Identification of glycosylated nucleosides in small synthetic glyco-RNAs.

Recently, the post-transcriptional modification of RNA with N-glycans was reported, changing the paradigm that RNAs are not commonly N-glycosylated. Moreover, glycan modifications of RNA are investigated for therapeutic targeting purposes. But the glyco-RNA field is in its infancy with many challenges to overcome. One question is how to accurately characterize glycosylated RNA constructs. Thus, we generated glycosylated forms of Y5 RNA mimics, a short non-coding RNA. The simple glycans lactose and sialyllactose were attached to the RNA backbone using azide-alkyne cycloadditions. Using nuclease digestion followed by LC-MS, we confirmed the presence of the glycosylated nucleosides, and characterized the chemical linkage. Next, we probed if glycosylation would affect the cellular response to Y5 RNA. We treated human foreskin fibroblasts in culture with the generated compounds. Key transcripts in the innate immune response were quantified by RT-qPCR. We found that under our experimental conditions, exposure of cells to the Y5 RNA did not trigger an interferon response, and glycosylation of this RNA did not have an impact. Thus, we have identified a successful approach to chemically characterize synthetic glyco-RNAs, which will be critical for further studies to elucidate how the presence of complex glycans on RNA affects the cellular response.

Entry of the Varicellovirus Canid herpesvirus 1 into Madin-Darby canine kidney epithelial cells is pH-independent and occurs via a macropinocytosis-like mechanism but without increase in fluid uptake.

Canid herpesvirus 1 (CHV-1) is a Varicellovirus that causes self-limiting infections in adult dogs but morbidity and mortality in puppies. Using a multipronged approach, we discovered the CHV-1 entry pathway into Madin-Darby canine kidney (MDCK) epithelial cells. We found that CHV-1 triggered extensive host cell membrane lamellipodial ruffling and rapid internalisation of virions in large, uncoated vacuoles, suggestive of macropinocytosis. Treatment with inhibitors targeting key macropinocytosis factors, including inhibitors of Na+ /H+ exchangers, F-actin, myosin light-chain kinase, protein kinase C, p21-activated kinase, phosphatidylinositol-3-kinase and focal adhesion kinase, significantly reduced viral replication. Moreover, the effect was restricted to exposure to the inhibitors early in infection, confirming a role for the macropinocytic machinery during entry. The profile of inhibitors also suggested a role for signalling via integrins and receptor tyrosine kinases in viral entry. In contrast, inhibitors of clathrin, caveolin, microtubules and endosomal acidification did not affect CHV-1 entry into MDCK cells. We found that the virus colocalised with the fluid-phase uptake marker dextran; however, surprisingly, CHV-1 infection did not enhance the uptake of dextran. Thus, our results indicate that CHV-1 uses a macropinocytosis-like, pH-independent entry pathway into MDCK cells, which nevertheless is not based on stimulation of fluid uptake. TAKE AWAYS: CHV-1 enters epithelial cells via a macropinocytosis-like mechanism. CHV-1 induces extensive lamellipodial ruffling. CHV-1 entry into MDCK cells is pH-independent.

Rel-Dependent Immune and Central Nervous System Mechanisms Control Viral Replication and Inflammation during Mouse Herpes Simplex Encephalitis.

Herpes simplex encephalitis (HSE), caused by HSV type 1 (HSV-1) infection, is an acute neuroinflammatory condition of the CNS and remains the most common type of sporadic viral encephalitis worldwide. Studies in humans have shown that susceptibility to HSE depends in part on the genetic make-up of the host, with deleterious mutations in the TLR3/type I IFN axis underlying some cases of childhood HSE. Using an in vivo chemical mutagenesis screen for HSV-1 susceptibility in mice, we identified a susceptible pedigree carrying a causal truncating mutation in the Rel gene (RelC307X ), encoding for the NF-κB transcription factor subunit c-Rel. Like Myd88-/- and Irf3-/- mice, RelC307X mice were susceptible to intranasal HSV-1 infection. Reciprocal bone marrow transfers into lethally irradiated hosts suggested that defects in both hematopoietic and CNS-resident cellular compartments contributed together to HSE susceptibility in RelC307X mice. Although the RelC307X mutation maintained cell-intrinsic antiviral control, it drove increased apoptotic cell death in infected fibroblasts. Moreover, reduced numbers of CD4+CD25+Foxp3+ T regulatory cells, and dysregulated NK cell and CD4+ effector T cell responses in infected RelC307X animals, indicated that protective immunity was also compromised in these mice. In the CNS, moribund RelC307X mice failed to control HSV-1 viral replication in the brainstem and cerebellum, triggering cell death and elevated expression of Ccl2, Il6, and Mmp8 characteristic of HSE neuroinflammation and pathology. In summary, our work implicates c-Rel in both CNS-resident cell survival and lymphocyte responses to HSV-1 infection and as a novel cause of HSE disease susceptibility in mice.

A Mutation in the UL24 Gene Abolishes Expression of the Newly Identified UL24.5 Protein of Herpes Simplex Virus 1 and Leads to an Increase in Pathogenicity in Mice.

Herpes simplex virus 1 (HSV-1) infects the host via epithelia and establishes latency in sensory neurons. The UL24 gene is conserved throughout the Herpesviridae family, and the UL24 protein is important for efficient viral replication and pathogenesis. Multiple transcripts are expressed from the UL24 gene. The presence of a transcription initiation site inside the open reading frame of UL24 and an ATG start codon in the same open reading frame led us to suspect that another protein was expressed from the UL24 locus. To test our hypothesis, we constructed a recombinant virus that expresses a hemagglutinin tag at the C terminus of UL24. Western blot analysis revealed the expression of an 18-kDa protein that is not a degradation product of the full-length UL24, which we refer to as UL24.5. Ectopically expressed UL24.5 did not induce the dispersal of nucleolar proteins, as seen for UL24. In order to characterize the role of UL24.5, we constructed a mutant virus encoding a substitution of the predicted initiation methionine to a valine. This substitution eliminated the expression of the 18-kDa polypeptide. Unlike the UL24-null mutant (UL24X), which exhibits reduced viral yields, the UL24.5-null mutant exhibited the same replication phenotype in cell culture as the parental strain. However, in a murine ocular infection model, we observed an increase in the incidence of neurological disorders with the UL24.5 mutant. Alignment of amino acid sequences for various herpesviruses revealed that the initiation site of UL24.5 is conserved among HSV-1 strains and is present in many herpesviruses.IMPORTANCE We discovered a new HSV-1 protein, UL24.5, which corresponds to the C-terminal portion of UL24. In contrast to the replication defects observed with HSV-1 strains that do not express full-length UL24, the absence of UL24.5 did not affect viral replication in cell culture. Moreover, in mice, the absence of UL24.5 did not affect viral titers in epithelia or trigeminal ganglia during acute infection; however, it was associated with a prolonged persistence of signs of inflammation. Strikingly, the absence of UL24.5 also led to an increase in the incidence of severe neurological impairment compared to results for wild-type control viruses. This increase in pathogenicity is in stark contrast to the reduction in clinical signs associated with the absence of full-length UL24. Bioinformatic analyses suggest that UL24.5 is conserved among all human alphaherpesviruses and in some nonhuman alphaherpesviruses. Thus, we have identified UL24.5 as a new HSV-1 determinant of pathogenesis.

Dok-1 and Dok-2 Are Required To Maintain Herpes Simplex Virus 1-Specific CD8+ T Cells in a Murine Model of Ocular Infection.

Dok-1 and Dok-2 negatively regulate responses downstream of several immune receptors in lymphoid and myeloid cells. Recent evidence showed that Dok proteins are essential in the formation of memory CD8+ T cells to an exogenous epitope expressed by vaccinia virus; however, the importance of Dok-1 and Dok-2 in the control of viral infection is unknown. Here, we investigated the role of Dok proteins in modulating the immune response against herpes simplex virus 1 (HSV-1) in a mouse model of ocular infection. During acute infection, viral titers in the eye were similar in wild-type (WT) and Dok-1 and Dok-2 double-knockout (DKO) mice, and the percentages of infiltrating leukocytes were similar in DKO and WT corneas and trigeminal ganglia (TG). DKO mice exhibited a diminished CD8+ T cell response to the immunodominant HSV-1 glycoprotein B (gB) epitope in the spleen and draining lymph nodes compared to WT mice during acute infection. Remarkably, gB-specific CD8+ T cells almost completely disappeared in the spleens of DKO mice during latency, and the reduction of CD8+ effector memory T (Tem) cells was more severe than that of CD8+ central memory T (Tcm) cells. The percentage of gB-specific CD8+ T cells in TG during latency was also dramatically reduced in DKO mice; however, they were phenotypically similar to those from WT mice. In ex vivo assays, reactivation was detected earlier in TG cultures from infected DKO versus WT mice. Thus, Dok-1 and Dok-2 promote survival of gB-specific CD8+ T cells in TG latently infected with HSV-1.IMPORTANCE HSV-1 establishes lifelong latency in sensory neurons of trigeminal ganglia (TG). In humans, HSV-1 is able to sporadically reactivate from latently infected neurons and establish a lytic infection at a site to which the neurons project. Most herpetic disease in humans is due to reactivation of HSV-1 from latency rather than to primary acute infection. CD8+ T cells are thought to play an important role in controlling recurrent infections. In this study, we examined the involvement of Dok-1 and Dok-2 signaling proteins in the control of HSV-1 infection. We provide evidence that Dok proteins are required to maintain a CD8+ T cell response against HSV-1 during latency-especially CD8+ Tem cells-and that they negatively affect HSV-1 reactivation from latency. Elucidating Dok-mediated mechanisms involved in the control of HSV-1 reactivation from latency might contribute to the development of therapeutic strategies to prevent recurrent HSV-1-induced pathology.

Herpes Simplex Virus 1 UL24 Abrogates the DNA Sensing Signal Pathway by Inhibiting NF-κB Activation.

Cyclic GMP-AMP synthase (cGAS) is a newly identified DNA sensor that recognizes foreign DNA, including the genome of herpes simplex virus 1 (HSV-1). Upon binding of viral DNA, cGAS produces cyclic GMP-AMP, which interacts with and activates stimulator of interferon genes (STING) to trigger the transcription of antiviral genes such as type I interferons (IFNs), and the production of inflammatory cytokines. HSV-1 UL24 is widely conserved among members of the herpesviruses family and is essential for efficient viral replication. In this study, we found that ectopically expressed UL24 could inhibit cGAS-STING-mediated promoter activation of IFN-ß and interleukin-6 (IL-6), and UL24 also inhibited interferon-stimulatory DNA-mediated IFN-ß and IL-6 production during HSV-1 infection. Furthermore, UL24 selectively blocked nuclear factor κB (NF-κB) but not IFN-regulatory factor 3 promoter activation. Coimmunoprecipitation analysis demonstrated that UL24 bound to the endogenous NF-κB subunits p65 and p50 in HSV-1-infected cells, and UL24 was also found to bind the Rel homology domains (RHDs) of these subunits. Furthermore, UL24 reduced the tumor necrosis factor alpha (TNF-α)-mediated nuclear translocation of p65 and p50. Finally, mutational analysis revealed that the region spanning amino acids (aa) 74 to 134 of UL24 [UL24(74-134)] is responsible for inhibiting cGAS-STING-mediated NF-κB promoter activity. For the first time, UL24 was shown to play an important role in immune evasion during HSV-1 infection.IMPORTANCE NF-κB is a critical component of the innate immune response and is strongly induced downstream of most pattern recognition receptors (PRRs), leading to the production of IFN-ß as well as a number of inflammatory chemokines and interleukins. To establish persistent infection, viruses have evolved various mechanisms to counteract the host NF-κB pathway. In the present study, for the first time, HSV-1 UL24 was demonstrated to inhibit the activation of NF-κB in the DNA sensing signal pathway via binding to the RHDs of the NF-κB subunits p65 and p50 and abolishing their nuclear translocation.

Mutation of UL24 impedes the dissemination of acute herpes simplex virus 1 infection from the cornea to neurons of trigeminal ganglia.

Herpes simplex virus 1 (human herpesvirus 1) initially infects epithelial cells of the mucosa and then goes on to infect sensory neurons leading ultimately to a latent infection in trigeminal ganglia (TG). UL24 is a core herpesvirus gene that has been identified as a determinant of pathogenesis in several Alphaherpesvirinae, although the underlying mechanisms are unknown. In a mouse model of ocular infection, a UL24-deficient virus exhibited a reduction in viral titres in tear films of 1 log10, whilst titres in TG are often below the level of detection. Moreover, the efficiency of reactivation from latency was also severely reduced. Herein, we investigated how UL24 contributed to acute infection of TG. Our results comparing the impact of UL24 on viral titres in eye tissue versus in tear films did not reveal a general defect in virus release from the cornea. We also found that the impairment of replication seen in mouse primary embryonic neurons with a UL24-deficient virus was not more severe than that observed in an epithelial cell line. Rather, in situ histological analyses revealed that infection with a UL24-deficient virus led to a significant reduction in the number of acutely infected neurons at 3 days post-infection (p.i.). Moreover, there was a significant reduction in the number of neurons positive for viral DNA at 2 days p.i. for the UL24-deficient virus as compared with that observed for WT or a rescue virus. Our results supported a model whereby UL24 functions in the dissemination of acute infection from the cornea to neurons in TG.

Genome-wide mouse mutagenesis reveals CD45-mediated T cell function as critical in protective immunity to HSV-1.

Herpes simplex encephalitis (HSE) is a lethal neurological disease resulting from infection with Herpes Simplex Virus 1 (HSV-1). Loss-of-function mutations in the UNC93B1, TLR3, TRIF, TRAF3, and TBK1 genes have been associated with a human genetic predisposition to HSE, demonstrating the UNC93B-TLR3-type I IFN pathway as critical in protective immunity to HSV-1. However, the TLR3, UNC93B1, and TRIF mutations exhibit incomplete penetrance and represent only a minority of HSE cases, perhaps reflecting the effects of additional host genetic factors. In order to identify new host genes, proteins and signaling pathways involved in HSV-1 and HSE susceptibility, we have implemented the first genome-wide mutagenesis screen in an in vivo HSV-1 infectious model. One pedigree (named P43) segregated a susceptible trait with a fully penetrant phenotype. Genetic mapping and whole exome sequencing led to the identification of the causative nonsense mutation L3X in the Receptor-type tyrosine-protein phosphatase C gene (Ptprc(L3X)), which encodes for the tyrosine phosphatase CD45. Expression of MCP1, IL-6, MMP3, MMP8, and the ICP4 viral gene were significantly increased in the brain stems of infected Ptprc(L3X) mice accounting for hyper-inflammation and pathological damages caused by viral replication. Ptprc(L3X) mutation drastically affects the early stages of thymocytes development but also the final stage of B cell maturation. Transfer of total splenocytes from heterozygous littermates into Ptprc(L3X) mice resulted in a complete HSV-1 protective effect. Furthermore, T cells were the only cell population to fully restore resistance to HSV-1 in the mutants, an effect that required both the CD4⁺ and CD8⁺ T cells and could be attributed to function of CD4⁺ T helper 1 (Th1) cells in CD8⁺ T cell recruitment to the site of infection. Altogether, these results revealed the CD45-mediated T cell function as potentially critical for infection and viral spread to the brain, and also for subsequent HSE development.

Identification of Risk Factors for Chemotherapy-Related 30-Day Readmissions.

PURPOSE: The goal of this study was to develop a method for practitioners to evaluate both quality of care delivered to patients receiving chemotherapy and illicit risk factors for 30-day chemotherapy-related readmissions (CRR). METHODS: Midas+ DataVision Readmission Tool Pack (Version 2.x) was used to retrospectively identify patients who received inpatient chemotherapy from April 2010 through May 2013. The population was screened for unscheduled admissions within 30 days after discharge. A multidisciplinary team was used to attribute readmissions to chemotherapy administration. Demographic information and oncology-specific characteristics were collected. The CRR rate and relative risk for readmission were calculated for each characteristic. RESULTS: A baseline CRR rate of 11.1% was established. Risk factors associated with an increased risk for experiencing a CRR included age of 65 years or older, hematologic cancer diagnosis, first cycle chemotherapy, Medicare coverage, discharge to a skilled nursing facility, and anthracycline administration. CONCLUSIONS: A baseline CRR rate was established. Institution-specific 30-day CRR risk factors were elucidated. Modifiable risk factors included discharge to a skilled nursing facility and administration of an anthracycline. Further investigation for opportunities for quality improvement in these 2 risk factors is a topic for future research. Expanded research into chemotherapy-related toxicities requiring inpatient admission/readmission outside of clinical trials is warranted.

The Disruption of a Nuclear Export Signal in the C-Terminus of the Herpes Simplex Virus 1 Determinant of Pathogenicity UL24 Protein Leads to a Syncytial Plaque Phenotype.

UL24 of herpes simplex virus 1 (HSV-1) has been shown to be a determinant of pathogenesis in mouse models of infection. The N-terminus of UL24 localizes to the nucleus and drives the redistribution of nucleolin and B23. In contrast, when expressed alone, the C-terminal domain of UL24 accumulates in the Golgi apparatus; its importance during infection is unknown. We generated a series of mammalian expression vectors encoding UL24 with nested deletions in the C-terminal domain. Interestingly, enhanced nuclear staining was observed for several UL24-deleted forms in transient transfection assays. The substitution of a threonine phosphorylation site had no effect on UL24 localization or viral titers in cell culture. In contrast, mutations targeting a predicted nuclear export signal (NES) significantly enhanced nuclear localization, indicating that UL24 is able to shuttle between the nucleus and the cytoplasm. Recombinant viruses that encode UL24-harboring substitutions in the NES led to the accumulation of UL24 in the nucleus. Treatment with the CRM-1-specific inhibitor leptomycin B blocked the nuclear export of UL24 in transfected cells but not in the context of infection. Viruses encoding UL24 with NES mutations resulted in a syncytial phenotype, but viral yield was unaffected. These results are consistent with a role for HSV-1 UL24 in late cytoplasmic events in HSV-1 replication.

The ribonucleotide reductase R1 subunits of herpes simplex virus 1 and 2 protect cells against poly(I · C)-induced apoptosis.

We recently provided evidence that the ribonucleotide reductase R1 subunits of herpes simplex virus types 1 and 2 (HSV-1 and -2) protect cells against tumor necrosis factor alpha- and Fas ligand-induced apoptosis by interacting with caspase 8. Double-stranded RNA (dsRNA) is a viral intermediate known to initiate innate antiviral responses. Poly(I · C), a synthetic analogue of viral dsRNA, rapidly triggers caspase 8 activation and apoptosis in HeLa cells. Here, we report that HeLa cells after HSV-1 and HSV-2 infection were quickly protected from apoptosis caused by either extracellular poly(I · C) combined with cycloheximide or transfected poly(I · C). Cells infected with the HSV-1 R1 deletion mutant ICP6Δ were killed by poly(I · C), indicating that HSV-1 R1 plays a key role in antiapoptotic responses to poly(I · C). Individually expressed HSV R1s counteracted caspase 8 activation by poly(I · C). In addition to their binding to caspase 8, HSV R1s also interacted constitutively with receptor-interacting protein 1 (RIP1) when expressed either individually or with other viral proteins during HSV infection. R1(1-834)-green fluorescent protein (GFP), an HSV-2 R1 deletion mutant protein devoid of antiapoptotic activity, did not interact with caspase 8 and RIP1, suggesting that these interactions are required for protection against poly(I · C). HSV-2 R1 inhibited the interaction between the Toll/interleukin-1 receptor domain-containing adaptor-inducing beta interferon (IFN-ß) (TRIF) and RIP1, an interaction that is essential for apoptosis triggered by extracellular poly(I · C) plus cycloheximide or TRIF overexpression. TRIF silencing reduced poly(I · C)-triggered caspase 8 activation in mock- and ICP6Δ-infected cells, confirming that TRIF is involved in poly(I · C)-induced apoptosis. Thus, by interacting with caspase 8 and RIP1, HSV R1s impair the apoptotic host defense mechanism prompted by dsRNA.

Canid herpesvirus 1 Preferentially Infects Polarized Madin-Darby Canine Kidney Cells from the Basolateral Surface.

Canid herpesvirus 1 (CHV-1) infects polarized canine epithelia. Herein, we present our initial work characterizing CHV-1 infection of Madin-Darby canine kidney (MDCK) cells that were polarized on trans-wells. We previously showed that infection of these cells in non-polarized cultures stimulated the formation of extensive lamellipodial membrane protrusions. Uninfected polarized MDCK cells already form extensive lamellipodial membrane protrusions on the apical surface in the absence of virus. Using scanning electron microscopy, we found that CHV-1 infection does not lead to a change in the form of the lamellipodial membrane protrusions on the apical surface of polarized MDCK cells. We found that CHV-1 was able to infect polarized cultures from either the apical or basolateral side; however, higher viral titers were produced upon infection of the basolateral side. Regardless of the side infected, titers of virus were higher in the apical compartment compared to the basal compartment; however, these differences were not statistically significant. In addition to cell-free virus that was recovered in the media, the highest amount of virus produced remained cell-associated over the course of the experiment. The efficiency of CHV-1 infection of the basolateral side of polarized epithelial cells is consistent with the pathobiology of this varicellovirus.

Relocalization of upstream binding factor to viral replication compartments is UL24 independent and follows the onset of herpes simplex virus 1 DNA synthesis.

Herpes simplex virus 1 (HSV-1) induces relocalization of several nucleolar proteins. We have found that, as for fibrillarin, the HSV-1-induced redistribution of two RNA polymerase I components, upstream binding factor (UBF) and RPA194, was independent of the viral protein UL24, which affects nucleolin localization. Nevertheless, the kinetics and sites of redistribution for fibrillarin and UBF differed. Interestingly, UBF remained associated with RPA194 during infection. Although UBF is redistributed to viral replication compartments during infection, we did not detect foci of UBF at early sites of viral DNA synthesis, suggesting that it may not be directly involved in this process at early times.

Conserved residues in the UL24 protein of herpes simplex virus 1 are important for dispersal of the nucleolar protein nucleolin.

The UL24 family of proteins is widely conserved among herpesviruses. We demonstrated previously that UL24 of herpes simplex virus 1 (HSV-1) is important for the dispersal of nucleolin from nucleolar foci throughout the nuclei of infected cells. Furthermore, the N-terminal portion of UL24 localizes to nuclei and can disperse nucleolin in the absence of any other viral proteins. In this study, we tested the hypothesis that highly conserved residues in UL24 are important for the ability of the protein to modify the nuclear distribution of nucleolin. We constructed a panel of substitution mutations in UL24 and tested their effects on nucleolin staining patterns. We found that modified UL24 proteins exhibited a range of subcellular distributions. Mutations associated with a wild-type localization pattern for UL24 correlated with high levels of nucleolin dispersal. Interestingly, mutations targeting two regions, namely, within the first homology domain and overlapping or near the previously identified PD-(D/E)XK endonuclease motif, caused the most altered UL24 localization pattern and the most drastic reduction in its ability to disperse nucleolin. Viral mutants corresponding to the substitutions G121A and E99A/K101A both exhibited a syncytial plaque phenotype at 39 degrees C. vUL24-E99A/K101A replicated to lower titers than did vUL24-G121A or KOS. Furthermore, the E99A/K101A mutation caused the greatest impairment of HSV-1-induced dispersal of nucleolin. Our results identified residues in UL24 that are critical for the ability of UL24 to alter nucleoli and further support the notion that the endonuclease motif is important for the function of UL24 during infection.

Differential importance of highly conserved residues in UL24 for herpes simplex virus 1 replication in vivo and reactivation.

The UL24 gene of herpes simplex virus 1 (HSV-1) is widely conserved among all subfamilies of the Herpesviridae. It is one of only four HSV-1 genes for which mutations have been mapped that confer a syncytial plaque phenotype. In a mouse model of infection, UL24-deficient viruses exhibit reduced titres, particularly in neurons, and an apparent defect in reactivation from latency. There are several highly conserved residues in UL24; however, their importance in the role of UL24 in vivo is unknown. In this study, we compared virus strains with substitution mutations corresponding to the PD-(D/E)XK endonuclease motif of UL24 (vUL24-E99A/K101A) or a substitution of another highly conserved residue (vUL24-G121A). Both mutant viruses cause the formation of syncytial plaques at 39 degrees C; however, we found that the viruses differed dramatically when tested in a mouse model of infection. vUL24-E99A/K101A exhibited titres in the eye that were 10-fold lower than those of the wild-type virus KOS, and titres in trigeminal ganglia (TG) that were more than 2 log10 lower. Clinical signs were barely detectable with vUL24-E99A/K101A. Furthermore, the percentage of TG from which virus reactivated was also significantly lower for this mutant than for KOS. In contrast, vUL24-G121A behaved similarly to the wild-type virus in mice. These results are consistent with the endonuclease motif being important for the role of UL24 in vivo and also imply that the UL24 temperature-dependent syncytial plaque phenotype can be separated genetically from several in vivo phenotypes.

Herpes simplex virus 1 infection of T cells causes VP11/12-dependent phosphorylation and degradation of the cellular protein Dok-2.

Previous studies have shown that HSV-1 infection of lymphocytes induces the tyrosine phosphorylation of several proteins that might correspond to viral or host proteins. VP11/12, a viral tegument protein, is the major HSV-induced tyrosine phosphorylated protein identified thus far. In this report, we demonstrated that the cellular adaptor proteins Dok-2 and Dok-1 are tyrosine phosphorylated upon HSV-1 infection. In addition, HSV-1 induced the selective degradation of Dok-2. Finally, we provide evidence that Dok-2 interacts with VP11/12, and that HSV-induced tyrosine phosphorylation and degradation of Dok-2 require VP11/12. Inactivation of either the Src Family Kinases binding motifs or the SHC binding motif of VP11/12 eliminated the interaction of Dok-2 with VP11/12. Elimination of the binding of Dok-2 to VP11/12 prevented Dok-2 phosphorylation and degradation. We propose that HSV-induced Dok phosphorylation and Dok-2 degradation is an immune evasion mechanism to inactivate T cells that might play an important role in HSV pathogenesis.

Regulation of viral gene expression by the herpes simplex virus 1UL24 protein (HSV-1UL24 inhibits accumulation of viral transcripts).

UL24 is conserved among all Herpesviridae. In herpes simplex virus 1 (HSV-1), UL24 mutations lead to reduced viral titers both in cell culture and in vivo, and reduced pathogenicity. The human cytomegalovirus ortholog of UL24 has a gene regulatory function; however, it is not known whether other UL24 orthologs also affect gene expression. We discovered that in co-transfection experiments, expression of UL24 correlated with a reduction in the expression of several viral proteins and transcripts. Substitution mutations targeting conserved residues in UL24 impaired this function. Reduced transcript levels did not appear attributable to changes in mRNA stability. The UL24 ortholog of Herpes B virus exhibited a similar activity. An HSV-1 mutant that does not express UL24 produced more viral R1 and R2 transcripts than the wild type or rescue virus relative to the amount of viral DNA. These results reveal a new role for HSV-1UL24 in regulating viral mRNA accumulation.

Upstream binding factor inhibits herpes simplex virus replication.

Herpes simplex virus 1 (HSV-1) infection induces changes to the host cell nucleus including relocalization of the cellular protein Upstream Binding Factor (UBF) from the nucleolus to viral replication compartments (VRCs). Herein, we tested the hypothesis that UBF is recruited to VRCs to promote viral DNA replication. Surprisingly, infection of UBF-depleted HeLa cells with HSV-1 or HSV-2 produced higher viral titers compared to controls. Reduced expression of UBF also led to a progressive increase in the relative amount of HSV-1 DNA versus controls, and increased levels of HSV-1 ICP27 and TK mRNA and protein, regardless of whether viral DNA replication was inhibited or not. Our results suggest that UBF can inhibit gene expression from viral DNA prior to its replication. A similar but smaller effect on viral titers was observed in human foreskin fibroblasts. This is the first report of UBF having a restrictive effect on replication of a virus.

Visualization of mouse neuronal ganglia infected by Herpes Simplex Virus 1 (HSV-1) using multimodal non-linear optical microscopy.

Herpes simplex virus 1 (HSV-1) is a neurotropic virus that causes skin lesions and goes on to enter a latent state in neurons of the trigeminal ganglia. Following stress, the virus may reactivate from latency leading to recurrent lesions. The in situ study of neuronal infections by HSV-1 is critical to understanding the mechanisms involved in the biology of this virus and how it causes disease; however, this normally requires fixation and sectioning of the target tissues followed by treatment with contrast agents to visualize key structures, which can lead to artifacts. To further our ability to study HSV-1 neuropathogenesis, we have generated a recombinant virus expressing a second generation red fluorescent protein (mCherry), which behaves like the parental virus in vivo. By optimizing the application of a multimodal non-linear optical microscopy platform, we have successfully visualized in unsectioned trigeminal ganglia of mice both infected cells by two-photon fluorescence microscopy, and myelinated axons of uninfected surrounding cells by coherent anti-Stokes Raman scattering (CARS) microscopy. These results represent the first report of CARS microscopy being combined with 2-photon fluorescence microscopy to visualize virus-infected cells deep within unsectioned explanted tissue, and demonstrate the application of multimodal non-linear optical microscopy for high spatial resolution biological imaging of tissues without the use of stains or fixatives.

The UL24 protein of herpes simplex virus 1 affects the sub-cellular distribution of viral glycoproteins involved in fusion.

Mutations in UL24 of herpes simplex virus type 1 can lead to a syncytial phenotype. We hypothesized that UL24 affects the sub-cellular distribution of viral glycoproteins involved in fusion. In non-immortalized human foreskin fibroblasts (HFFs) we detected viral glycoproteins B (gB), gD, gH and gL present in extended blotches throughout the cytoplasm with limited nuclear membrane staining; however, in HFFs infected with a UL24-deficient virus (UL24X), staining for the viral glycoproteins appeared as long, thin streaks running across the cell. Interestingly, there was a decrease in co-localized staining of gB and gD with F-actin at late times in UL24X-infected HFFs. Treatment with chemical agents that perturbed the actin cytoskeleton hindered the formation of UL24X-induced syncytia in these cells. These data support a model whereby the UL24 syncytial phenotype results from a mislocalization of viral glycoproteins late in infection.