Herpes simplex virus infects most cell types in vitro: clues to its success

Herpes simplex virus (HSV) type-1 and type-2 have evolved numerous strategies to infect a wide range of hosts and cell types. The result is a very successful prevalence of the virus in the human population infecting 40-80% of people worldwide. HSV entry into host cell is a multistep process that involves the interaction of the viral glycoproteins with various cell surface receptors. Based on the cell type, HSV enter into host cell using different modes of entry. The combination of various receptors and entry modes has resulted in a virus that is capable of infecting virtually all cell types. Identifying the common rate limiting steps of the infection may help the development of antiviral agents that are capable of preventing the virus entry into host cell. In this review we describe the major features of HSV entry that have contributed to the wide susceptibility of cells to HSV infection

An Important Role for Syndecan-1 in Herpes Simplex Virus Type-1 Induced Cell-to-Cell Fusion and Virus Spread

Herpes simplex virus type-1 (HSV-1) is a common human pathogen that relies heavily on cell-to-cell spread for establishing a lifelong latent infection. Molecular aspects of HSV-1 entry into host cells have been well studied; however, the molecular details of the spread of the virus from cell-to-cell remain poorly understood. In the past, the role of heparan sulfate proteoglycans (HSPG) during HSV-1 infection has focused solely on the role of HS chains as an attachment receptor for the virus, while the core protein has been assumed to perform a passive role of only carrying the HS chains. Likewise, very little is known about the involvement of any specific HSPGs in HSV-1 lifecycle. Here we demonstrate that a HSPG, syndecan-1, plays an important role in HSV-1 induced membrane fusion and cell-to-cell spread. Interestingly, the functions of syndecan-1 in fusion and spread are independent of the presence of HS on the core protein. Using a mutant CHO-K1 cell line that lacks all glycosaminoglycans (GAGs) on its surface (CHO-745) we demonstrate that the core protein of syndecan-1 possesses the ability to modulate membrane fusion and viral spread. Altogether, we identify a new role for syndecan-1 in HSV-1 pathogenesis and demonstrate HS-independent functions of its core protein in viral spread

Protease, Growth Factor, and Heparanase-Mediated Syndecan-1 Shedding Leads to Enhanced HSV-1 Egress

Heparan sulfate (HS) and heparan sulfate proteoglycans (HSPGs) are considered important for the entry of many different viruses. Previously, we demonstrated that heparanase (HPSE), the host enzyme responsible for cleaving HS chains, is upregulated by herpes simplex virus-1 (HSV-1) infection. Higher levels of HPSE accelerate HS removal from the cell surface, facilitating viral release from infected cells. Here, we study the effects of overexpressing HPSE on viral entry, cell-to-cell fusion, plaque formation, and viral egress. We provide new information that higher levels of HPSE reduce syncytial plaque formation while promoting egress and extracellular release of the virions. We also found that transiently enhanced expression of HPSE did not affect HSV-1 entry into host cells or HSV-1-induced cell-to-cell fusion, suggesting that HPSE activation is tightly regulated and facilitates extracellular release of the maturing virions. We demonstrate that an HSPG-shedding agonist, PMA; a protease, thrombin; and a growth factor, EGF as well as bacterially produced recombinant heparinases resulted in enhanced HSV-1 release from HeLa and human corneal epithelial (HCE) cells. Our findings here underscore the significance of syndecan-1 functions in the HSV-1 lifecycle, provide evidence that the shedding of syndecan-1 ectodomain is another way HPSE works to facilitate HSV-1 release, and add new evidence on the significance of various HSPG shedding agonists in HSV-1 release from infected cells

Modulation of Heparan Sulfate Proteoglycans During Herpes Simplex Virus Type-1 Infection

Herpes simplex virus type-1 (HSV-1) is an important human pathogen that relies heavily on cell-to-cell spread for establishing a lifelong latent infection. The result is a very successful prevalence of the virus in the human population infecting 40-80% of people worldwide. It causes oral and ocular lesions and more serious diseases, such as blindness, meningitis, and encephalitis. HSV-1 is a leading cause of viral corneal blindness and viral encephalitis in developed countries. HSV-1 entry into host cell is a multistep process that involves the interaction of the viral glycoproteins with various cell surface receptors. The role of heparan sulfate proteoglycans (HSPG) during HSV-1 infection has focused solely on the role of HS chains as an attachment receptor for the virus, while the core protein has been assumed to perform a passive role of only carrying the HS chains. Likewise, very little is known about the involvement of any specific HSPGs in HSV-1 lifecycle. The purpose of this study was to further analyze the contribution and modulation of HSPG during HSV-1 infection. Syndecan-1 is predominantly expressed in epithelial cells which are prime targets for HSV-1 initial infection. Therefore, this study is focused primarily on the contribution of this family member on HSV-1 infection. Important findings in this study include: (i) The core protein of syndecan-1 facilitates HSV-1 induced cell-to-cell fusion. CHO-745 cells, which are deficient in glycosaminoglycans’ (GAGs) synthesis including HS, were utilized to evaluate HS-independent contribution of syndecan-1 to cell fusion. (ii) Syndecan-1 facilitates HSV-1 cell-to-cell spread, as evident by determining HSV-1 plaque size, and utilizing a spread assay after changing syndecan-1 expression level on the cell surface through syndecan-1expression plasmid and syndecan-1 specific siRNA transfection. (iii) Modulating HSPG by Heparanase induces HSV-1 release from infected cells. Heparanase modulation was achieved by enhancing Heparanase expression, and by treating cells with exogenous recombinant Heparinases. (iv) HSV-1 infection induces active Heparanase expression, which is associated with a reduction in inactive Heparanase expression. Together, our study has expanded our understanding of HSPG role in HSV-1 infection, and has provided new insights into the modulation of HSPG during HSV-1 infection which might help the development of new antiviral agents or an effective HSV-1 vaccine

Herpes simplex virus infects most cell types in vitro: clues to its success

Abstract Herpes simplex virus (HSV) type-1 and type-2 have evolved numerous strategies to infect a wide range of hosts and cell types. The result is a very successful prevalence of the virus in the human population infecting 40-80% of people worldwide. HSV entry into host cell is a multistep process that involves the interaction of the viral glycoproteins with various cell surface receptors. Based on the cell type, HSV enter into host cell using different modes of entry. The combination of various receptors and entry modes has resulted in a virus that is capable of infecting virtually all cell types. Identifying the common rate limiting steps of the infection may help the development of antiviral agents that are capable of preventing the virus entry into host cell. In this review we describe the major features of HSV entry that have contributed to the wide susceptibility of cells to HSV infection.</p

Syndecan-1 knockdown or overexpression do not affect cell viability.

<p>(A). CHO-K1, CHO-745, and HCE cells were transfected with scrambled (scr) siRNA or syndecan-1 (SDC1) siRNA. 72–96 h after transfection, immunoblots of cell lysates were prepared and probed with anti-SDC1 polyclonal Ab. β-actin protein level was measured as loading control. Representative blots are shown. Protein bands were quantified using ImageQuant TL image analysis software (version: 7). SDC1 protein expression (mean ± 1SD), normalized to that of β-actin, of at least three independent experiments was quantified by calculating the relative intensity of each syndecan-1 band relative to the control scrambled siRNA treated bands, and presented as bar graph. (B) Cells were grown in 6-well plates, mock treated or transfected with human SDC1 plasmid for 48 h. Cell surface level of SDC1 was evaluated by flowcytomety. FITC stained cells were used as background control. Results are representative of two independent experiments (C, D). Cells were grown in 96-well plates, transfected with scrambled siRNA or SDC1 siRNA for 48 h (C), or transfected with control GFP plasmid or human SDC1 plasmid for 24 h (D). Triplicate wells were evaluated for cell viability using MTS assay. Results are expressed as 100% wild type (wt) viability where they represent the percent corrected absorbance after subtracting the background absorbance, relative to scrambled siRNA transfected cells (C), or relative to GFP transfected cells (D), and are mean ± 1SD of at least 2 independent experiments.</p

Downregulation of Syndecan-1 results in reduced production of infectious virus.

<p>HCE cells were transfected with either control scrambled siRNA or syndecan-1 siRNA. 72 h post-transfection cells were infected with HSV-1 (KOS) at an MOI of 0.1. At 0, 5, 24, 48, and 72 h post-infection, infectious virus was quantified by a standard plaque assay on HCE cell monolayers. The titers shown are the mean ± 1 SD of a representative experiment of two independent experiments performed in duplicates. <i>SDC1</i>, syndecan-1.</p

Comparison of syncytia number and nuclei count after syndecan-1 overexpression in each CHO cell type.

<p>Average number of syncytial cells, as well as, the average number of nuclei per syncytia was counted in CHO-K1 and CHO-745 cells after overexpressing syndecan-1 on target cells or effector cells. Positive controls are target and effector cells expressing normal levels of syndecan-1. Syncytial cells were counted 72 h post mixing. Syncytia were classified as any red cell having two or more nuclei. Number of syncytia was normalized to the number of syncytia detected in the negative control wells where the effector cell population lacks gB. The average is based on results from two independent experiment performed in duplicate (mean ± 1SD).</p

Syndecan-1 knockdown reduces plaque formation in HCE cells.

<p>(A). Monolayers of HCE cells were transfected with either control plasmid GFP, or with human syndecan-1. 24 h post-transfection cells were infected with serial dilution of HSV-1(KOS) stocks. (B). 50% confluent HCE cells were transfected with either control scrambled siRNA or syndecan-1 specific siRNA. 72 h post-transfection, cells were infected with serial dilution of HSV-1(KOS) stocks. (A, B). 72 h post-infection cells were fixed and stained with crystal violet stain. Infectivity was measured by the number of plaque forming units (PFUs). Number of PFUs was counted at the 10× objective (Zeiss Axiovert 200). Plaques consist of 15 or more nuclei were counted. Results are means ± 1 SD of three independent experiments conducted in duplicate. <i>SDC1</i>, syndecan-1.</p

syndecan-1 ectodomain and cytoplasmic domains are important for inhibiting cell fusion when overexpressed on effector cells.

<p>(A). Syndecan-1 truncation and mutants used in the study are illustrated including the full-length wild type (<i>wt</i>) core protein syndecan-1 (SDC1) that includes an extracellular domain, transmembrane domain (TM), and COOH-terminal cytoplasmic domain. Also illustrated are the construct FcR^ectohS1 that is a chimera comprised of the ectodomain of human IgG Fcγ receptor Ia/CD64 fused to the transmembrane and cytoplasmic domains of human syndecan-1, the construct hS1^pLeu™ that has the transmembrane domain replaced with leucine residues, and a truncation mutant hS1^Δcyto that lacks the 33 C-terminal amino acids. (B) Cells were grown in 96-well plates, transfected with control GFP plasmid, full-length <i>wt</i> human SDC1 plasmid, the construct FcR^ectohS1, the construct hS1^pLeu™, or the construct hS1^Δcyto for 24 h. Triplicate wells were evaluated for cell viability using MTS assay. Results are expressed as 100% wild type (wt) viability where they represent the percent corrected absorbance after subtracting the background absorbance, relative to control GFP plasmid transfected cells, and are mean ± 1SD of at least 3 independent experiments. (C). Effector cells for CHO-K1 and CHO-745 cells were transfected with either control GFP plasmid, full-length <i>wt</i> syndecan-1, the construct FcR^ectohS1, the construct hS1^pLeu™, or the construct hS1^Δcyto and mixed with the target cells 24 h post-transfection. Fusion was measured 16 h post mixing. Results are presented as mean ± 1 SD of at least 3 independent experiments. As a negative control, target cells were mixed with effector cells lacking HSV-1 gB.</p