Faculty Bibliography

HYPOTHESIS: Reactivation of herpes simplex virus type 1 (HSV-1) in geniculate ganglion neurons (GGNs) is an etiologic mechanism of Bell's palsy (BP) and delayed facial palsy (DFP) after otologic surgery. BACKGROUND: Several clinical studies, including temporal bone studies, antibody, titers, and intraoperative studies, suggest that reactivation of HSV-1 from latently infected GGNs may lead to both BP and DFP. However, it is difficult to study these processes in humans or live animals. METHODS: Primary cultures of GGNs were latently infected with Patton strain HSV-1 expressing a green fluorescent protein-late lytic gene chimera. Four days later, these cultures were treated with trichostatin A (TSA), a known chemical reactivator of HSV-1 in other neurons. Cultures were monitored daily by fluorescent microscopy. Titers of media from lytic, latent, and latent/TSA treated GGN cultures were obtained using plaque assays on Vero cells. RNA was harvested from latently infected GGN cultures and examined for the presence of viral transcripts using reverse transcription-polymerase chain reaction. RESULTS: Latently infected GGN cultures displayed latency-associated transcripts only, whereas lytically infected and reactivated latent cultures produced other viral transcripts, as well. The GGN cultures displayed a reactivation rate of 65% after treatment with TSA. Media from latently infected cultures contained no detectable infectious HSV-1, whereas infectious virus was observed in both lytically and latently infected/TSA-treated culture media. CONCLUSION: We have shown that cultured GGNs can be latently infected with HSV-1, and HSV-1 in these latently infected neurons can be reactivated using TSA, yielding infectious virus. These results have implications for the cause of both BP and DFP

OBJECTIVES/HYPOTHESIS: Vestibular neuritis is a common cause of both acute and chronic vestibular dysfunction. Multiple pathologies have been hypothesized to be the causative agent of vestibular neuritis; however, whether herpes simplex type I (HSV1) reactivation occurs within the vestibular ganglion has not been demonstrated previously by experimental evidence. We developed an in vitro system to study HSV1 infection of vestibular ganglion neurons (VGNs) using a cell culture model system. STUDY DESIGN: basic science study. RESULTS: Lytic infection of cultured rat VGNs was observed following low viral multiplicity of infection (MOI). Inclusion of acyclovir suppressed lytic replication and allowed latency to be established. Upon removal of acyclovir, latent infection was confirmed with reverse-transcription polymerase chain reaction and by RNA fluorescent in situ hybridization for the latency-associated transcript (LAT). A total of 29% cells in latently infected cultures were LAT positive. The lytic ICP27 transcript was not detected by reverse-transcription polymerase chain reaction (RT-PCR). Reactivation of HSV1 occurred at a high frequency in latently infected cultures following treatment with trichostatin A (TSA), a histone deactylase inhibitor. CONCLUSIONS: VGNs can be both lytically and latently infected with HSV1. Furthermore, latently infected VGNs can be induced to reactivate using TSA. This demonstrates that reactivation of latent HSV1 infection in the vestibular ganglion can occur in a cell culture model, and suggests that reactivation of HSV1 infection a plausible etiologic mechanism of vestibular neuritis

Kaposi's sarcoma-associated herpesvirus (KSHV) infected cells express the latency-associated nuclear antigen (LANA) involved in regulation of host and viral gene expression and maintenance of the KSHV latent episome. Performance of these diverse functions involves a 7-amino acid chromatin-binding motif (CBM) situated at the amino terminus of LANA that is capable of binding directly to nucleosomes. LANA interacts with additional chromatin components including methyl-CpG binding protein 2 (MeCP2). Here we show that the carboxy-terminal DNA-binding/dimerization domain of LANA provides the principal interaction with MeCP2 but that this association is modulated by the CBM. Both domains are required for LANA to co-localize with MeCP2 at chromocenters, regions of extensive pericentric heterochromatin that can be imaged by fluorescence microscopy. Within MeCP2, the methyl-CpG binding domain (MBD) is the primary determinant for chromatin localization and acts together with the adjacent repression domain (CRID/TRD) to redirect LANA to chromocenters. MeCP2 facilitates repression by LANA bound to the KSHV terminal repeats, and requires the MeCP2 C terminus in addition to the MBD and CRID/TRD. LANA and MeCP2 can also cooperate to stimulate transcription of the human E2F1 promoter which lacks a LANA DNA-binding sequence but requires both the N and C terminus of LANA. The ability of LANA to establish multivalent interactions with histones and chromatin-binding proteins such as MeCP2, would enable LANA to direct regulatory complexes to specific chromosomal sites and thereby achieve stable reprogramming of cellular gene expression in latently-infected cells

For Kaposi's sarcoma-associated herpesvirus (KSHV, HHV8), the switch from latency into active lytic replication requires RTA, the product of open reading frame 50 (ORF50). RTA activates transcription from nearly 40 early and delayed-early viral promoters mainly through interactions with cellular DNA binding proteins such as CSL/RBP-Jkappa, Oct-1, C/EBPalpha and c-Jun. Reliance on cellular co-regulators may allow KSHV to adjust its lytic program to suit different cellular contexts or interpret signals from the outside. CSL is a key component of the Notch signaling pathway and is targeted by several viruses. A search with known CSL binding sequences from cellular genes found at least 260 matches in the KSHV genome, many from regions containing known or suspected to lytic promoters. Analysis of clustered sites located immediately upstream of ORF70 (thymidylate synthase), ORF19 (tegument protein) and ORF47 (glycoprotein L) uncovered RTA-responsive promoters that were validated using mRNA isolated from KSHV-infected cells undergoing lytic reactivation. Notably, ORF19 behaves as a true-late gene, indicating that RTA regulates all three phases of the lytic program. For each new promoter, the response to RTA was dependent on CSL, and five of the ten candidate sites were shown to bind CSL in vitro. Analysis of individuals sites highlighted the importance of a cytosine residue flanking the core CSL binding sequence. These findings broaden the role for CSL in coordinating the KSHV lytic gene expression program and help define a signature motif for functional CSL sites within the viral genome

Herpes simplex virus-1 (HSV-1) establishes life-long latency in peripheral neurons where productive replication is suppressed. While periodic reactivation results in virus production, the molecular basis of neuronal latency remains incompletely understood. Using a primary neuronal culture system of HSV-1 latency and reactivation, we show that continuous signaling through the phosphatidylinositol 3-kinase (PI3-K) pathway triggered by nerve growth factor (NGF)-binding to the TrkA receptor tyrosine kinase (RTK) is instrumental in maintaining latent HSV-1. The PI3-K p110alpha catalytic subunit, but not the beta or delta isoforms, is specifically required to activate 3-phosphoinositide-dependent protein kinase-1 (PDK1) and sustain latency. Disrupting this pathway leads to virus reactivation. EGF and GDNF, two other growth factors capable of activating PI3-K and PDK1 but that differ from NGF in their ability to persistently activate Akt, do not fully support HSV-1 latency. Thus, the nature of RTK signaling is a critical host parameter that regulates the HSV-1 latent-lytic switch

In response to numerous signals, latent herpesvirus genomes abruptly switch their developmental program, aborting stable host-cell colonization in favor of productive viral replication that ultimately destroys the cell. To achieve a rapid gene expression transition, newly minted capped, polyadenylated viral mRNAs must engage and reprogram the cellular translational apparatus. While transcriptional responses of viral genomes undergoing lytic reactivation have been amply documented, roles for cellular translational control pathways in enabling the latent-lytic switch have not been described. Using PEL-derived B-cells naturally infected with KSHV as a model, we define efficient reactivation conditions and demonstrate that reactivation substantially changes the protein synthesis profile. New polypeptide synthesis correlates with 4E-BP1 translational repressor inactivation, nuclear PABP accumulation, eIF4F assembly, and phosphorylation of the cap-binding protein eIF4E by Mnk1. Significantly, inhibiting Mnk1 reduces accumulation of the critical viral transactivator RTA through a post-transcriptional mechanism, limiting downstream lytic protein production, and impairs reactivation efficiency. Thus, herpesvirus reactivation from latency activates the host cap-dependent translation machinery, illustrating the importance of translational regulation in implementing new developmental instructions that drastically alter cell fate

Protein ubiquitination provides an efficient and reversible mechanism to regulate cell cycle progression and checkpoint control. Numerous regulatory proteins direct the addition of ubiquitin to lysine residues on target proteins and these are countered by an army of deubiquitinating enzymes (DUBs). BRCA1-associated protein-1 (Bap1) is a ubiquitin carboxy-terminal hydrolase and is frequently mutated in lung and sporadic breast tumors. Bap1 can suppress growth of lung cancer cells in athymic nude mice and this requires its DUB activity. Here we show that Bap1 interacts with host cell factor-1 (HCF-1), a transcriptional cofactor found in a number of important regulatory complexes. Bap1 binds to the HCF-1 beta-propeller using a variant of the HCF-binding motif (HBM) found in herpes simplex virus VP16 and other HCF-interacting proteins. HCF-1 is K48 and K63 ubiquitinated, with a major site of linkage at lysines-1807/1808 in the HCF-1C subunit. Expression of a catalytically inactive version of Bap1 results in the selective accumulation of K48 ubiquitinated polypeptides. Depletion of Bap1 using siRNA results in a modest accumulation of HCF-1C suggesting that Bap1 helps to control cell proliferation by regulating HCF-1 protein levels and by associating with genes involved in the G1-S transition

In a recent issue of Molecular Cell, Tyagi et al. (2007) show that E2F1, a positive regulator of S phase entry, recruits cofactor HCF-1 and associated hSet1/MLL histone H3 lysine 4 methyltransferase complex, facilitating the activation of genes required for proliferation

Kaposi's sarcoma-associated herpesvirus (KSHV) is the causative agent of three malignancies associated with AIDS and immunosuppression. Tumor cells harbor latent virus and express kaposin (open reading frame [ORF] K12), v-FLIP (ORF 71), v-Cyclin (ORF 72), and latency-associated nuclear antigen (LANA; ORF 73). ORFs 71 to 73 are transcribed as multicistronic RNAs initiating from adjacent constitutive and inducible promoters upstream of ORF 73. Here we characterize a third promoter embedded within the ORF 71-to-73 transcription unit specifying transcripts that encode ORF 71/72 or K12. These transcripts may also be the source of 11 microRNAs arranged as a cluster between K12 and ORF 71. Our studies reveal a complex arrangement of interlaced transcription units, incorporating four important protein-encoding genes required for latency and pathogenesis and the entire KSHV microRNA repertoire

During latency, the Kaposi's sarcoma-associated herpesvirus genome is maintained as a circular episome, replicating in synchrony with host chromosomes. Replication requires the latency-associated nuclear antigen (LANA) and an origin of latent DNA replication located in the viral terminal repeats, consisting of two LANA binding sites (LBSs) and a GC-rich sequence. Here, we show that the recruitment of a LANA dimer to high-affinity site LBS-1 bends DNA by 57 degrees and towards the major groove. The cooccupancy of LBS-1 and lower-affinity LBS-2 induces a symmetrical bend of 110 degrees . By changing the origin architecture, LANA may help to assemble a specific nucleoprotein structure important for the initiation of DNA replication