Egress Of Herpes Simplex Virus Research Articles

The Egress of Herpes Simplex Virus from the Infected Cell

The Egress of Herpes Simplex Virus from the Infected Cell

Role of Vesicle-Associated Membrane Protein-Associated Proteins (VAP) A and VAPB in Nuclear Egress of the Alphaherpesvirus Pseudorabies Virus.

The molecular mechanism affecting translocation of newly synthesized herpesvirus nucleocapsids from the nucleus into the cytoplasm is still not fully understood. The viral nuclear egress complex (NEC) mediates budding at and scission from the inner nuclear membrane, but the NEC is not sufficient for efficient fusion of the primary virion envelope with the outer nuclear membrane. Since no other viral protein was found to be essential for this process, it was suggested that a cellular machinery is recruited by viral proteins. However, knowledge on fusion mechanisms involving the nuclear membranes is rare. Recently, vesicle-associated membrane protein-associated protein B (VAPB) was shown to play a role in nuclear egress of herpes simplex virus 1 (HSV-1). To test this for the related alphaherpesvirus pseudorabies virus (PrV), we mutated genes encoding VAPB and VAPA by CRISPR/Cas9-based genome editing in our standard rabbit kidney cells (RK13), either individually or in combination. Single as well as double knockout cells were tested for virus propagation and for defects in nuclear egress. However, no deficiency in virus replication nor any effect on nuclear egress was obvious suggesting that VAPB and VAPA do not play a significant role in this process during PrV infection in RK13 cells.

Host and Viral Factors Involved in Nuclear Egress of Herpes Simplex Virus 1.

Herpes simplex virus 1 (HSV-1) replicates its genome and packages it into capsids within the nucleus. HSV-1 has evolved a complex mechanism of nuclear egress whereby nascent capsids bud on the inner nuclear membrane to form perinuclear virions that subsequently fuse with the outer nuclear membrane, releasing capsids into the cytosol. The viral-encoded nuclear egress complex (NEC) plays a crucial role in this vesicle-mediated nucleocytoplasmic transport. Nevertheless, similar system mediates the movement of other cellular macromolecular complexes in normal cells. Therefore, HSV-1 may utilize viral proteins to hijack the cellular machinery in order to facilitate capsid transport. However, little is known about the molecular mechanisms underlying this phenomenon. This review summarizes our current understanding of the cellular and viral factors involved in the nuclear egress of HSV-1 capsids.

Differentiating the Roles of UL16, UL21, and Us3 in the Nuclear Egress of Herpes Simplex Virus Capsids

Viral proteins pUL16 and pUL21 are required for efficient nuclear egress of herpes simplex virus 2 capsids. To better understand the role of these proteins in nuclear egress, we established whether nuclear egress complex (NEC) distribution and/or function was altered in the absence of either pUL16 or pUL21. NEC distribution in cells infected with pUL16-deficient viruses was indistinguishable from that observed in cells infected with wild-type viruses. In contrast, NEC distribution was aberrant in cells infected with pUL21-deficient virus and, instead, showed some similarity to the aberrant NEC distribution pattern observed in cells infected with pUs3-deficient virus. These results indicated that pUL16 plays a role in nuclear egress that is distinct from that of pUL21 and pUs3. Higher-resolution examination of nuclear envelope ultrastructure in cells infected with pUL21-deficient viruses by transmission electron microscopy showed different types of nuclear envelope perturbations, including some that were not observed in cells infected with pUs3 deficient virus. The formation of the nuclear envelope perturbations observed in pUL21-deficient virus infections was dependent on a functional NEC, revealing a novel role for pUL21 in regulating NEC activity. The results of comparisons of nuclear envelope ultrastructure in cells infected with viruses lacking pUs3, pUL16, or both pUs3 and pUL16 were consistent with a role for pUL16 in advance of primary capsid envelopment and shed new light on how pUs3 functions in nuclear egress.IMPORTANCE The membrane deformation activity of the herpesvirus nuclear egress complex (NEC) allows capsids to transit through both nuclear membranes into the cytoplasm. NEC activity must be precisely controlled during viral infection, and yet our knowledge of how NEC activity is controlled is incomplete. To determine how pUL16 and pUL21, two viral proteins required for nuclear egress of herpes simplex virus 2, function in nuclear egress, we examined how the lack of each protein impacted NEC distribution. These analyses revealed a function of pUL16 in nuclear egress distinct from that of pUL21, uncovered a novel role for pUL21 in regulating NEC activity, and shed new light on how a viral kinase, pUs3, regulates nuclear egress. Nuclear egress of capsids is required for all herpesviruses. A complete understanding of all aspects of nuclear egress, including how viral NEC activity is controlled, may yield strategies to disrupt this process and aid the development of herpes-specific antiviral therapies.

Molecular mechanisms of entry and egress of herpes simplex virus 1

Molecular mechanisms of entry and egress of herpes simplex virus 1

Prediction of conserved sites and domains in glycoproteins B, C and D of herpes viruses

Glycoprotein B (gB), C (gC) and D (gD) of herpes simplex virus are implicated in virus adsorption and penetration. The gB, gC and gD are glycoproteins for different processes of virus binding and attachment to the host cells. Moreover, their expression is necessary and sufficient to induce cell fusion in the absence of other glycoproteins. Egress of herpes simplex virus (HSV) and other herpes viruses from cells involves extensive modification of cellular membranes and sequential envelopment, de-envelopment and re-envelopment steps. Viral glycoproteins are important in these processes, and frequently two or more glycoproteins can largely suffice in any step. Hence, we target the 3 important glycoproteins (B, C and D) of eight different herpes viruses of different species. These species include human (HSV1 and 2), bovine (BHV1), equine (EHV1 and 4), chicken (ILT1 and MDV2) and pig (PRV1). By applying different bioinformatics tools, we highlighted the conserved sites in these glycoproteins which might be most significant regarding attachment and infection of the viruses. Moreover the conserved domains in these glycoproteins are also highlighted. From this study, we will able to analyze the role of different viral glycoproteins of different species during herpes virus adsorption and penetration. Moreover, this study will help to construct the antivirals that target the glycoproteins of different herpes viruses.

Virus Assembly and Egress of HSV.

The assembly and egress of herpes simplex virus (HSV) is a complicated multistage process that involves several different cellular compartments and the activity of many viral and cellular proteins. The process begins in the nucleus, with capsid assembly followed by genome packaging into the preformed capsids. The DNA-filled capsids (nucleocapsids) then exit the nucleus by a process of envelopment at the inner nuclear membrane followed by fusion with the outer nuclear membrane. In the cytoplasm nucleocapsids associate with tegument proteins, which form a complicated protein network that links the nucleocapsid to the cytoplasmic domains of viral envelope proteins. Nucleocapsids and associated tegument then undergo secondary envelopment at intracellular membranes originating from late secretory pathway and endosomal compartments. This leads to assembled virions in the lumen of large cytoplasmic vesicles, which are then transported to the cell periphery to fuse with the plasma membrane and release virus particles from the cell. The details of this multifaceted process are described in this chapter.

The Torsin Activator LULL1 Is Required for Efficient Growth of Herpes Simplex Virus 1.

TorsinA is a membrane-tethered AAA+ ATPase implicated in nuclear envelope dynamics as well as the nuclear egress of herpes simplex virus 1 (HSV-1). The activity of TorsinA and the related ATPase TorsinB strictly depends on LAP1 and LULL1, type II transmembrane proteins that are integral parts of the Torsin/cofactor AAA ring, forming a composite, membrane-spanning assembly. Here, we use CRISPR/Cas9-mediated genome engineering to create single- and double knockout (KO) cell lines of TorA and TorB as well as their activators, LAP1 and LULL1, to investigate the effect on HSV-1 production. Consistent with LULL1 being the more potent Torsin activator, a LULL1 KO reduces HSV-1 growth by one order of magnitude, while the deletion of other components of the Torsin system in combination causes subtle defects. Notably, LULL1 deficiency leads to a 10-fold decrease in the number of viral genomes per host cell without affecting viral protein production, allowing us to tentatively assign LULL1 to an unexpected role that precedes HSV-1 nuclear egress. In this study, we conduct the first comprehensive genetic and phenotypic analysis of the Torsin/cofactor system in the context of HSV-1 infection, establishing LULL1 as the most important component of the Torsin system with respect to viral production.

Myosin Va Enhances Secretion of Herpes Simplex Virus 1 Virions and Cell Surface Expression of Viral Glycoproteins

The final step in the egress of herpes simplex virus (HSV) virions requires virion-laden vesicles to bypass cortical actin and fuse with the plasma membrane, releasing virions into the extracellular space. Little is known about the host or viral proteins involved. In the current study, we noted that the conformation of myosin Va (myoVa), a protein known to be involved in melanosome and secretory granule trafficking to the plasma membrane in melanocytes and neuroendocrine cells, respectively, was altered by 4 h after infection with HSV-1 such that an N-terminal epitope expected to be masked in its inactive state was rendered immunoreactive. Wild-type myoVa localized throughout the cytoplasm and to a limited extent in the nuclei of HSV-infected cells. Two different dominant negative myoVa molecules containing cargo-binding domains but lacking the lever arms and actin-binding domains colocalized with markers of the trans-Golgi network (TGN). Expression of dominant negative myoVa isoforms reduced secretion of HSV-1 infectivity into the medium by 50 to 75%, reduced surface expression of glycoproteins B, M, and D, and increased intracellular virus infectivity to levels consistent with increased retention of virions in the cytoplasm. These data suggest that myoVa is activated during HSV-1 infection to help transport virion- and glycoprotein-laden vesicles from the TGN, through the cortical actin, to the plasma membrane. We cannot exclude a role for myoVa in promoting fusion of these vesicles with the inner surface of the plasma membrane. These data also indicate that myoVa is involved in exocytosis in human epithelial cells as well as other cell types.

Transport and egress of herpes simplex virus in neurons

The mechanisms of axonal transport of the alphaherpesviruses, HSV and pseudorabies virus (PrV), in neuronal axons are of fundamental interest, particularly in comparison with other viruses, and offer potential sites for antiviral intervention or development of gene therapy vectors. These herpesviruses are transported rapidly along microtubules (MTs) in the retrograde direction from the axon terminus to the dorsal root ganglion and then anterogradely in the opposite direction. Retrograde transport follows fusion and deenvelopment of the viral capsid at the axonal membrane followed by loss of most of the tegument proteins and then binding of the capsid via one or more viral proteins (VPs) to the retrograde molecular motor dynein. The HSV capsid protein pUL35 has been shown to bind to the dynein light chain Tctex1 but is likely to be accompanied by additional dynein binding of an inner tegument protein. The mechanism of anterograde transport is much more controversial with different processes being claimed for PrV and HSV: separate transport of HSV capsid/tegument and glycoproteins versus PrV transport as an enveloped virion. The controversy has not been resolved despite application, in several laboratories, of confocal microscopy (CFM), real-time fluorescence with viruses dual labelled on capsid and glycoprotein, electron microscopy in situ and immuno-electron microscopy. Different processes for each virus seem counterintuitive although they are the most divergent in the alphaherpesvirus subfamily. Current hypotheses suggest that unenveloped HSV capsids complete assembly in the axonal growth cones and varicosities, whereas with PrV unenveloped capsids are only found travelling in a retrograde direction.

Herpes simplex virus gene products: the accessories reflect her lifestyle well

Herpes simplex virus (HSV) genomic DNA contains at least 74 distinct genes. A set of 40 genes, termed the 'core genes', is commonly found in all herpesviruses; their products include four capsid proteins, six DNA replication proteins, seven DNA packaging/cleavage proteins, four envelope glycoproteins, as well as several others. Although approximately half of the HSV genes are not essential for replication in cell cultures, all accessory gene products are predicted to play indispensable roles for viral replication and dissemination in vivo. Intensive studies have been undertaken to elucidate the functions and roles of HSV gene products, and we are now able to address, at least partially, the biological aspects of all HSV encoded proteins. This article is a brief summary of our present knowledge of the functions and roles of HSV gene products with special attention focused on UL14, UL34, UL51, UL56 and US3, all of which are thought to be involved in HSV egress. Furthermore, efforts are discussed to generate replication-competent HSV lacking a single or multiple accessory gene(s) for potential use in gene therapy or as anti-cancer therapeutics. Finally, specific HSV gene products are being explored as therapeutic agents.

Antigenic Variation of Varicella Zoster Virus Fc Receptor gE: Loss of a Major B Cell Epitope in the Ectodomain

Varicella zoster virus (VZV) is considered to possess a genetically stable genome; only one serotype is recognized around the world. The 125-kbp genome contains ∼70 open reading frames. One that has received particular attention is open reading frame 68, which codes for glycoprotein gE, the predominant 623-residue viral envelope product that harbors both B and T cell epitopes. This report describes the initial characterization of a community-acquired VZV isolate that was a distinguishable second serotype (i.e., it had lost a major B cell epitope defined on the gE ectodomain by a murine monoclonal antibody called mAb 3B3). The mAb 3B3 epitope was found not only on the prototype sequenced Dumas strain from Holland and all previously tested North American isolates but also on the varicella vaccine Oka strain originally attenuated in Japan. Sequencing of the mutated gE ectodomain demonstrated that codon 150 exhibited a single base change that led to an amino acid change (aspartic acid to asparagine). Observation of the monolayers infected with the mutant VZV strain also led to the surprising discovery that the topography of egress was altered. Wild-type VZV emerges along distinctive viral highways, whereas the mutant strain virions were nearly uniformly distributed over the cell surface in a pattern more closely resembling egress of herpes simplex virus 1. The mutant VZV strain was designated VZV-MSP because it was isolated in Minnesota.

Monensin inhibits the processing of herpes simplex virus glycoproteins, their transport to the cell surface, and the egress of virions from infected cells

HEp-2 cells or Vero cells infected with herpes simplex virus type 1 were exposed to the ionophore monensin, which is thought to block the transit of membrane vesicles from the Golgi apparatus to the cell surface. We found that yields of extracellular virus were reduced to less than 0.5% of control values by 0.2 microM monensin under conditions that permitted accumulation of cell-associated infectious virus at about 20% of control values. Viral protein synthesis was not inhibited by monensin, whereas late stages in the post-translational processing of the viral glycoproteins were blocked. The transport of viral glycoproteins to the cell surface was also blocked by monensin. Although the assembly of nucleocapsids appeared to be somewhat inhibited in monensin-treated cells, electron microscopy revealed that nucleocapsids were enveloped to yield virions, and electrophoretic analyses showed that the isolated virions contained immature forms of the envelope glycoproteins. Most of the virions which were assembled in monensin-treated cells accumulated in large intracytoplasmic vacuoles, whereas most of the virions produced by and associated with untreated cells were found attached to the cell surface. Our results implicate the Golgi apparatus in the egress of herpes simplex virus from infected cells and also suggest that complete processing of the viral envelope glycoproteins is not essential for nucleocapsid envelopment or for virion infectivity.

Concerning the egress of herpes simplex virus from infected cells: Electron and light microscope observations

Electron microscopic studies of the cytoplasm of HEp-2 cells productively infected with herpes simplex virus revealed a network of branched tubules 65 nm in diameter. The membranes limiting the tubules were continuous at one end with the outer lamellae of the nuclear membrane and at the other end with the cytoplasmic membrane. At 8 hours after infection enveloped nucleocapsids appeared at the junction of the tubules with the nucleus; tubules were filled with enveloped nucleocapsids 16 hours after infection. Branched fibrils were seen in infected cells with phase optics; they stained with conjugated human convalescent antibody in immunofluorescence tests. The ducts as seen by the electron microscope and fibrils as seen by light microscopy were absent from uninfected cells and from DK cells abortively infected with the same virus. Cross sections of the tubules appear as vacuoles; the finding of “vacuoles” containing virus led in the past to the erroneous conclusion that they serve to transport enveloped virus to the extracellular fluid by “reverse phagocytosis.” It is suggested that the tubules function as a third compartment of the cell to (1) prevent the uncoating of the virus in the cytoplasm and (2) furnish a pathway for egress from the site of envelopment to the extracellular fluid.