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Identification of 22 Novel Motifs of the Cell Entry Fusion Glycoprotein B of Oncolytic Herpes Simplex Viruses: Sequence Analysis and Literature Review.

ABSTRACT: Objective: Herpes simplex viruses (HSVs) are widely spread throughout the world, causing infections from oral, and genital mucous membrane ulcerations to severe viral encephalitis. Glycoprotein B (gB) was the first HSV envelope glycoprotein identified to induce cell fusion. This glycoprotein initiates viral entry and thereby determines the infectivity of HSV, as well as oncolytic HSV (oHSV). Clarifying its molecular characterization and enlarging its motif reservoir will help to engineer oHSV and in cancer treatment applications. Only in recent years has the importance of gB been acknowledged in HSV infection and oHSV engineering. Although gB-modified oHSVs have been developed, the detailed molecular biology of gB needs to be illustrated more clearly in order to construct more effective oHSVs. Method: Here, we performed a systematic comparative sequence analysis of gBs from the 9 HSV-1 and 2 HSV-2 strains, including HSV-1-LXMW, which was isolated by our lab. Online software was implemented to predict gB secondary structure and motifs. Based on extensive literature reviews, a functional analysis of the predicted motifs was performed. Results: Here, we reported the DNA and predicted amino acid sequences of our recently isolated HSV-1-LXMW and found that the strain was evolutionarily close to HSV-1 strains F, H129, and SC16 based on gB analysis. The 22 novel motifs of HSV gB were identified for the first time. An amino acid sequence alignment of the 11 HSV strains showed that the gB motifs are conserved among HSV strains, suggesting that they are functional in vivo. Additionally, we found that certain amino acids within the 13 motifs out of the 22 were reported to be functional in vivo. Furthermore, the gB mutants and gB-engineered oHSVs were also summarized. Conclusion: Our identification of the 22 novel motifs shed light on HSV gB biology and provide new options for gB engineering to improve the efficiency and safety of oHSVs.

SUBMITTER: Shi F

PROVIDER: S-EPMC7466406 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature

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Identification of 22 Novel Motifs of the Cell Entry Fusion Glycoprotein B of Oncolytic Herpes Simplex Viruses: Sequence Analysis and Literature Review.

Shi Fang F Xin Victoria W VW Liu Xiao-Qin XQ Wang Ying-Ying YY Zhang Ying Y Cheng Jun-Ting JT Cai Wen-Qi WQ Xiang Ying Y Peng Xiao-Chun XC Wang Xianwang X Xin Hong-Wu HW

Frontiers in oncology 20200819

<b>Objective:</b> Herpes simplex viruses (HSVs) are widely spread throughout the world, causing infections from oral, and genital mucous membrane ulcerations to severe viral encephalitis. Glycoprotein B (gB) was the first HSV envelope glycoprotein identified to induce cell fusion. This glycoprotein initiates viral entry and thereby determines the infectivity of HSV, as well as oncolytic HSV (oHSV). Clarifying its molecular characterization and enlarging its motif reservoir will help to engineer ...[more]

PMID: 32974139

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Project description:The oncolytic herpes simplex virus (HSV) that has been approved for clinical practice and those HSVs in clinical trials are attenuated viruses, often with the neurovirulence gene γ134.5 and additional genes deleted. One strategy to engineer nonattenuated oncolytic HSVs consists of retargeting the viral tropism to a cancer-specific receptor of choice, exemplified by HER2 (human epidermal growth factor receptor 2), which is present in breast, ovary, and other cancers, and in detargeting from the natural receptors. Because the HER2-retargeted HSVs strictly depend on this receptor for infection, the viruses employed in preclinical studies were cultivated in HER2-positive cancer cells. The production of clinical-grade viruses destined for humans should avoid the use of cancer cells. Here, we engineered the R-213 recombinant, by insertion of a 20-amino-acid (aa) short peptide (named GCN4) in the gH of R-LM113; this recombinant was retargeted to HER2 through insertion in gD of a single-chain antibody (scFv) to HER2. Next, we generated a Vero cell line expressing an artificial receptor (GCN4R) whose N terminus consists of an scFv to GCN4 and therefore is capable of interacting with GCN4 present in gH of R-213. R-213 replicated as well as R-LM113 in SK-OV-3 cells, implying that addition of the GCN4 peptide was not detrimental to gH. R-213 grew to relatively high titers in Vero-GCN4R cells, efficiently spread from cell to cell, and killed both Vero-GCN4R and SK-OV-3 cells, as expected for an oncolytic virus. Altogether, Vero-GCN4R cells represent an efficient system for cultivation of retargeted oncolytic HSVs in non-cancer cells.IMPORTANCE There is growing interest in viruses as oncolytic agents, which can be administered in combination with immunotherapeutic compounds, including immune checkpoint inhibitors. The oncolytic HSV approved for clinical practice and those in clinical trials are attenuated viruses. An alternative to attenuation is a cancer specificity achieved by tropism retargeting to selected cancer receptors. However, the retargeted oncolytic HSVs strictly depend on cancer receptors for infection. Here, we devised a strategy for in vitro cultivation of retargeted HSVs in non-cancer cells. The strategy envisions a double-retargeting approach: one retargeting is via gD to the cancer receptor, and the second retargeting is via gH to an artificial receptor expressed in Vero cells. The double-retargeted HSV uses alternatively the two receptors to infect cancer cells or producer cells. A universal non-cancer cell line for growth of clinical-grade retargeted HSVs represents a step forward in the translational phase.

Project description:Herpesviruses-ubiquitous pathogens that cause persistent infections-have some of the most complex cell entry mechanisms. Entry of the prototypical herpes simplex virus 1 (HSV-1) requires coordinated efforts of 4 glycoproteins, gB, gD, gH, and gL. The current model posits that the glycoproteins do not interact before receptor engagement and that binding of gD to its receptor causes a "cascade" of sequential pairwise interactions, first activating the gH/gL complex and subsequently activating gB, the viral fusogen. But how these glycoproteins interact remains unresolved. Here, using a quantitative split-luciferase approach, we show that pairwise HSV-1 glycoprotein complexes form before fusion, interact at a steady level throughout fusion, and do not depend on the presence of the cellular receptor. Based on our findings, we propose a revised "conformational cascade" model of HSV-1 entry. We hypothesize that all 4 glycoproteins assemble into a complex before fusion, with gH/gL positioned between gD and gB. Once gD binds to a cognate receptor, the proximity of the glycoproteins within this complex allows for efficient transmission of the activating signal from the receptor-activated gD to gH/gL to gB through sequential conformational changes, ultimately triggering the fusogenic refolding of gB. Our results also highlight previously unappreciated contributions of the transmembrane and cytoplasmic domains to glycoprotein interactions and fusion. Similar principles could be at play in other multicomponent viral entry systems, and the split-luciferase approach used here is a powerful tool for investigating protein-protein interactions in these and a variety of other systems. IMPORTANCE Herpes simplex virus 1 (HSV-1) infects the majority of humans for life and can cause diseases ranging from painful sores to deadly brain inflammation. No vaccines or curative treatments currently exist. HSV-1 infection of target cells requires coordinated efforts of four viral glycoproteins. But how these glycoproteins interact remains unclear. Using a quantitative protein interaction assay, we found that HSV-1 glycoproteins form receptor-independent complexes and interact at a steady level. We propose that the 4 proteins form a complex, which could facilitate transmission of the entry-triggering signal from the receptor-binding component to the membrane fusogen component through sequential conformational changes. Similar principles could be applicable across other multicomponent protein systems. A revised model of HSV-1 entry could facilitate the development of therapeutics targeting this process.

Dataset Information

Identification of 22 Novel Motifs of the Cell Entry Fusion Glycoprotein B of Oncolytic Herpes Simplex Viruses: Sequence Analysis and Literature Review.

Publications

Identification of 22 Novel Motifs of the Cell Entry Fusion Glycoprotein B of Oncolytic Herpes Simplex Viruses: Sequence Analysis and Literature Review.

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