morphologically, three forms of influenza virus particles, spherical, regular, and irregular rods, and long filamentous particles, were demonstrated. However, the spherical form was the most predominant morphological type and accounted for more than 80% of the virus populations examined. In addition, the viral entry and exit steps including incomplete particles in infected Madin-Darby canine kidney cells were visualized. Our analyses did not find any M amino acid residues that might influence the viral morphology. Conclusion: Of all virus isolates studied, we demonstrated that the spherical particles were the major population observed regardless of virus subtype, host of origin, virus virulence, or passage history. Our study suggested that the morphology of influenza virus particles released, might not be strongly influenced by M gene polymorphism. Keywords: Electron micrographs, highly pathogenic avian influenza H5N1 virus, influenza virus, low pathogenic avian influenza virus, M amino acid sequence, virus morphology

The B cell response to influenza infection is limited by CD4 T cell help (P4305)

Abstract Seasonal influenza and the risk of widespread influenza pandemics are an ongoing major world health concern. In the United States alone, influenza infection results in more than 200,000 hospitalizations and up to 41,000 deaths each year. The current vaccine strategies focus on eliciting neutralizing antibodies against hemagglutinin and neuraminidase, the major surface glycoproteins involved with viral entry and exit from a host cell. Because of the highly mutable nature of influenza, the vaccine must be reformulated each year - highlighting the necessity for an alternate approach that provides broad and long-lasting protection. We have approached this problem by asking if CD4 T cell memory, elicited by peptide priming, is sufficient to accelerate T cell and B cell responses to influenza infection. After immunization with immunogenic peptides derived from A/New Caledonia/20/99 followed by infection with the same virus, we observed an increased frequency of virus specific CD4 T cells 7 days post infection. Additionally, there was an enhanced B cell response both kinetically and in magnitude. We have also observed a strong correlation between the frequencies of follicular T helper cells and germinal center B cells in the peptide primed/infected cohorts that are not seen in the control primed/infected group. Our results suggest that T cell help for a primary B cell response to infection is limiting, and that peptide priming is sufficient to generate a functional memory CD4 pool.

VP16‐HCF interaction is required for herpes simplex virus exit from latency in neurons in vivo

A mechanistic understanding of viral and host functions regulating herpes simplex virus (HSV) latency and reactivation remains a major goal. We have shown that the interaction of VP16 and Oct‐1 is required very early during entry into the lytic cycle from the latent state and have proposed a VP16‐dependent model of reactivation in vivo. HCF‐1 has important roles in promoting HSV replication and a model has been proposed in which stress induces translocation of HCF‐1 into the neuronal nucleus and reactivation is regulated by direct recruitment of HCF to viral IE genes. To investigate whether HCF‐1 can precipitate viral gene expression in latently infected neurons in the absence of binding to VP16, a mutation in VP16 at Y364A, shown previously to disrupt VP16‐ HCF interaction (but not interaction with Oct‐1) was engineered into HSV‐1. Three independent isolates and a genomically restored virus were examined in vitro and in the mouse ocular model. Our findings demonstrate that the interaction of VP16 with HCF‐1 is required for efficient infection at low moi, efficient viral replication in vivo, and exit from latency/entry into the lytic cycle in trigeminal ganglion neurons in mice following hyperthermic stress. In the absence of VP16 binding, nuclear translocation of HCF is not sufficient to initiate the viral lytic cycle, demonstrating the key role of VP16 in regulating in vivo reactivation.

Matriptase proteolytically activates influenza virus and promotes multicycle replication in the human airway epithelium.

Influenza viruses do not encode any proteases and must rely on host proteases for the proteolytic activation of their surface hemagglutinin proteins in order to fuse with the infected host cells. Recent progress in the understanding of human proteases responsible for influenza virus hemagglutinin activation has led to the identification of members of the type II transmembrane serine proteases TMPRSS2 and TMPRSS4 and human airway trypsin-like protease; however, none has proved to be the sole enzyme responsible for hemagglutinin cleavage. In this study, we identify and characterize matriptase as an influenza virus-activating protease capable of supporting multicycle viral replication in the human respiratory epithelium. Using confocal microscopy, we found matriptase to colocalize with hemagglutinin at the apical surface of human epithelial cells and within endosomes, and we showed that the soluble form of the protease was able to specifically cleave hemagglutinins from H1 virus, but not from H2 and H3 viruses, in a broad pH range. We showed that small interfering RNA (siRNA) knockdown of matriptase in human bronchial epithelial cells significantly blocked influenza virus replication in these cells. Lastly, we provide a selective, slow, tight-binding inhibitor of matriptase that significantly reduces viral replication (by 1.5 log) of H1N1 influenza virus, including the 2009 pandemic virus. Our study establishes a three-pronged model for the action of matriptase: activation of incoming viruses in the extracellular space in its shed form, upon viral attachment or exit in its membrane-bound and/or shed forms at the apical surface of epithelial cells, and within endosomes by its membrane-bound form where viral fusion takes place.

Mapping the Phosphoproteome of Influenza A and B Viruses by Mass Spectrometry

Protein phosphorylation is a common post-translational modification in eukaryotic cells and has a wide range of functional effects. Here, we used mass spectrometry to search for phosphorylated residues in all the proteins of influenza A and B viruses – to the best of our knowledge, the first time such a comprehensive approach has been applied to a virus. We identified 36 novel phosphorylation sites, as well as confirming 3 previously-identified sites. N-terminal processing and ubiquitination of viral proteins was also detected. Phosphorylation was detected in the polymerase proteins (PB2, PB1 and PA), glycoproteins (HA and NA), nucleoprotein (NP), matrix protein (M1), ion channel (M2), non-structural protein (NS1) and nuclear export protein (NEP). Many of the phosphorylation sites detected were conserved between influenza virus genera, indicating the fundamental importance of phosphorylation for all influenza viruses. Their structural context indicates roles for phosphorylation in regulating viral entry and exit (HA and NA); nuclear localisation (PB2, M1, NP, NS1 and, through NP and NEP, of the viral RNA genome); and protein multimerisation (NS1 dimers, M2 tetramers and NP oligomers). Using reverse genetics we show that for NP of influenza A viruses phosphorylation sites in the N-terminal NLS are important for viral growth, whereas mutating sites in the C-terminus has little or no effect. Mutating phosphorylation sites in the oligomerisation domains of NP inhibits viral growth and in some cases transcription and replication of the viral RNA genome. However, constitutive phosphorylation of these sites is not optimal. Taken together, the conservation, structural context and functional significance of phosphorylation sites implies a key role for phosphorylation in influenza biology. By identifying phosphorylation sites throughout the proteomes of influenza A and B viruses we provide a framework for further study of phosphorylation events in the viral life cycle and suggest a range of potential antiviral targets.

Tetraspanin 6 (TSPAN6) Negatively Regulates Retinoic Acid-inducible Gene I-like Receptor-mediated Immune Signaling in a Ubiquitination-dependent Manner

The recognition between retinoic acid-inducible gene I-like receptors (RLRs) and viral RNA triggers an intracellular cascade of signaling to induce the expression of type I IFNs. Both positive and negative regulation of the RLR signaling pathway are important for the host antiviral immune response. Here, we demonstrate that the tetraspanin protein TSPAN6 inhibits RLR signaling by affecting the formation of the adaptor MAVS (mitochondrial antiviral signaling)-centered signalosome. We found that overexpression of TSPAN6 impaired RLR-mediated activation of IFN-stimulated response element, NF-κB, and IFN-β promoters, whereas knockdown of TSPAN6 enhanced the RLR-mediated signaling pathway. Interestingly, as the RLR pathway was activated, TSPAN6 underwent Lys-63-linked ubiquitination, which promoted its association with MAVS. The interaction of TSPAN6 and MAVS interfered with the recruitment of RLR downstream molecules TRAF3, MITA, and IRF3 to MAVS. Further study revealed that the first transmembrane domain of TSPAN6 is critical for its ubiquitination and association with MAVS as well as its inhibitory effect on RLR signaling. We concluded that TSPAN6 functions as a negative regulator of the RLR pathway by interacting with MAVS in a ubiquitination-dependent manner.

Identification of the HIV-1 NC Binding Interface in Alix Bro1 Reveals a Role for RNA

HIV-1 recruits members of ESCRT, the cell membrane fission machinery that promotes virus exit. HIV-1 Gag protein gains access to ESCRT directly by binding Alix, an ESCRT-associated protein that promotes budding. The Alix Bro1 and V domains bind Gag NC and p6 regions, respectively. Whereas V-p6 binding and function are well characterized, residues in Bro1 that interact with NC and their functional contribution to Alix-mediated HIV-1 budding are unknown. We mapped Bro1 residues that constitute the NC binding interface and found that they are critical for function. Intriguingly, residues involved in interactions on both sides of the Bro1-NC interface are positively charged, suggesting the involvement of a negatively charged cellular factor serving as a bridge. Nuclease treatment eliminated Bro1-NC interactions, revealing the involvement of RNA. These findings establish a direct role for NC in mediating interactions with ESCRT necessary for virus release and report the first evidence of RNA involvement in such recruitments.

The ATF6 branch of unfolded protein response and apoptosis are activated to promote African swine fever virus infection

African swine fever virus (ASFV) infection induces apoptosis in the infected cell; however, the consequences of this activation on virus replication have not been defined. In order to identify the role of apoptosis in ASFV infection, we analyzed caspase induction during the infection and the impact of caspase inhibition on viral production. Caspases 3, 9 and 12 were activated from 16 h post-infection, but not caspase 8. Indeed, caspase 3 activation during the early stages of the infection appeared to be crucial for efficient virus exit. In addition, the inhibition of membrane blebbing reduced the release of virus particles from the cell. ASFV uses the endoplasmic reticulum (ER) as a site of replication and this process can trigger ER stress and the unfolded protein response (UPR) of the host cell. In addition to caspase 12 activation, indicators of ER stress include the upregulation of the chaperones calnexin and calreticulin upon virus infection. Moreover, ASFV induces transcription factor 6 signaling pathway of the UPR, but not the protein kinase-like ER kinase or the inositol-requiring enzyme 1 pathways. Thus, the capacity of ASFV to regulate the UPR may prevent early apoptosis and ensure viral replication.

Viral Product Trafficking to Mitochondria, Mechanisms and Roles in Pathogenesis

A wide variety of viruses cause significant morbidity and mortality in humans. However, targeted antiviral therapies have been developed for only a subset of these viruses, with the majority of currently licensed antiviral drugs targeting viral entry, replication or exit steps during the viral life cycle. Due to increasing emergence of antiviral drug resistant viruses, the isolation of multiple viral subtypes, and toxicities of existing therapies, there remains an urgent need for the timely development of novel antiviral agents, including those targeting host factors essential for viral replication. This review summarizes viral products that target mitochondria and their effects on common mitochondria regulated pathways. These viral products and the mitochondrial pathways affected by them provide potential novel targets for the rational design of antiviral drugs. Viral products alter oxidative balance, mitochondrial permeability transition pore, mitochondrial membrane potential, electron transport and energy production. Moreover, viruses may cause the Warburg Effect, in which metabolism is reprogrammed to aerobic glycolysis as the main source of energy. Finally, viral products affect proapoptotic and antiapoptotic signaling, as well as mitochondrial innate immune signaling. Because of their importance for the generation of metabolic intermediates and energy as well as cell survival, mitochondrial pathways are targeted by multiple independent viral products. Structural modifications of existing drugs targeted to mitochondrial pathways may lead to the development of novel antiviral drugs with improved efficacy and reduced toxicity.

Alternative Roles of Neuraminidase in Influenza a Viral Propagation

Alternative Roles of Neuraminidase in Influenza a Viral Propagation Influenza A is an envelope virus containing a segmented negative RNA genome. The viral envelope has two major surface glycoproteins, Hemagglutinin (HA) and Neuraminidase (NA) of which there are 16 and 9 subtypes respectively. While HA has been well characterized and shown to be essential for viral fusion and entry into host cells, the functional roles of NA in viral replication are much less understood. Some previous reports suggest that NA exerts its primary role during viral exit from the cell by enzymatically cleaving sialic acid and facilitating the release of viral particles from the host cell. Indeed this role is supported by the observation, that NA-defective viruses, or wild type (WT) viruses in the presence of NA inhibitors, form aggregations on the apical surface of the cells.

VP16 serine 375 is a critical determinant of herpes simplex virus exit from latency in vivo

Development of novel prevention and treatment strategies for herpes simplex virus (HSV) mediated diseases is dependent upon an accurate understanding of the central molecular events underlying the regulation of latency and reactivation. We have recently shown that the transactivation function of the virion protein VP16 is a critical determinant in the exit from latency in vivo. HSV-1 strain SJO2 carries a single serine to alanine substitution at position 375 in VP16 which disrupts its interaction with its essential co-activator Oct-1. Here we report that SJO2 is severely impaired in its ability to exit latency in vivo. This result reinforces our prior observations with VP16 transactivation mutant, in1814, in which VP16 interaction with Oct-1 is also disrupted and solidifies the importance of the VP16-Oct-1 interaction in the early steps in HSV-1 reactivation.

Emerging paramyxoviruses: molecular mechanisms and antiviral strategies

In recent years, several paramyxoviruses have emerged to infect humans, including previously unidentified zoonoses. Hendra and Nipah viruses (henipaviruses within this family) were first identified in the 1990s in Australia, Malaysia and Singapore, causing epidemics with high mortality and morbidity rates in affected animals and humans. Other paramyxoviruses, such as Menangle virus, Tioman virus, human metapneumovirus and avian paramyxovirus 1, which cause less morbidity in humans, have also been recently identified. Although the Paramyxoviridae family of viruses has been previously recognised as biomedically and veterinarily important, the recent emergence of these paramyxoviruses has focused our attention on this family. Antiviral drugs can be designed to target specific important determinants of the viral life cycle. Therefore, identifying and understanding the mechanistic underpinnings of viral entry, replication, assembly and budding will be critical in the development of antiviral therapeutic agents. This review focuses on the molecular mechanisms discovered and the antiviral strategies pursued in recent years for emerging paramyxoviruses, with particular emphasis on viral entry and exit mechanisms.

Low-dose paclitaxel synergizes with oncolytic adenoviruses via mitotic slippage and apoptosis in ovarian cancer

The microtubule-stabilizing drug paclitaxel has activity in relapsed ovarian cancer. dl922-947, an oncolytic adenovirus with a 24-bp deletion in E1A CR2, replicates selectively within and lyses cells with a dysregulated Rb pathway and has efficacy in ovarian cancer. In the aggressive A2780CP xenograft, combination treatment with weekly dl922-947 and paclitaxel has significantly greater efficacy than either treatment alone and can produce complete tumor eradication in some animals. We investigated the mechanisms of paclitaxel's synergy with dl922-947 in ovarian cancer. The host-cell microtubule network is grossly rearranged and stabilized following adenovirus infection, but paclitaxel does not increase this significantly. Paclitaxel does not synergize by increasing infectivity, viral protein expression or virus release. However, destabilizing the microtubule network with nocodazole reduces viral exit, revealing a novel microtubule-dependent pathway for non-lytic adenoviral exit. dl922-947 can override multiple cell cycle checkpoints but induces cell death by a non-apoptotic mechanism. In combination, dl922-947 and low-dose paclitaxel induces aberrant, multipolar mitoses, mitotic slippage and multinucleation, triggering an apoptotic cell death.

New Insights into Measles Virus Propagation: From Entry to Shedding

The last decade of research on measles virus entry has provided new clues and concepts on the molecular mechanisms that drive virus entry in distinct tissues, virus propagation in vivo throughout multiple organs and virus exit to ensure epidemic propagation. Novel biochemical and structural information on viral glycoproteins have shed light on how a genotypically variable RNA virus with error-prone RNA polymerase can behave as an immunologically invariant virus for over half a century worldwide. Moreover, the viral attachment protein was found to support efficient retargeting to unnatural cellular receptors, and this led to the design of the first class of retargeted viral vectors possessing an envelope.

The long-awaited demonstration of protein pyrophosphorylation by IP7 in vivo?

> Being wanted, he may be more wonder’d at… . > > William Shakespeare, Henry IV , Part 1 The “high-energy” of inositol pyrophosphates such as IP7 has long been considered as their raison d’etre (see ref. 1). Unsurprisingly, therefore, the excitement was palpable when these pyrophosphates were shown to transphosphorylate proteins in vitro (1). This observation was hailed as “a new mechanism in second messenger biology” (2). However, there is also a more circumspect viewpoint which requests evidence that this transphosphorylation actually occurs in vivo (2). Thus, the demonstration by Azevedo et al. (3) that IP7 hinders viral exit from cells by perturbing the interaction between a molecular … 1E-mail: shears{at}niehs.nih.gov.

Meeting of conventional and unconventional pathways at the TGN

The biosynthetic pathway allows the transport of newly synthesized proteins to the TGN via the reticulum endoplasmic and Golgi apparatus. However, many large particles reach the TGN by unconventional means. For instance, Herpes simplex virus type 1 capsids assemble within the nucleus, bud into the perinuclear space, fuse with the outer nuclear membrane and finally travel unenveloped towards the TGN. Given the central role of protein kinase D in the transport of small cargo from the TGN to the cell surface, we probed its potential contribution in HSV-1 egress, as a model for studying large cargo exiting from the TGN. Using a synchronized infection, we show that inactivation of protein kinase D with pharmacological inhibitors, a kinase dead mutant or siRNA all causes the retention of HSV-1 at the TGN. This highlights the role of PKD in viral exit and a dependence of the virus on the classical host cell machinery to leave the TGN, unlike its previous transport steps. Conceptually, this supports a model in which the TGN is a meeting point where conventional and unconventional routes encounter.

The N-terminal Amphipathic α-Helix of Viperin Mediates Localization to the Cytosolic Face of the Endoplasmic Reticulum and Inhibits Protein Secretion

Viperin is an evolutionarily conserved interferon-inducible protein that localizes to the endoplasmic reticulum (ER) and inhibits a number of DNA and RNA viruses. In this study, we report that viperin specifically localizes to the cytoplasmic face of the ER and that an amphipathic α-helix at its N terminus is necessary for the ER localization of viperin and sufficient to promote ER localization of a reporter protein, dsRed. Overexpression of intact viperin but not the amphipathic α-helix fused to dsRed induced crystalloid ER. Consistent with other proteins that induce crystalloid ER, viperin self-associates, and it does so independently of the amphipathic α-helix. Viperin expression also affected the transport of soluble but not membrane-associated proteins. Expression of intact viperin or an N-terminal α-helix-dsRed fusion protein significantly reduced secretion of soluble alkaline phosphatase and reduced its rate of ER-to-Golgi trafficking. Similarly, viperin expression inhibited bulk protein secretion and secretion of endogenous α1-antitrypsin and serum albumin from HepG2 cells. Converting hydrophobic residues in the N-terminal α-helix to acidic residues partially or completely restored normal transport of soluble alkaline phosphatase, suggesting that the extended amphipathic nature of the N-terminal α-helical domain is essential for inhibiting protein secretion.

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.

The entry of the Picornaviridae virus family in resident macrophages

For viruses, the following mechanisms of penetration into cells are typical: clathrin- or dinaminmediated endocytosis, the formation of caveolae, local lysis of cell membranes, and macropinocytosis. It is accepted that (those nonenveloped viruses in the Picornaviridae family) enter cells mostly through the local lysis of their membranes. The purpose of the present study is to research the mechanisms of penetration into resident macrophages of viruses of the indicated family, including poliovirus, Echol1 and Coxsackie B1 viruses, and Type 71 enterovirus. It has been detected that, at the adhesion sites of the Coxsackie B1 virus and Type 71 enterovirus on a macrophage surface, invaginations of the plasma membranes of cells appear, followed by the consequent formation of endocytoplasmatic vesicles, i.e., caveolae. The penetration of poliovirus into macrophages occurs both through the formation of caveolae and the local lysis of the plasmolemma of cells; during the later terms (after 45 min), macropinocytosis is observed in the viral particles during the first 15 min after the Echol1 virus penetrated the cytoplasm of macrophages through the local lysis of their plasmolemma. Thereafter, the formation of endocytic vacuolae including viral particles was observed in the cytoplasm of infected macrophages. The exit of the Echol1 virus from endocytic vacuoles was performed by the local lysis of cell membrane.

Vaccinia Virus Entry, Exit, and Interaction with Differentiated Human Airway Epithelia

Vaccinia Virus Entry, Exit, and Interaction with Differentiated Human Airway Epithelia