Reconstitution of herpes simplex virus type 1 nuclear capsid egress in vitro

Newly assembled herpesvirus capsids travel from the nucleus to the plasma membrane by a mechanism that is poorly understood. Furthermore, the contribution of cellular proteins to this egress has yet to be clarified. To address these issues, an in vitro nuclear egress assay that reproduces the exit of herpes simplex virus type 1 (HSV-1) capsids from nuclei isolated from infected cells was established. As expected, the assay has all the hallmarks of intracellular transport assays, namely, a dependence on time, energy, and temperature. Surprisingly, it is also dependent on cytosol and was slightly enhanced by infected cytosol, suggesting an implication of both host and viral proteins in the process. The capsids escaped these nuclei by budding through the inner nuclear membrane, accumulated as enveloped capsids between the two nuclear membranes, and were released in cytosol exclusively as naked capsids, exactly as in intact cells. This is most consistent with the view that the virus escapes by crossing the two nuclear membranes rather than through nuclear pores. Unexpectedly, nuclei isolated at the nonpermissive temperature from cells infected with a U(L)26 thermosensitive protease mutant (V701) supported capsid egress. Although electron microscopy, biochemical, and PCR analyses hinted at a likely reconstitution of capsid maturation, DNA encapsidation could not be confirmed by a traditional SQ test. This assay should prove very useful for identification of the molecular players involved in HSV-1 nuclear egress.     

Herpes simplex type 1 activation by Epstein-Barr virus nuclear antigen 1

We have investigated the effect of Epstein-Barr virus nuclear antigen 1 (EBNA-1), a nuclear protein encoded by EBV, on herpes simplex virus type 1 (HSV-1) infection either in cells constitutively expressing EBNA-1 or in transient expression assays. Rat-1 cells and rat embryo fibroblasts (REF) immortalized by c-myc or E1A were transfected with a specific EBV DNA fragment coding for EBNA-1. Cloned cell lines which constitutively expressed this antigen were infected with HSV-1. Our results indicate that in EBNA-1-expressing cells, virus growth was higher than in control cells for different virus strains or rodent cell lines. This increase was maximal when cells were infected at low multiplicity, as determined by virus growth, and correlated with the stimulation of viral DNA synthesis. REF + c-myc and Vero cells were contransfected by an EBNA-1 expression vector driven by Moloney murine leukaemia virus LTR and HSV-1 immediate-early (α0) or early thymidine kinase upstream promoter regulatory regions linked to chloramphenicol acetyltransferase (CAT) coding sequences as effectors. In both cell lines, stimulation of CAT expression by EBNA-1 was observed only with the immediate-early promoter. These results suggest that EBNA-1 can transactivate immediate-early HSV-1 expression.     

The mode of entry of herpes simplex virus type 1 into Vero cells

The mode of entry of herpes simplex virus type 1 (HSV-1) into Vero cells was investigated quantitatively with biological techniques. The entry of virus occurred rapidly when the virus-adsorbed cells were incubated at 37 C. The kinetics of virus entry was found to be similar to that of the process of uncoating, indicating that the uncoating of HSV-1 occurs simultaneously with the entry of virus into the cell. Experiments with ammonium chloride revealed that acidity in endosomes is not necessary for the entry or uncoating of HSV-1, in contrast with the cases of enveloped RNA viruses. In addition, endocytosis of the virus seems to be one of the processes of entry for HSV-1. However, the kinetics of endocytosis showed that the cell-bound virus is endocytosed gradually and suggested that the endocytosis of HSV-1 does not lead the virus to an uncoating process. These results are most consistent with a mechanism of entry for HSV-1 involving fusion of the viral envelope with the plasma membrane of the host cell.     

Single hepatitis-B virus core capsid binding to individual nuclear pore complexes in Hela cells

We investigate the interaction of hepatitis B virus capsids lacking a nuclear localization signal with nuclear pore complexes (NPCs) in permeabilized HeLa cells. Confocal and wide-field optical images of the nuclear envelope show well-spaced individual NPCs. Specific interactions of capsids with single NPCs are characterized by extended residence times of capsids in the focal volume which are characterized by fluorescence correlation spectroscopy. In addition, single-capsid-tracking experiments using fast wide-field fluorescence microscopy at 50 frames/s allow us to directly observe specific binding via a dual-color colocalization of capsids and NPCs. We find that binding occurs with high probability on the nuclear-pore ring moiety, at 44 ± 9 nm radial distance from the central axis.     

Herpes simplex virus ICP27 protein directly interacts with the nuclear pore complex through Nup62, inhibiting host nucleocytoplasmic transport pathways

The herpes simplex virus ICP27 protein is important for the expression and nuclear export of viral mRNAs. Although several binding sites have been mapped along the ICP27 sequence for various RNA and protein partners, including the transport receptor TAP of the host cell nuclear transport machinery, several aspects of ICP27 trafficking through the nuclear pore complex remain unclear. We investigated if ICP27 could interact directly with the nuclear pore complex itself, finding that ICP27 directly binds the core nucleoporin Nup62. This is confirmed through co-immunoprecipitation and in vitro binding assays with purified components. Mapping with ICP27 deletion and point mutants further shows that the interaction requires sequences in both the N and C termini of ICP27. Expression of wild type ICP27 protein inhibited both classical, importin α/β-dependent and transportin-dependent nuclear import. In contrast, an ICP27 point mutant that does not interact with Nup62 had no such inhibitory effect. We suggest that ICP27 association with Nup62 provides additional binding sites at the nuclear pore for ICP27 shuttling, thus supporting ICP27-mediated transport. We propose that ICP27 competes with some host cell transport receptors for binding, resulting in inhibition of those host transport pathways.     

Kinesin-1-mediated capsid disassembly and disruption of the nuclear pore complex promote virus infection

Many viruses deliver their genomes into the host cell nucleus for replication. However, the size restrictions of the nuclear pore complex (NPC), which regulates the passage of proteins, nucleic acids, and solutes through the nuclear envelope, require virus capsid uncoating before viral DNA can access the nucleus. We report a microtubule motor kinesin-1-mediated and NPC-supported mechanism of adenovirus uncoating. The capsid binds to the NPC filament protein Nup214 and kinesin-1 light-chain Klc1/2. The nucleoporin Nup358, which is bound to Nup214/Nup88, interacts with the kinesin-1 heavy-chain Kif5c to indirectly link the capsid to the kinesin motor. Kinesin-1 disrupts capsids docked at Nup214, which compromises the NPC and dislocates nucleoporins and capsid fragments into the cytoplasm. NPC disruption increases nuclear envelope permeability as indicated by the nuclear influx of large cytoplasmic?dextran polymers. Thus, kinesin-1 uncoats viral DNA?and compromises NPC integrity, allowing viral genomes nuclear access to promote infection.     

Herpes simplex virus type 1 DNA is immunostimulatory in vitro and in vivo

Recently, prokaryotic DNAs containing unmethylated CpG motifs have been shown to be intrinsically immunostimulatory both in vitro and in vivo, tending to promote Th1-like responses. In contrast, CpG dinucleotides in mammalian DNAs are extensively methylated on cytosines and hence immunologically inert. Since the herpes simplex virus (HSV) genome is unmethylated and G+C rich, we predicted that CpG motifs would be highly prevalent in the HSV genome; hence, we examined the immunostimulatory potential of purified HSV DNA in vitro and in vivo. Mouse splenocyte cultures treated with HSV DNA or HSV-derived oligodeoxyribonucleotides (ODNs) showed strong proliferative responses and production of inflammatory cytokines (gamma interferon [IFN-γ], tumor necrosis factor [TNF], and interleukin-6 [IL-6]) in vitro, whereas splenocytes treated with mammalian CV-1 DNA or non-CpG ODN did not. After immunization with ovalbumin (OVA), only splenocytes from mice immunized with HSV DNA or HSV-ODN as the adjuvants proliferated strongly and produced typical Th1 responses, including CD8⁺ cytotoxic T-lymphocyte responses, upon restimulation with OVA. Furthermore, HSV-ODN synergized with IFN-γ to induce nitric oxide (NO), IL-6, and TNF production from macrophages. These results demonstrate that HSV DNA and HSV-ODN are immunostimulatory, driving potent Th1 responses both in vitro and in vivo. Considering that HSV DNA has been found to persist in nonneuronal cells, these results fuel speculation that HSV DNA might play a role in pathogenesis, in particular, in diseases like herpes stromal keratitis (HSK) that involve chronic inflammatory responses in the absence of virus or viral antigens.     

Herpes simplex virus binding and entry modulate cell surface protein mobility.

Fluorescence photobleaching recovery measurements showed that herpes simplex virus type 1 attachment to target cells rapidly induced an anchorage modulation of cell surface protein mobility, an activity mediated by the cytoskeleton and associated with the multivalent attachment of other ligands (e.g., cells, lectins, or anti-immunoglobulin) to cell surfaces. The restriction in cell surface protein mobility was released concurrently with virus penetration. The effects of attachment and penetration on cell surface protein mobility and cytoskeletal function are some of the earliest cellular changes induced by herpes simplex virus infection.     

Herpes simplex virus entry mediator associates in infected cells in a complex with viral proteins gD and at least gH

We examined herpes simplex virus (HSV)-infected human HEp-2 cells or porcine cells that express herpes virus entry mediator (HVEM) for virus and receptor protein interactions. Antibody to HVEM, or its viral ligand gD, coimmunoprecipitated several similar proteins. A prominent 110-kDa protein that coprecipitated was identified as gH. The HVEM/gD/gH complex was detected with mild or stringent cell lysis conditions. It did not form in cells infected with HSV-1(KOS)Rid1 virus or with null virus lacking gD, gH, or gL. Thus, in cells a complex forms through physical associations of HVEM, gD, and at least gH.     

Subunit stoichiometry of human immunodeficiency virus type 1 envelope glycoprotein trimers during virus entry into host cells

The envelope glycoproteins of human immunodeficiency virus type 1 (HIV-1) function as a homotrimer of gp120/gp41 heterodimers to support virus entry. During the process of virus entry, an individual HIV-1 envelope glycoprotein trimer binds the cellular receptors CD4 and CCR5/CXCR4 and mediates the fusion of the viral and the target cellular membranes. By studying the function of heterotrimers between wild-type and nonfunctional mutant envelope glycoproteins, we found that two wild-type subunits within an envelope glycoprotein trimer are required to support virus entry. Complementation between HIV-1 envelope glycoprotein mutants defective in different functions to allow virus entry was not evident. These results assist our understanding of the mechanisms whereby the HIV-1 envelope glycoproteins mediate virus entry and membrane fusion and guide attempts to inhibit these processes.     

Herpes simplex virus type 1 helicase-primase: DNA binding and consequent protein oligomerization and primase activation

The heterotrimeric helicase-primase complex of herpes simplex virus type I (HSV-1), consisting of UL5, UL8, and UL52, possesses 5' to 3' helicase, single-stranded DNA (ssDNA)-dependent ATPase, primase, and DNA binding activities. In this study we confirm that the UL5-UL8-UL52 complex has higher affinity for forked DNA than for ssDNA and fails to bind to fully annealed double-stranded DNA substrates. In addition, we show that a single-stranded overhang of greater than 6 nucleotides is required for efficient enzyme loading and unwinding. Electrophoretic mobility shift assays and surface plasmon resonance analysis provide additional quantitative information about how the UL5-UL8-UL52 complex associates with the replication fork. Although it has previously been reported that in the absence of DNA and nucleoside triphosphates the UL5-UL8-UL52 complex exists as a monomer in solution, we now present evidence that in the presence of forked DNA and AMP-PNP, higher-order complexes can form. Electrophoretic mobility shift assays reveal two discrete complexes with different mobilities only when helicase-primase is bound to DNA containing a single-stranded region, and surface plasmon resonance analysis confirms larger amounts of the complex bound to forked substrates than to single-overhang substrates. Furthermore, we show that primase activity exhibits a cooperative dependence on protein concentration while ATPase and helicase activities do not. Taken together, these data suggest that the primase activity of the helicase-primase requires formation of a dimer or higher-order structure while ATPase activity does not. Importantly, this provides a simple mechanism for generating a two-polymerase replisome at the replication fork.     

Herpes simplex virus: receptors and ligands for cell entry

Entry of herpes simplex virus (HSV) into cells depends upon multiple cell surface receptors and multiple proteins on the surface of the virion. The cell surface receptors include heparan sulphate chains on cell surface proteoglycans, a member of the tumor necrosis factor (TNF) receptor family and two members of the immunoglobulin superfamily related to the poliovirus receptor. The HSV ligands for these receptors are the envelope glycoproteins gB and gC for heparan sulphate and gD for the protein receptors and specific sites in heparan sulphate generated by certain 3-O-sulfotransferases. HSV gC also binds to the C3b component of complement and can block complement-mediated neutralization of virus. The purposes of this review are to summarize available information about these cell surface receptors and the viral ligands, gC and gD, and to discuss roles of these viral glycoproteins in immune evasion and cellular responses as well as in viral entry.     

In vitro nuclear egress of herpes simplex virus type 1 capsids

During their life cycles, viruses typically undergo many transport events throughout the cell. These events depend on a variety of both viral and host proteins and are often not fully understood. Such studies are often complicated by asynchronous infections and the concurrent presence of various viral intermediates in the cells, making it difficult to molecularly define each step. In the case of the herpes simplex virus type 1, the etiological agent of cold sores and many other illnesses, the viral particles undergo an intricate series of transport steps during its life cycle. Upon entry by fusion with a cellular membrane, they travel to the host cell nucleus where the virus replicates and assembles new viral particles. These particles then travel across the two nuclear envelopes and transit through the trans-Golgi network before finally being transported to and released at the cell surface. Though viral components and some host proteins modulating these numerous transport events have been identified, the details of these processes remain to be elucidated. To specifically address how the virus escapes the nucleus, we set up an in vitro model that reproduces the unconventional route used by herpes simplex type 1 virus to leave nuclei. This has not only allowed us to clarify the route of capsid egress of the virus but is now useful to define it at the molecular level.     

Mechanics of enveloped virus entry into host cells

Enveloped viruses such as HIV-1 enter their hosts by first establishing a contact region at the cell surface, which is stabilized by the formation of receptor-ligand complexes. We show that the favorable contact energy stemming from the formation of the receptor complexes in the interaction zone is sufficient to drive the engulfment of the virus by the cell. Using a continuum model, we show that the equilibrium engulfment depth and the force driving the engulfment are functions of the virus size and the complex formation energy. Resistance to engulfment is dominated by the elastic deformation of the cytoskeleton.     

Phospholipid scramblase 1 mediates hepatitis C virus entry into host cells

Hepatitis C virus (HCV) infects human hepatocytes through several host factors. However, other prerequisite factors for viral entry remain to be identified. Using a yeast two-hybrid screen, we found that human phospholipid scramblase 1 interacts with HCV envelope proteins E1 and E2. These physical interactions were confirmed by co-immunoprecipitation and GST pull-down assays. Knocking down the expression of PLSCR1 inhibited the entry of HCV pseudoparticles. Moreover, PLSCR1 was required for the initial attachment of HCV onto hepatoma cells, where it specifically interacted with entry factor OCLN. We show that PLSCR1 is a novel attachment factor for HCV entry.     

Herpes simplex virus type 1 origin-specific binding protein: oriS-binding properties and effects of cellular proteins.

The herpes simplex virus type 1 (HSV-1) origin of replication, oriS, contains three highly homologous sequences, sites I, II, and III. The HSV-1 origin-binding protein (OBP), the product of the UL9 gene, has been shown to bind specifically to sites I and II. In this study, gel shift analysis was used to characterize interactions between site I DNA and proteins in infected and uninfected cell extracts. The formation of two protein-DNA complexes, bands A and B, was demonstrated with infected cell extracts, and one predominant protein-DNA complex, band M, was identified with mock-infected extracts. Protein interactions with the highly homologous site II and III DNAs were also characterized. Incubation of infected cell extracts with the lower-affinity site II DNA as a probe resulted in the appearance of two protein-DNA complexes with mobilities identical to those of the A and B complexes, while incubation with site III DNA resulted in the formation of a single complex with the mobility of band B; no A-like band was observed. Incubation of high concentrations of partially purified OBP with site I DNA resulted in the formation of two novel complexes, bands 9-1 and 9-2. Addition of uninfected or HSV-1-infected cell extracts to the purified OBP-site I DNA mix significantly enhanced the formation of complex 9-1. The enhanced formation of complex 9-1 by uninfected cell extracts implicates a cellular factor or factors in the formation or stabilization of the OBP-site I DNA complex.