Genome-wide engineering of an infectious clone of herpes simplex virus type 1 using synthetic genomics assembly methods.

Here, we present a transformational approach to genome engineering of herpes simplex virus type 1 (HSV-1), which has a large DNA genome, using synthetic genomics tools. We believe this method will enable more rapid and complex modifications of HSV-1 and other large DNA viruses than previous technologies, facilitating many useful applications. Yeast transformation-associated recombination was used to clone 11 fragments comprising the HSV-1 strain KOS 152 kb genome. Using overlapping sequences between the adjacent pieces, we assembled the fragments into a complete virus genome in yeast, transferred it into an Escherichia coli host, and reconstituted infectious virus following transfection into mammalian cells. The virus derived from this yeast-assembled genome, KOSYA, replicated with kinetics similar to wild-type virus. We demonstrated the utility of this modular assembly technology by making numerous modifications to a single gene, making changes to two genes at the same time and, finally, generating individual and combinatorial deletions to a set of five conserved genes that encode virion structural proteins. While the ability to perform genome-wide editing through assembly methods in large DNA virus genomes raises dual-use concerns, we believe the incremental risks are outweighed by potential benefits. These include enhanced functional studies, generation of oncolytic virus vectors, development of delivery platforms of genes for vaccines or therapy, as well as more rapid development of countermeasures against potential biothreats.

Efficient size-independent chromosome delivery from yeast to cultured cell lines.

The delivery of large DNA vectors (>100 000 bp) remains a limiting step in the engineering of mammalian cells and the development of human artificial chromosomes (HACs). Yeast is commonly used to assemble genetic constructs in the megabase size range, and has previously been used to transfer constructs directly into cultured cells. We improved this method to efficiently deliver large (1.1 Mb) synthetic yeast centromeric plasmids (YCps) to cultured cell lines at rates similar to that of 12 kb YCps. Synchronizing cells in mitosis improved the delivery efficiency by 10-fold and a statistical design of experiments approach was employed to boost the vector delivery rate by nearly 300-fold from 1/250 000 to 1/840 cells, and subsequently optimize the delivery process for multiple mammalian, avian, and insect cell lines. We adapted this method to rapidly deliver a 152 kb herpes simplex virus 1 genome cloned in yeast into mammalian cells to produce infectious virus.

A hydrophobic domain within the small capsid protein of Kaposi's sarcoma-associated herpesvirus is required for assembly.

Kaposi's sarcoma-associated herpesvirus (KSHV) capsids can be produced in insect cells using recombinant baculoviruses for protein expression. All six capsid proteins are required for this process to occur and, unlike for alphaherpesviruses, the small capsid protein (SCP) ORF65 is essential for this process. This protein decorates the capsid shell by virtue of its interaction with the capsomeres. In this study, we have explored the SCP interaction with the major capsid protein (MCP) using GFP fusions. The assembly site within the nucleus of infected cells was visualized by light microscopy using fluorescence produced by the SCP-GFP polypeptide, and the relocalization of the SCP to these sites was evident only when the MCP and the scaffold protein were also present - indicative of an interaction between these proteins that ensures delivery of the SCP to assembly sites. Biochemical assays demonstrated a physical interaction between the SCP and MCP, and also between this complex and the scaffold protein. Self-assembly of capsids with the SCP-GFP polypeptide was evident. Potentially, this result can be used to engineer fluorescent KSHV particles. A similar SCP-His6 polypeptide was used to purify capsids from infected cell lysates using immobilized affinity chromatography and to directly label this protein in capsids using chemically derivatized gold particles. Additional studies with SCP-GFP polypeptide truncation mutants identified a domain residing between aa 50 and 60 of ORF65 that was required for the relocalization of SCP-GFP to nuclear assembly sites. Substitution of residues in this region and specifically at residue 54 with a polar amino acid (lysine) disrupted or abolished this localization as well as capsid assembly, whereas substitution with non-polar residues did not affect the interaction. Thus, this study identified a small conserved hydrophobic domain that is important for the SCP-MCP interaction.

ONCR-177, an Oncolytic HSV-1 Designed to Potently Activate Systemic Antitumor Immunity.

ONCR-177 is an engineered recombinant oncolytic herpes simplex virus (HSV) with complementary safety mechanisms, including tissue-specific miRNA attenuation and mutant UL37 to inhibit replication, neuropathic activity, and latency in normal cells. ONCR-177 is armed with five transgenes for IL12, FLT3LG (extracellular domain), CCL4, and antagonists to immune checkpoints PD-1 and CTLA-4. In vitro assays demonstrated that targeted miRNAs could efficiently suppress ONCR-177 replication and transgene expression, as could the HSV-1 standard-of-care therapy acyclovir. Although ONCR-177 was oncolytic across a panel of human cancer cell lines, including in the presence of type I IFN, replication was suppressed in human pluripotent stem cell-derived neurons, cardiomyocytes, and hepatocytes. Dendritic cells activated with ONCR-177 tumor lysates efficiently stimulated tumor antigen-specific CD8+ T-cell responses. In vivo, biodistribution analyses suggested that viral copy number and transgene expression peaked approximately 24 to 72 hours after injection and remained primarily within the injected tumor. Intratumoral administration of ONCR-177 mouse surrogate virus, mONCR-171, was efficacious across a panel of syngeneic bilateral mouse tumor models, resulting in partial or complete tumor regressions that translated into significant survival benefits and to the elicitation of a protective memory response. Antitumor effects correlated with local and distant intratumoral infiltration of several immune effector cell types, consistent with the proposed functions of the transgenes. The addition of systemic anti-PD-1 augmented the efficacy of mONCR-171, particularly for abscopal tumors. Based in part upon these preclinical results, ONCR-177 is being evaluated in patients with metastatic cancer (ONCR-177-101, NCT04348916).

Design of an Interferon-Resistant Oncolytic HSV-1 Incorporating Redundant Safety Modalities for Improved Tolerability.

Development of next-generation oncolytic viruses requires the design of vectors that are potently oncolytic, immunogenic in human tumors, and well tolerated in patients. Starting with a joint-region deleted herpes simplex virus 1 (HSV-1) to create large transgene capability, we retained a single copy of the ICP34.5 gene, introduced mutations in UL37 to inhibit retrograde axonal transport, and inserted cell-type-specific microRNA (miRNA) target cassettes in HSV-1 genes essential for replication or neurovirulence. Ten miRNA candidates highly expressed in normal tissues and with low or absent expression in malignancies were selected from a comprehensive profile of 800 miRNAs with an emphasis on protection of the nervous system. Among the genes essential for viral replication identified using a small interfering RNA (siRNA) screen, we selected ICP4, ICP27, and UL8 for miRNA attenuation where a single miRNA is sufficient to potently attenuate viral replication. Additionally, a neuron-specific miRNA target cassette was introduced to control ICP34.5 expression. This vector is resistant to type I interferon compared to ICP34.5-deleted oncolytic HSVs, and in cancer cell lines, the oncolytic activity of the modified vector is equivalent to its parental virus. In vivo, this vector potently inhibits tumor growth while being well tolerated, even at high intravenous doses, compared to parental wild-type HSV-1.

Functional Domains of the Herpes Simplex Virus Type 1 Tegument Protein pUL37: The Amino Terminus is Dispensable for Virus Replication in Tissue Culture.

The herpes simplex virus type 1 (HSV-1) UL37 gene encodes for a multifunctional component of the virion tegument, which is necessary for secondary envelopment in the cytoplasm of infected cells, for motility of the viral particle, and for the first steps in the initiation of virus infection. This 120 kDa protein has several known viral interacting partners, including pUL36, gK/pUL20, pUS10, and VP26, and cellular interacting proteins which include TRAF6, RIG-I, and dystonin. These interactions are likely important for the functions of pUL37 at both early and late stages of infection. We employed a genetic approach to determine essential domains and amino acid residues of pUL37 and their associated functions in cellular localization and virion morphogenesis. Using marker-rescue/marker-transfer methods, we generated a library of GFP-tagged pUL37 mutations in the HSV-1 strain KOS genome. Through viral growth and ultra-structural analysis, we discovered that the C-terminus is essential for replication. The N-terminal 480 amino acids are dispensable for replication in cell culture, although serve some non-essential function as viral titers are reduced in the presence of this truncation. Furthermore, the C-terminal 133 amino acids are important in so much that their absence leads to a lethal phenotype. We further probed the carboxy terminal half of pUL37 by alanine scanning mutagenesis of conserved residues among alphaherpesviruses. Mutant viruses were screened for the inability to form plaques-or greatly reduced plaque size-on Vero cells, of which 22 mutations were chosen for additional analysis. Viruses discovered to have the greatest reduction in viral titers on Vero cells were examined by electron microscopy (EM) and by confocal light microscopy for pUL37-EGFP cellular localization. This genetic approach identified both essential and non-essential domains and residues of the HSV-1 UL37 gene product. The mutations identified in this study are recognized as significant candidates for further analysis of the pUL37 function and may unveil previously undiscovered roles and interactions of this essential tegument gene.

Rapid and efficient in vitro excision of BAC sequences from herpesvirus genomes using Cre-mediated recombination.

Cre-mediated recombination is a widely used technique for the re-arrangement of DNA sequences that are bracketed by loxP recognition sites. This bacteriophage P1 enzyme is commonly used to excise the bacterial artificial chromosome (BAC) sequence, a vector sequence used for large herpesvirus genomes for the purposes of propagation and manipulation in Escherichia coli. Most methods utilize cell lines that can be induced for the expression of Cre enzyme to facilitate this excision. In addition, methods have been developed that express Cre from the virus genome and enable auto-excision of the BAC plasmid. We report a versatile and rapid in vitro method based on purified Cre enzyme to carry out the same process in a test tube and does not require cell line generation or cloning into the virus genome. This method greatly increases the repertoire of methods available to modify the genome prior to reconstitution of virus infectivity in a mammalian host.

Current perspectives on the optimal management of infantile hemangioma.

Infantile hemangiomas (IHs) are the most common benign tumor of infancy. As our understanding of their pathobiology has evolved, treatment has become more focused and tailored to specifically treat IH while minimizing adverse effects. Propranolol has gained FDA approval as the first medical therapy for a traditionally surgical disease. This review provides readers with an overview of IH, treatment modalities, and addresses specific considerations in IH disease management.

Incorporation of the Kaposi's sarcoma-associated herpesvirus capsid vertex-specific component (CVSC) into self-assembled capsids.

Self-assembly of herpesvirus capsids can be accomplished in heterologous expression systems provided all six capsid proteins are present. We have demonstrated the assembly of icosahedral Kaposi's sarcoma-associated herpesvirus (KSHV) capsids in insect cells using the baculovirus expression system. Using this self-assembly system we investigated whether we could add additional capsid associated proteins and determine their incorporation into the assembled capsid. We chose the capsid vertex-specific component (CVSC) proteins encoded by open reading frames (ORFs) 19 and 32 to test this. This complex sits on the capsid vertex and is important for capsid maturation in herpesvirus-infected cells. Co-immunoprecipitation assays were used to initially confirm a bi-molecular interaction between ORF19 and ORF32. Both proteins also precipitated the triplex proteins of the capsid shell (ORF26 and ORF62) as well as the major capsid protein (ORF25). Capsid immunoprecipitation assays revealed the incorporation of ORF19 as well as ORF32 into assembled capsids. Similar experiments also showed that the incorporation of each protein occurred independent of the other. These studies reveal biochemically how the KSHV CVSC interacts with the capsid shell.

Visualization of herpes simplex virus type 1 virions using fluorescent colors.

Our laboratory was one of the first to engineer a live fluorescent tag, enhanced green fluorescent protein (eGFP), that marked the capsid of herpes simplex virus type 1 (HSV-1) and subsequently maturing virus as the particle made its way to the cell surface. In the present study we sought to increase the repertoire of colors available as fusion to the small capsid protein, VP26, so that they can be used alone or in conjunction with other fluorescent tags (fused to other HSV proteins) to follow the virus as it enters and replicates within the cell. We have now generated viruses expressing VP26 fusions with Cerulean, Venus, mOrange, tdTomato, mCherry, and Dronpa3 fluorescent proteins. These fusions were made in a repaired UL35 gene (VP26) background. These fusions do not affect the replication properties of the virus expressing the fusion polypeptide and the fusion tag was stably associated with intranuclear capsids and mature virions. Of note we could not isolate viruses expressing fusions with fluorescent proteins that have a tendency to dimerize.

NOTCH3 regulates stem-to-mural cell differentiation in infantile hemangioma.

Infantile hemangioma (IH) is a vascular tumor that begins with rapid vascular proliferation shortly after birth, followed by vascular involution in early childhood. We have found that NOTCH3, a critical regulator of mural cell differentiation and maturation, is expressed in hemangioma stem cells (HemSCs), suggesting that NOTCH3 may function in HemSC-to-mural cell differentiation and pathological vessel stabilization. Here, we demonstrate that NOTCH3 is expressed in NG2+PDGFRß+ perivascular HemSCs and CD31+GLUT1+ hemangioma endothelial cells (HemECs) in proliferating IHs and becomes mostly restricted to the αSMA+NG2loPDGFRßlo mural cells in involuting IHs. NOTCH3 knockdown in HemSCs inhibited in vitro mural cell differentiation and perturbed αSMA expression. In a mouse model of IH, NOTCH3 knockdown or systemic expression of the NOTCH3 inhibitor, NOTCH3 Decoy, significantly decreased IH blood flow, vessel caliber, and αSMA+ perivascular cell coverage. Thus, NOTCH3 is necessary for HemSC-to-mural cell differentiation, and adequate perivascular cell coverage of IH vessels is required for IH vessel stability.

An Unusual Cause of Dyspnea.

Sialadenoma papilliferum is a tumor of salivary gland origin. It is believed that sialadenoma papilliferum is a benign lesion usually known to occur in the hard palate, retromolar fat pads, lips, and parotid glands. Its origin in the bronchial tree is an extremely rare occurrence. We report only the fourth known case of bronchial sialadenoma papilliferum and the first to originate in the trachea. The patient underwent successful resection of the lesion, with complete relief of his dyspnea.