Inactivation of the UL37 Deamidase Enhances Virus Replication and Spread of the HSV-1(VC2) Oncolytic Vaccine Strain and Secretion of GM-CSF.

The HSV-1 (VC2) live-attenuated vaccine strain was engineered with specific deletions in the amino termini of glycoprotein K (gK) and membrane protein UL20, rendering the virus unable to enter neurons and establish latency. VC2 replicates efficiently in epithelial cell culture but produces lower viral titers and smaller viral plaques than its parental HSV-1 (F) wild-type virus. VC2 is an effective live-attenuated vaccine against HSV-1 and HSV-2 infections in mice and guinea pigs and an anti-tumor immunotherapeutic and oncolytic virus against melanoma and breast cancer in mouse models. Previously, we reported that the gK/UL20 complex interacts with the UL37 tegument protein, and this interaction is essential for virion intracellular envelopment and egress. To investigate the potential role of the UL37 deamidase functions, the recombinant virus FC819S and VC2C819S were constructed with a C819S substitution to inactivate the UL37 predicted deamidase active site on an HSV-1(F) and HSV-1(VC2) genetic background, respectively. FC819S replicated to similar levels with HSV-1(F) and produced similar size viral plaques. In contrast, VC2C819S replication was enhanced, and viral plaques increased in size, approaching those of the wild-type HSV-1(F) virus. FC819S infection of cell cultures caused enhanced GM-CSF secretion in comparison to HSV-1(F) across several cell lines, including HEp2 cells and cancer cell lines, DU145 (prostate) and Panc 04.03 (pancreas), and primary mouse peritoneal cells. VC2 infection of these cell lines caused GM-CSF secretion at similar levels to FC819S infection. However, the VC2C819S virus did not exhibit any further enhancement of GM-CSF secretion compared to the VC2 virus. These results suggest that the UL37 deamidation functions in conjunction with the gK/UL20 complex to facilitate virus replication and GM-CSF secretion.

Predicted Structure and Functions of the Prototypic Alphaherpesvirus Herpes Simplex Virus Type-1 UL37 Tegument Protein.

The alphaherpesvirus UL37 tegument protein is a highly conserved, multi-functional protein. Mutagenesis analysis delineated the UL37 domains necessary for retrograde transport and viral replication. Specifically, the amino-terminal 480 amino acids are dispensable for virus replication in epithelial cell culture, but it is unknown whether this amino-terminal deletion affects UL37 structure and intracellular transport in epithelial cells and neurons. To investigate the structure and function of UL37, we utilized multiple computational approaches to predict and characterize the secondary and tertiary structure and other functional features. The structure of HSV-1 UL37 and Δ481N were deduced using publicly available predictive algorithms. The predicted model of HSV-1 UL37 is a stable, multi-functional, globular monomer, rich in alpha helices, with unfolded regions within the linker and the C-tail domains. The highly flexible C-tail contains predicted binding sites to the dynein intermediate chain, as well as DNA and RNA. Predicted interactions with the cytoplasmic surface of the lipid membrane suggest UL37 is a peripheral membrane protein. The Δ481N truncation did not alter the predicted structure of the UL37 C-terminus protein and its predicted interaction with dynein. We validated these models by examining the replication kinetics and transport of the Δ481N virus toward the nuclei of infected epithelial and neuronal cells. The Δ481N virus had substantial defects in virus spread; however, it exhibited no apparent defects in virus entry and intracellular transport. Using computational analyses, we identified several key features of UL37, particularly the flexible unstructured tail; we then demonstrated that the UL37 C-terminus alone is sufficient to effectively transport the virus towards the nucleus of infected epithelial and neuronal cells.

Two Sides to Every Story: Herpes Simplex Type-1 Viral Glycoproteins gB, gD, gH/gL, gK, and Cellular Receptors Function as Key Players in Membrane Fusion.

Herpes simplex virus type-1 (HSV-1) and type-2 (HSV-2) are prototypical alphaherpesviruses that are characterized by their unique properties to infect trigeminal and dorsal root ganglionic neurons, respectively, and establish life-long latent infections. These viruses initially infect mucosal epithelial tissues and subsequently spread to neurons. They are associated with a significant disease spectrum, including orofacial and ocular infections for HSV-1 and genital and neonatal infections for HSV-2. Viral glycoproteins within the virion envelope bind to specific cellular receptors to mediate virus entry into cells. This is achieved by the fusion of the viral envelope with the plasma membrane. Similarly, viral glycoproteins expressed on cell surfaces mediate cell-to-cell fusion and facilitate virus spread. An interactive complex of viral glycoproteins gB, gD/gH/gL, and gK and other proteins mediate these membrane fusion phenomena with glycoprotein B (gB), the principal membrane fusogen. The requirement for the virion to enter neuronal axons suggests that the heterodimeric protein complex of gK and membrane protein UL20, found only in alphaherpesviruses, constitute a critical determinant for neuronal entry. This hypothesis was substantiated by the observation that a small deletion in the amino terminus of gK prevents entry into neuronal axons while allowing entry into other cells via endocytosis. Cellular receptors and receptor-mediated signaling synergize with the viral membrane fusion machinery to facilitate virus entry and intercellular spread. Unraveling the underlying interactions among viral glycoproteins, envelope proteins, and cellular receptors will provide new innovative approaches for antiviral therapy against herpesviruses and other neurotropic viruses.

Intramuscular vaccination of mice with the human herpes simplex virus type-1(HSV-1) VC2 vaccine, but not its parental strain HSV-1(F) confers full protection against lethal ocular HSV-1 (McKrae) pathogenesis.

Herpes simplex virus type-1 (HSV-1) can cause severe ocular infection and blindness. We have previously shown that the HSV-1 VC2 vaccine strain is protective in mice and guinea pigs against genital herpes infection following vaginal challenge with HSV-1 or HSV-2. In this study, we evaluated the efficacy of VC2 intramuscular vaccination in mice against herpetic keratitis following ocular challenge with lethal human clinical strain HSV-1(McKrae). VC2 vaccination in mice produced superior protection and morbidity control in comparison to its parental strain HSV-1(F). Specifically, after HSV-1(McKrae) ocular challenge, all VC2 vaccinated- mice survived, while 30% of the HSV-1(F)- vaccinated and 100% of the mock-vaccinated mice died post challenge. VC2-vaccinated mice did not exhibit any symptoms of ocular infection and completely recovered from initial conjunctivitis. In contrast, HSV-1(F)-vaccinated mice developed time-dependent progressive keratitis characterized by corneal opacification, while mock-vaccinated animals exhibited more severe stromal keratitis characterized by immune cell infiltration and neovascularization in corneal stroma with corneal opacification. Cornea in VC2-immunized mice exhibited significantly increased infiltration of CD3+ T lymphocytes and decreased infiltration of Iba1+ macrophages in comparison to mock- or HSV-1(F)-vaccinated groups. VC2 immunization produced higher virus neutralization titers than HSV-1(F) post challenge. Furthermore, VC-vaccination significantly increased the CD4 T central memory (TCM) subsets and CD8 T effector memory (TEM) subsets in the draining lymph nodes following ocular HSV-1 (McKrae) challenge, then mock- or HSV-1(F)-vaccination. These results indicate that VC2 vaccination produces a protective immune response at the site of challenge to protect against HSV-1-induced ocular pathogenesis.

Proximity ligation assay to study protein-protein interactions of proteins on two different cells.

Protein-protein interactions (PPI) by homo-, hetero- or oligo-merization in the cellular environment regulate cellular processes. PPI can be inhibited by antibodies, small molecules or peptides, and this inhibition has therapeutic value. A recently developed method, the proximity ligation assay (PLA), provides detection of PPI in the cellular environment. However, most applications using this assay are for proteins expressed in the same cell. We employ PLA for the first time to study PPI of cell surface proteins on two different cells. Inhibition of PPI using a peptide inhibitor is also quantified using this assay; PLA is used to detect PPI of CD2 and CD58 between Jurkat cells (T cells) and human fibroblast-like synoviocyte-rheumatoid arthritis cells that are important in the immune response in the autoimmune disease rheumatoid arthritis. This assay provides direct evidence of inhibition of PPI of two proteins on different cell surfaces.

The Amino Terminus of Herpes Simplex Virus 1 Glycoprotein K (gK) Is Required for gB Binding to Akt, Release of Intracellular Calcium, and Fusion of the Viral Envelope with Plasma Membranes.

Previously, we have shown that the amino terminus of glycoprotein K (gK) binds to the amino terminus of gB and that deletion of the amino-terminal 38 amino acids of gK prevents herpes simplex virus 1 (HSV-1) infection of mouse trigeminal ganglia after ocular infection and virus entry into neuronal axons. Recently, it has been shown that gB binds to Akt during virus entry and induces Akt phosphorylation and intracellular calcium release. Proximity ligation and two-way immunoprecipitation assays using monoclonal antibodies against gB and Akt-1 phosphorylated at S473 [Akt-1(S473)] confirmed that HSV-1(McKrae) gB interacted with Akt-1(S473) during virus entry into human neuroblastoma (SK-N-SH) cells and induced the release of intracellular calcium. In contrast, the gB specified by HSV-1(McKrae) gKΔ31-68, lacking the amino-terminal 38 amino acids of gK, failed to interact with Akt-1(S473) and induce intracellular calcium release. The Akt inhibitor miltefosine inhibited the entry of McKrae but not the gKΔ31-68 mutant into SK-N-SH cells. Importantly, the entry of the gKΔ31-68 mutant but not McKrae into SK-N-SH cells treated with the endocytosis inhibitors pitstop-2 and dynasore hydrate was significantly inhibited, indicating that McKrae gKΔ31-68 entered via endocytosis. These results suggest that the amino terminus of gK functions to regulate the fusion of the viral envelope with cellular plasma membranes.IMPORTANCE HSV-1 glycoprotein B (gB) functions in the fusion of the viral envelope with cellular membranes during virus entry. Herein, we show that a deletion in the amino terminus of glycoprotein K (gK) inhibits gB binding to Akt-1(S473), the release of intracellular calcium, and virus entry via fusion of the viral envelope with cellular plasma membranes.

Herpes Simplex Virus 1 UL37 Protein Tyrosine Residues Conserved among All Alphaherpesviruses Are Required for Interactions with Glycoprotein K, Cytoplasmic Virion Envelopment, and Infectious Virus Production.

The herpes simplex virus 1 (HSV-1) UL37 protein functions in virion envelopment at trans-Golgi membranes, as well as in retrograde and anterograde transport of virion capsids. Recently, we reported that UL37 interacts with glycoprotein K (gK) and its interacting partner protein UL20 (N. Jambunathan, D. Chouljenko, P. Desai, A. S. Charles, R. Subramanian, V. N. Chouljenko, and K. G. Kousoulas, J Virol 88:5927-5935, 2014, http://dx.doi.org/10.1128/JVI.00278-14), facilitating cytoplasmic virion envelopment. Alignment of UL37 homologs encoded by alphaherpesviruses revealed the presence of highly conserved residues in the central portion of the UL37 protein. A cadre of nine UL37 site-specific mutations were produced and tested for their ability to inhibit virion envelopment and infectious virus production. Complementation analysis revealed that replacement of tyrosines 474 and 480 with alanine failed to complement the UL37-null virus, while all other mutated UL37 genes complemented the virus efficiently. The recombinant virus DC474-480 constructed with tyrosines 474, 476, 477, and 480 mutated to alanine residues produced a gK-null-like phenotype characterized by the production of very small plaques and accumulation of capsids in the cytoplasm of infected cells. Recombinant viruses having either tyrosine 476 or 477 replaced with alanine produced a wild-type phenotype. Immunoprecipitation assays revealed that replacement of all four tyrosines with alanines substantially reduced the ability of gK to interact with UL37. Alignment of HSV UL37 with the human cytomegalovirus and Epstein-Barr virus UL37 homologs revealed that Y480 was conserved only for alphaherpesviruses. Collectively, these results suggest that the UL37 conserved tyrosine 480 residue plays a crucial role in interactions with gK to facilitate cytoplasmic virion envelopment and infectious virus production. IMPORTANCE: The HSV-1 UL37 protein is conserved among all herpesviruses, functions in both retrograde and anterograde transport of virion capsids, and plays critical roles in cytoplasmic virion envelopment by interacting with gK. We show here that UL37 tyrosine residues conserved among all alphaherpesviruses serve critical roles in cytoplasmic virion envelopment and interactions with gK.

Deletion of a Predicted ß-Sheet Domain within the Amino Terminus of Herpes Simplex Virus Glycoprotein K Conserved among Alphaherpesviruses Prevents Virus Entry into Neuronal Axons.

UNLABELLED: We have shown previously that herpes simplex virus 1 (HSV-1) lacking expression of the entire glycoprotein K (gK) or expressing gK with a 38-amino-acid deletion (gKΔ31-68 mutation) failed to infect ganglionic neurons after ocular infection of mice. We constructed a new model for the predicted three-dimensional structure of gK, revealing that the gKΔ31-68 mutation spans a well-defined ß-sheet structure within the amino terminus of gK, which is conserved among alphaherpesviruses. The HSV-1(McKrae) gKΔ31-68 virus was tested for the ability to enter into ganglionic neuronal axons in cell culture of explanted rat ganglia using a novel virus entry proximity ligation assay (VEPLA). In this assay, cell surface-bound virions were detected by the colocalization of gD and its cognate receptor nectin-1 on infected neuronal surfaces. Capsids that have entered into the cytoplasm were detected by the colocalization of the virion tegument protein UL37, with dynein required for loading of virion capsids onto microtubules for retrograde transport to the nucleus. HSV-1(McKrae) gKΔ31-68 attached to cell surfaces of Vero cells and ganglionic axons in cell culture as efficiently as wild-type HSV-1(McKrae). However, unlike the wild-type virus, the mutant virus failed to enter into the axoplasm of ganglionic neurons. This work suggests that the amino terminus of gK is a critical determinant for entry into neuronal axons and may serve similar conserved functions for other alphaherpesviruses. IMPORTANCE: Alphaherpesviruses, unlike beta- and gammaherpesviruses, have the unique ability to infect and establish latency in neurons. Glycoprotein K (gK) and the membrane protein UL20 are conserved among all alphaherpesviruses. We show here that a predicted ß-sheet domain, which is conserved among alphaherpesviruses, functions in HSV-1 entry into neuronal axons, suggesting that it may serve similar functions for other herpesviruses. These results are in agreement with our previous observations that deletion of this gK domain prevents the virus from successfully infecting ganglionic neurons after ocular infection of mice.

Phenylalanine residues at the carboxyl terminus of the herpes simplex virus 1 UL20 membrane protein regulate cytoplasmic virion envelopment and infectious virus production.

UNLABELLED: The herpes simplex virus type 1 (HSV-1) UL20 gene encodes a 222-amino-acid nonglycosylated envelope protein which forms a complex with viral glycoprotein K (gK) that functions in virion envelopment, egress, and virus-induced cell fusion. To investigate the role of the carboxyl terminus of the UL20 protein (UL20p) in cytoplasmic virion envelopment, a cadre of mutant viruses was constructed and characterized. The deletion of six amino acids from the carboxyl terminus of UL20p caused an approximately 1-log reduction in infectious virus production compared to that of the wild-type virus. Surprisingly, a phenylalanine-to-alanine replacement at amino acid position 210 caused a gain-of-function phenotype, increasing infectious virus production up to 1 log more than in the wild-type virus. In contrast, the replacement of two membrane-proximal phenylalanines with alanines caused drastic inhibition of infectious virion production and cytoplasmic virion envelopment. Prediction of the membrane topology of UL20p revealed that these two amino acid changes cause retraction of the carboxyl terminus of UL20p from the intracellular space. Confocal microscopy revealed that none of the engineered UL20 mutations affected intracellular transport of UL20p to trans-Golgi network membranes. In addition, a proximity ligation assay showed that none of the UL20 mutations affected UL20p colocalization and potential interactions with the UL37 protein recently found to interact with the gK/UL20 protein complex. Collectively, these studies show that phenylalanine residues within the carboxyl terminus of UL20p are involved in the regulation of cytoplasmic virion envelopment and infectious virus production. IMPORTANCE: We have shown previously that the UL20/gK protein complex serves crucial roles in cytoplasmic virion envelopment and that it interacts with the UL37 tegument protein to facilitate cytoplasmic virion envelopment. In this study, we investigated the role of phenylalanine residues within the carboxyl terminus of UL20p, since aromatic and hydrophobic amino acids are known to be involved in protein-protein interactions through stacking of their aromatic structures. Characterization of mutant viruses carrying phenylalanine (Phe)-to-alanine (Ala) mutations revealed that the two membrane-proximal Phe residues were critical for the proper UL20p membrane topology and efficient virion envelopment and infectious virus production. Surprisingly, a Phe-to-Ala change located approximately in the middle of the UL20p carboxyl terminus substantially enhanced cytoplasmic envelopment and overall production of infectious virions. This work revealed that Phe residues within the UL20p carboxyl terminus are involved in the regulation of cytoplasmic virion envelopment and infectious virus production.

Herpes simplex virus 1 protein UL37 interacts with viral glycoprotein gK and membrane protein UL20 and functions in cytoplasmic virion envelopment.

UNLABELLED: We have shown that glycoprotein K (gK) and its interacting partner, the UL20 protein, play crucial roles in virion envelopment. Specifically, virions lacking either gK or UL20 fail to acquire an envelope, thus causing accumulation of capsids in the cytoplasm of infected cells. The herpes simplex virus 1 (HSV-1) UL37 protein has also been implicated in cytoplasmic virion envelopment. To further investigate the role of UL37 in virion envelopment, the recombinant virus DC480 was constructed by insertion of a 12-amino-acid protein C (protC) epitope tag within the UL37 amino acid sequence immediately after amino acid 480. The DC480 mutant virus expressed full-size UL37 as detected by the anti-protC antibody in Western immunoblots, accumulated unenveloped capsids in the cytoplasm of infected cells, and produced very small plaques on African green monkey kidney (Vero) cells that were similar in size to those produced by the UL20-null and UL37-null viruses. The DC480 virus replicated nearly 4 log less efficiently than the parental wild-type virus when grown on Vero cells. However, DC480 mutant virus titers increased nearly 20-fold when the virus was grown on FRT cells engineered to express the UL20 gene in comparison to the titers on Vero cells, while the UL37-null virus replicated approximately 20-fold less efficiently than the DC480 virus on FRT cells. Coimmunoprecipitation experiments and proximity ligation assays showed that gK and UL20 interact with the UL37 protein in infected cells. Collectively, these results indicate that UL37 interacts with the gK-UL20 protein complex to facilitate cytoplasmic virion envelopment. IMPORTANCE: Herpes simplex viruses acquire their final envelopes by budding into cytoplasmic membranes derived from the trans-Golgi network (TGN). The tegument proteins UL36 and UL37 are known to be transported to the TGN sites of virus envelopment and to function in virion envelopment, since mutants lacking UL37 accumulate capsids in the cytoplasm that are unable to bud into TGN membranes. Viral glycoprotein K (gK) also functions in cytoplasmic envelopment, in a protein complex with the membrane-associated protein UL20 (UL20mp). This work shows for the first time that the UL37 protein functionally interacts with gK and UL20 to facilitate cytoplasmic virion envelopment. This work may lead to the design of specific drugs that can interrupt UL37 interactions with the gK-UL20 protein complex, providing new ways to combat herpesviral infections.

Site-specific proteolytic cleavage of the amino terminus of herpes simplex virus glycoprotein K on virion particles inhibits virus entry.

Herpes simplex virus 1 (HSV-1) glycoprotein K (gK) is expressed on virions and functions in entry, inasmuch as HSV-1(KOS) virions devoid of gK enter cells substantially slower than is the case for the parental KOS virus (T. P. Foster, G. V. Rybachuk, and K. G. Kousoulas, J. Virol. 75:12431-12438, 2001). Deletion of the amino-terminal 68-amino-acid (aa) portion of gK caused a reduction in efficiency and kinetics of virus entry similar to that of the gK-null virus in comparison to the HSV-1(F) parental virus. The UL20 membrane protein and gK were readily detected on double-gradient-purified virion preparations. Immuno-electron microscopy confirmed the presence of gK and UL20 on purified virions. Coimmunoprecipitation experiments using purified virions revealed that gK interacted with UL20, as has been shown in virus-infected cells (T. P. Foster, V. N. Chouljenko, and K. G. Kousoulas, J. Virol. 82:6310-6323, 2008). Scanning of the HSV-1(F) viral genome revealed the presence of a single putative tobacco etch virus (TEV) protease site within gD, while additional TEV predicted sites were found within the UL5 (helicase-primase helicase subunit), UL23 (thymidine kinase), UL25 (DNA packaging tegument protein), and UL52 (helicase-primase primase subunit) proteins. The recombinant virus gDΔTEV was engineered to eliminate the single predicted gD TEV protease site without appreciably affecting its replication characteristics. The mutant virus gK-V5-TEV was subsequently constructed by insertion of a gene sequence encoding a V5 epitope tag in frame with the TEV protease site immediately after gK amino acid 68. The gK-V5-TEV, R-gK-V5-TEV (revertant virus), and gDΔTEV viruses exhibited similar plaque morphologies and replication characteristics. Treatment of the gK-V5-TEV virions with TEV protease caused approximately 32 to 34% reduction of virus entry, while treatment of gDΔTEV virions caused slightly increased virus entry. These results provide direct evidence that the gK and UL20 proteins, which are genetically and functionally linked to gB-mediated virus-induced cell fusion, are structural components of virions and function in virus entry. Site-specific cleavage of viral glycoproteins on mature and fully infectious virions utilizing unique protease sites may serve as a generalizable method of uncoupling the roles of viral glycoproteins in virus entry and virion assembly.

Multiple bromodomain genes are involved in restricting the spread of heterochromatic silencing at the Saccharomyces cerevisiae HMR-tRNA boundary.

The transfer RNA gene downstream from the HMR locus in S. cerevisiae functions as part of a boundary (barrier) element that restricts the spread of heterochromatic gene silencing into the downstream region of chromosome III. A genetic screen for identifying additional genes that, when mutated, allow inappropriate spreading of silencing from HMR through the tRNA gene was performed. YTA7, a gene containing bromodomain and ATPase homologies, was identified multiple times. Previously, others had shown that the bromodomain protein Bdf1p functions to restrict silencing at yeast euchromatin-heterochromatin boundaries; therefore we deleted nonessential bromodomain-containing genes to test their effects on heterochromatin spreading. Deletion of RSC2, coding for a component of the RSC chromatin-remodeling complex, resulted in a significant spread of silencing at HMR. Since the bromodomain of YTA7 lacks a key tyrosine residue shown to be important for acetyllysine binding in other bromodomains, we confirmed that a GST-Yta7p bromodomain fusion was capable of binding to histones in vitro. Epistasis analysis suggests that YTA7 and the HMR-tRNA function independently to restrict the spread of silencing, while RSC2 may function through the tRNA element. Our results suggest that multiple bromodomain proteins are involved in restricting the propagation of heterochromatin at HMR.

The Saccharomyces cerevisiae TRT2 tRNAThr gene upstream of STE6 is a barrier to repression in MATalpha cells and exerts a potential tRNA position effect in MATa cells.

A growing body of evidence suggests that genes transcribed by RNA polymerase III exhibit multiple functions within a chromosome. While the predominant function of these genes is the synthesis of RNA molecules, certain RNA polymerase III genes also function as genomic landmarks. Transfer RNA genes are known to exhibit extra-transcriptional activities such as directing Ty element integration, pausing of replication forks, overriding nucleosome positioning sequences, repressing neighboring genes (tRNA position effect), and acting as a barrier to the spread of repressive chromatin. This study was designed to identify other tRNA loci that may act as barriers to chromatin-mediated repression, and focused on TRT2, a tRNA(Thr) adjacent to the STE6 alpha2 operator. We show that TRT2 acts as a barrier to repression, protecting the upstream CBT1 gene from the influence of the STE6 alpha2 operator in MATalpha cells. Interestingly, deletion of TRT2 results in an increase in CBT1 mRNA levels in MATa cells, indicating a potential tRNA position effect. The transcription of TRT2 itself is unaffected by the presence of the alpha2 operator, suggesting a hierarchy that favors assembly of the RNA polymerase III complex versus assembly of adjacent alpha2 operator-mediated repressed chromatin structures. This proposed hierarchy could explain how tRNA genes function as barriers to the propagation of repressive chromatin.