Entry of Herpes Simplex Virus Type 1 (HSV-1) into the Distal Axons of Trigeminal Neurons Favors the Onset of Nonproductive, Silent Infection

Following productive, lytic infection in epithelia, herpes simplex virus type 1 (HSV-1) establishes a lifelong latent infection in sensory neurons that is interrupted by episodes of reactivation. In order to better understand what triggers this lytic/latent decision in neurons, we set up an organotypic model based on chicken embryonic trigeminal ganglia explants (TGEs) in a double chamber system. Adding HSV-1 to the ganglion compartment (GC) resulted in a productive infection in the explants. By contrast, selective application of the virus to distal axons led to a largely nonproductive infection that was characterized by the poor expression of lytic genes and the presence of high levels of the 2.0-kb major latency-associated transcript (LAT) RNA. Treatment of the explants with the immediate-early (IE) gene transcriptional inducer hexamethylene bisacetamide, and simultaneous co-infection of the GC with HSV-1, herpes simplex virus type 2 (HSV-2) or pseudorabies virus (PrV) helper virus significantly enhanced the ability of HSV-1 to productively infect sensory neurons upon axonal entry. Helper-virus-induced transactivation of HSV-1 IE gene expression in axonally-infected TGEs in the absence of de novo protein synthesis was dependent on the presence of functional tegument protein VP16 in HSV-1 helper virus particles. After the establishment of a LAT-positive silent infection in TGEs, HSV-1 was refractory to transactivation by superinfection of the GC with HSV-1 but not with HSV-2 and PrV helper virus. In conclusion, the site of entry appears to be a critical determinant in the lytic/latent decision in sensory neurons. HSV-1 entry into distal axons results in an insufficient transactivation of IE gene expression and favors the establishment of a nonproductive, silent infection in trigeminal neurons

Deletion of the Herpes Simplex Virus VP22-Encoding Gene (UL49) Alters the Expression, Localization, and Virion Incorporation of ICP0

The role of the herpes simplex virus tegument protein VP22 is not yet known. Here we describe the characterization of a virus in which the entire VP22 open reading frame has been deleted. We show that VP22 is not essential for virus growth but that virus lacking VP22 (Δ22) displays a cell-specific replication defect in epithelial MDBK cells. Virus particles assembled in the absence of VP22 show few obvious differences to wild-type (WT) particles, except for a moderate reduction in glycoproteins gD and gB. In addition, the Δ22 virus exhibits a general delay in the initiation of virus protein synthesis, but this is not due to a glycoprotein-related defect in virus entry. Intriguingly, however, the absence of VP22 has an obvious effect on the intracellular level of the immediate-early (IE) protein ICP0. Moreover, following translocation from the nucleus to the cytoplasm, ICP0 is unable to localize to the characteristic cytoplasmic sites observed in a WT infection. We demonstrate that, in WT-infected cells, VP22 and ICP0 are concentrated in the same cytoplasmic sites. Furthermore, we show that, while ICP0 and ICP4 are components of WT extracellular virions, the altered localization of ICP0 in the cytoplasm of Δ22-infected cells correlates with an absence of both ICP0 and ICP4 from Δ22 virions. Hence, while a role has not yet been defined for virion IE proteins in virus infection, our results suggest that their incorporation is a specific event requiring the tegument protein VP22. This report provides the first direct evidence that VP22 influences virus assembly

Herpes Simplex Virus Tegument Protein VP22 Contains an Internal VP16 Interaction Domain and a C-Terminal Domain That Are Both Required for VP22 Assembly into the Virus Particle

Many steps along the herpesvirus assembly and maturation pathway remain unclear. In particular, the acquisition of the virus tegument is a poorly understood process, and the molecular interactions involved in tegument assembly have not yet been defined. Previously we have shown that the two major herpes simplex virus tegument proteins VP22 and VP16 are able to interact, although the relevance of this to virus assembly is not clear. Here we have constructed a number of recombinant viruses expressing N- and C-terminal truncations of VP22 and have used them to identify regions of the protein involved in its assembly into the virus structure. Analysis of the packaging of these VP22 variants into extracellular virions revealed that the C terminus of VP22 is absolutely required for this process, with removal of the C-terminal 89 residues abrogating its incorporation. However, while these 89 residues alone were sufficient for specific incorporation of small amounts of VP22 into the tegument, efficient packaging of VP22 to the levels of full-length protein required an additional 52 residues of the protein. Coimmunoprecipitation assays indicated that these 52 residues also contained the interaction domain for VP16. Furthermore, analysis of the subcellular localization of the mutant forms of VP22 revealed that only those truncations that were efficiently assembled formed characteristic cytoplasmic trafficking complexes, suggesting that these complexes may represent the cellular location for VP22 assembly into the virus. Taken together, these results suggest that there are two determinants involved in the packaging of VP22—a C-terminal domain and an internal VP16 interaction domain, both of which are required for the efficient recruitment of VP22 to sites of virus assembly

Evaluation of Confirmatory Strategies for Detection of Type-Specific Antibodies against Herpes Simplex Virus Type 2

In this study, the optimal combination of three commercial glycoprotein G-2 (gG-2)-based herpes simplex virus type 2 (HSV-2) type-specific enzyme-linked immunosorbent assays (Euroimmun anti-HSV-2 immunoglobulin G [IgG] ELISA [Eu2], Gull HSV-2-specific IgG ELISA [Gu2], and Radim HSV-2 IgG ELISA [Ra2]) and one gG-2-based HSV-2-specific immunoblot (Euroimmun anti-HSV-1/HSV-2 gG Western blot [EuW]) was determined with regard to diagnostic performance and cost efficiency. Two hundred fifty serum samples were included in this study, 194 of which were from female prostitutes. When a formal primary “gold standard” was defined based on majority agreement of the commercial tests, with EuW being decisive in stand-off situations, the sensitivity and specificity of the assays in the samples from prostitutes were as follows: Eu2, 100 and 89.22%; Gu2, 94.44 and 96.08%; Ra2, 61.18 and 95.10%; and EuW, 98.90 and 100%. The most cost-effective confirmatory strategy in the samples from prostitutes was screening with Eu2, retesting positive and equivocal samples with Gu2, and resolving the remaining discordant results with EuW (estimated additional costs per sample, 79.02%; sensitivity, 100%; positive predictive value, 96.81%). Applying a self-developed gG-2-independent assay to the discordant and concordant negative samples in the samples from prostitutes suggested that the primary gold standard may have missed six HSV-2-positive samples. In conclusion, confirmatory strategies based on commercial gG-2-dependent seroassays result in an increase in the specificity of HSV-2-specific serology. However, further improvement of the sensitivity of current HSV-2-specific serology may require the additional exploitation of the gG-2-independent type-specific antibody response

Cutaneous RANK–RANKL Signaling Upregulates CD8-Mediated Antiviral Immunity during Herpes simplex Virus Infection by Preventing Virus-Induced Langerhans Cell Apoptosis

Herpes simplex virus-type 1 (HSV-1) causes the majority of cutaneous viral infections. Viral infections are controlled by the immune system, and CD8+ cytotoxic T-lymphocytes (CTLs) have been shown to be crucial during the clearance of HSV-1 infections. Although epidermal Langerhans cells (LCs) are the first dendritic cells (DCs) to come into contact with the virus, it has been shown that the processing of viral antigens and the differentiation of antiviral CTLs are mediated by migratory CD103+ dermal DCs and CD8α+ lymph node–resident DCs. In vivo regulatory T-cells (Tregs) are implicated in the regulation of antiviral immunity and we have shown that signaling via the receptor activator of NF-κB (RANK) and its ligand RANKL mediates the peripheral expansion of Tregs. However, in addition to expanding Tregs, RANK–RANKL interactions are involved in the control of antimicrobial immunity by upregulating the priming of CD4+ effector T cells in LCMV infection or by the generation of parasite-specific CD8+ T cells in Trypanosoma cruzi infection. Here, we demonstrate that cutaneous RANK–RANKL signaling is critical for the induction of CD8-mediated antiviral immune responses during HSV-1 infection of the skin by preventing virus-induced LC apoptosis, improving antigen transport to regional lymph nodes, and increasing the CTL priming capacity of lymph node DCs

Broadly Applicable, Virus-Free Dual Reporter Assay to Identify Compounds Interfering with Membrane Fusion: Performance for HSV-1 and SARS-CoV-2

Membrane fusion constitutes an essential step in the replication cycle of numerous viral pathogens, hence it represents an important druggable target. In the present study, we established a virus-free, stable reporter fusion inhibition assay (SRFIA) specifically designed to identify compounds interfering with virus-induced membrane fusion. The dual reporter assay is based on two stable Vero cell lines harboring the third-generation tetracycline (Tet3G) transactivator and a bicistronic reporter gene cassette under the control of the tetracycline responsive element (TRE3G), respectively. Cell–cell fusion by the transient transfection of viral fusogens in the presence of doxycycline results in the expression of the reporter enzyme secreted alkaline phosphatase (SEAP) and the fluorescent nuclear localization marker EYFPNuc. A constitutively expressed, secreted form of nanoluciferase (secNLuc) functioned as the internal control. The performance of the SRFIA was tested for the quantification of SARS-CoV-2- and HSV-1-induced cell–cell fusion, respectively, showing high sensitivity and specificity, as well as the reliable identification of known fusion inhibitors. Parallel quantification of secNLuc enabled the detection of cytotoxic compounds or insufficient transfection efficacy. In conclusion, the SRFIA reported here is well suited for high-throughput screening for new antiviral agents and essentially will be applicable to all viral fusogens causing cell–cell fusion in Vero cells

3-O-Galloylated Procyanidins from <i>Rumex acetosa</i> L. Inhibit the Attachment of Influenza A Virus

<div><p>Infections by influenza A viruses (IAV) are a major health burden to mankind. The current antiviral arsenal against IAV is limited and novel drugs are urgently required. Medicinal plants are known as an abundant source for bioactive compounds, including antiviral agents. The aim of the present study was to characterize the anti-IAV potential of a proanthocyanidin-enriched extract derived from the aerial parts of <i>Rumex acetosa</i> (RA), and to identify active compounds of RA, their mode of action, and structural features conferring anti-IAV activity. In a modified MTT (MTT_IAV) assay, RA was shown to inhibit growth of the IAV strain PR8 (H1N1) and a clinical isolate of IAV(H1N1)pdm09 with a half-maximal inhibitory concentration (IC₅₀) of 2.5 µg/mL and 2.2 µg/mL, and a selectivity index (SI) (half-maximal cytotoxic concentration (CC₅₀)/IC₅₀)) of 32 and 36, respectively. At RA concentrations>1 µg/mL plaque formation of IAV(H1N1)pdm09 was abrogated. RA was also active against an oseltamivir-resistant isolate of IAV(H1N1)pdm09. TNF-α and EGF-induced signal transduction in A549 cells was not affected by RA. The dimeric proanthocyanidin epicatechin-3-O-gallate-(4β→8)-epicatechin-3′-O-gallate (procyanidin B2-di-gallate) was identified as the main active principle of RA (IC₅₀ approx. 15 µM, SI≥13). RA and procyanidin B2-di-gallate blocked attachment of IAV and interfered with viral penetration at higher concentrations. Galloylation of the procyanidin core structure was shown to be a prerequisite for anti-IAV activity; <i>o</i>-trihydroxylation in the B-ring increased the anti-IAV activity. <i>In silico</i> docking studies indicated that procyanidin B2-di-gallate is able to interact with the receptor binding site of IAV(H1N1)pdm09 hemagglutinin (HA). In conclusion, the proanthocyanidin-enriched extract RA and its main active constituent procyanidin B2-di-gallate protect cells from IAV infection by inhibiting viral entry into the host cell. RA and procyanidin B2-di-gallate appear to be a promising expansion of the currently available anti-influenza agents.</p></div

Effect of EGCG (6) (A, B) and procyanidin B2-di-gallate (8) (C, D) on electrophoretic mobility and detection of HA by immunoblotting.

<p>Recombinant soluble HA was either mock-treated (lanes 1), incubated with EGCG (6) (2.18 mM) or procyanidin B2-di-gallate (8) (1.13 mM) dissolved in PBS for the times indicated (lanes 3 to 9) or incubated with PBS only (lanes 10 to 13); EGCG (6) (2.18 mM) and procyanidin B2-di-gallate (8) (1.13 mM) incubated in the absence of HA served as control (lanes 2). <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0110089#pone-0110089-g006" target="_blank">Figure 6A, C:</a> Coomassie-stained SDS-PAGE. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0110089#pone-0110089-g006" target="_blank">Figure 6B, D:</a> Detection of HA by immunoblot using a penta-His-specific monoclonal antibody. The expected position of monomeric (approx. 75 kDa) and dimeric HA (approx. 150 kDa) is indicated. Required parameters are missing or incorrect.</p

Structural features of flavan-3-ols, oligomeric proanthocyanidins, hydrolyzable tannins, depsides and building blocks of tannins tested for antiviral activity; compounds isolated from <i>Rumex acetosa</i> extract RA are marked by asterisk.

<p>Structural features of flavan-3-ols, oligomeric proanthocyanidins, hydrolyzable tannins, depsides and building blocks of tannins tested for antiviral activity; compounds isolated from <i>Rumex acetosa</i> extract RA are marked by asterisk.</p