Rozanne M. Sandri-Goldin - US grants

The regulation of herpes simplex virus type 1 (HSV-1) gene expression is a highly complex process. Following primary exposure or reactivation, HSV-1 undergoes lytic growth, during which it can cause a wide spectrum of human diseases ranging from minor skin ulcerations to fatal neonatal and central nervous system diseases. On the other hand, the virus can remain latent and undetectable in the trigeminal ganglion for the life of an individual. It is clear that HSV-1 gene expression is regulated differently during these two phases of viral infection. In addition, lytic HSV-1 infection of tissue culture cells in vitro is regulated in a complex manner. Viral encoded inducers act on specific sequences in the promoter-regulatory regions of HSV-1 genes to modulate expression of those genes. To identify the specific sequences involved in the regulation of an HSV-1 early gene and to analyze the interaction of specific inducers with those sequences, the regulation of the glycoprotein D (gD) gene will be studied. To do this, a series of alteration will be introduced into the regulatory region of the gD gene. The effects of these regulatory alterations will be determined by examining transient expression of gD following transfer of the gD gene to mammalian cells, by analyzing gD expression in stably transformed cell lines, and by studying gD regulation during viral infection. Induction of GD expression in the transient expression assays and in the stable cell lines will be accomplished by infection with HSV-1. During such infection, it is known that the oc gene product ICP4 is the primary inducer, so the sequences that interact with ICP4 will be investigated. However, preliminary studies suggest that gD may also be induced by two other oc products, ICP22 and ICP47, even though no inducer activity has previously been demonstrated for either of these two genes. In stable cell lines containing the genes for ICP22 and ICP47 along with the gene for gD, ICP22 and/or ICP47 induced gD expression. We will continue these studies to determine which of these proteins acts as the inducer, and if the sequences in the gD regulatory region which respond to ICP22/ICP47 induction are the same ones which respond to ICP4 induction.

DESCRIPTION (provided by applicant): Herpes simplex virus type 1 (HSV-1) immediate-early protein ICP27 is a multifunctional regulatory protein that is essential for viral lytic infection. ICP27 contributes to the shut off of host protein synthesis and is required for the appropriate expression of viral early and late gene products. While ICP27 has been shown to affect the transcription of late genes, it acts predominantly at the post-transcriptional level affecting RNA processing and export. ICP27 performs its activities by interacting with RNA and with a myriad of proteins. ICP27 binds viral RNAs to facilitate their export. Further, ICP27 interacts with itself to form multimers, and with HSV-1 proteins ICP4 and ICP8, as well as with cellular proteins including: SR splicing proteins, SR protein kinase 1, spliceosomal protein SAP145, hnRNP K, CK2, RNA export protein Aly/REF, mRNA export receptor TAP/NXF1 and RNA polymerase II. The regions of ICP27 involved in these interactions have been broadly mapped to protein motifs in both the N-terminal and C-terminal halves of the molecule. Although several protein motifs were identified based upon sequence comparisons, no structural information on ICP27 has been generated. The goals of this project are to elucidate the structure of the functional domains of ICP27 and to chart the complete array of its dynamic interactions during viral infection. The specific aims are: 1) To perform a structural analysis of the functional domains of ICP27 by probing the solution structure of the N-terminal leucine-rich NES-like sequence, the highly acidic region, the NLS, the RGG box RNA-binding domain and R2, a second arginine-rich region; and the C-terminal KH RNA binding domains and zinc-finger domain by nuclear magnetic resonance; 2) to create mutations in critical residues that will inactivate the structure of each domain and to test those mutations for their effects on the activities and interactions of ICP27 during infection; and 3) to chart the dynamics of the interactions of ICP27 during viral infection with wild type and mutant viruses bearing targeted structural motif mutations using fluorescence microscopy strategies to view the interactions of ICP27 with cellular and viral factors throughout the infectious cycle in living cells; and to elucidate the full array of ICP27 interacting partners and its participation in different protein complexes by purifying and analyzing these complexes biochemically and by Mass Spectrometry.

DESCRIPTION (provided by applicant): The Viruses and Cells Gordon Research Conference (GRC) is the premier conference in the field of virology, which covers all aspects of virus infection, from entry into the cell through replication and assembly, to uncovering the molecular basis of pathogenesis, to prevention and therapy. The 2009 meeting will held at the GRC conference site, Il Ciocco in Barga, Italy from June 7-12, 2009. The invited speakers will present cutting edge findings on the dynamic interactions of viruses with the cells and organisms they infect as well as host counter responses to infection. Discussion leaders, who are expert in the topics covered in each session, will lead lively and open discussions following each presentation, as has always been the central theme of Gordon Conferences. In addition to the invited speakers, shorter talks will be chosen from among the abstracts that are submitted for poster sessions at the time of registration. Selections will be made to highlight exciting new developments in virus research with the emphasis on the selection of younger scientists for these presentations, including new investigators, postdoctoral fellows and graduate students. The short talks will also be followed by active, open discussions. The evening poster sessions are a prominent and important aspect of this conference providing yet another opportunity for participants to present and discuss their research findings. The meeting participants will include well known established investigators who have made seminal contributions to the field, young investigators launching their independent research careers, as well as postdoctoral fellows and graduate students and investigators from industry. Funding is requested to provide support to cover conference registration fees and/or travel costs for participants, and principally for young investigators who have been invited as speakers or discussion leaders, as well as for promising new investigators, postdoctoral fellows, graduate students and investigators from developing nations who present short talks.

DESCRIPTION (provided by applicant): Herpes simplex virus 1 (HSV-1) causes diseases that range from painful skin lesions to keratitis and encephalitis. HSV-1 encodes an essential protein called ICP27 that is involved in a diversity of functions during viral infection. ICP27 has the intriguing ability to interact with viral mRNA and a multiplicity of host cell proteins, hijacking hem to benefit virus production. Thus, ICP27 could conceivably serve as a target for antiviral intervention. The future development of antivirals requires an understanding of how the cooperative assembly of these essential multicomponent complexes occurs and how the assembly is regulated. We will study the role of cooperativity in protein-protein and protein-RNA interactions mediated by ICP27 and characterize the binding interfaces of its complexes to determine how ICP27 is assembled in different complexes during viral infection, with the goal of revealing the molecular mechanism of its function. The structural information will be used to explore the role of interaction interface residues in ICP27 function during infection. ICP27 consists of a number of structured as well as intrinsically-unstructured domains, which participate in a large number of diverse protein-protein and protein-RNA interactions. We hypothesize that these interactions may be multi-site and therefore cooperative, mediated by regions that may be distant in the primary sequence. We postulate that transient interactions also are possible, especially when unfolded regions are involved. We hypothesize that at least some of ICP27's interactions are regulated by phosphorylation and/or arginine methylation. We will test these hypotheses in two specific aims: 1) To study the direct binding interfaces of ICP27 with cellular proteins and viral mRNA by generating shorter protein constructs in which structural stability is not perturbed, and expressing them in amounts sufficient for structural biology studie including traditional and novel Isotopically-Discriminated (IDIS) NMR spectroscopy as well as biophysical approaches. Cooperativity will be analyzed in interactions between short functional fragments of intrinsically unstructured N-terminal ICP27 and partners that interact within this region, including viral RNA and host cell proteins. In vitro post-translational modifications of ICP27 will be performed to explore how ICP27-multi-protein complex assembly is affected by phosphorylation and arginine methylation. We will also endeavor to perform structural studies of folded domains from the C-terminal part of ICP27 and to characterize interactions with its cellular protein partners. Because ICP27 undergoes a head-to-tail intramolecular interaction in vivo, a chimera containing appropriate fragments of the N- and C-termini could be created to study how these regions interact. 2) To explore the role of interaction interface residues in ICP27 function during viral infection, recombinant viruses will be constructed bearing point mutations at interaction interfaces and will be characterized for in vivo interactions and ICP27 functional activities and effects on viral infection.

Project summary: Herpes simplex virus 1 (HSV-1) is a highly contagious pathogen that causes a number of diseases ranging from painful skin lesions to keratitis and encephalitis. The gene expression program of HSV-1 has been extensively studied for the past several decades. Despite the intensive effort, however, it remains unclear how HSV-1 suppresses host gene expression to allow efficient viral replication. Genetic studies have demonstrated that ICP27, encoded by an essential immediate early gene, plays an essential role in the inhibition of host mRNA biogenesis. Based on in vitro assays, earlier studies have suggested that ICP27 blocks transcription and splicing of host genes. Through high throughput analyses of host gene expression following HSV-1 infection or ICP27 overexpression, however, recent studies detected no global inhibition of either transcription or splicing, but only splicing changes in a small number of genes. Interestingly, these studies found that there was widespread transcription termination defect in HSV-1-infected cells and that this inhibition was specific to host genes. Although these recent studies provided important insight into HSV-1-induced host shutoff, the molecular mechanisms underlying HSV-1-mediated block of transcription termination remain completely unknown and it is unclear whether such a block is required for efficient viral replication. We aim to address these important questions in this proposal. In our preliminary studies, we found that ICP27 specifically interacts with the essential mRNA 3' processing factor CPSF and that ICP27 blocks mRNA 3' processing. Since mRNA 3' processing is required for transcription termination, we will test the hypothesis that ICP27 inhibits host transcription termination by blocking mRNA 3' processing via CPSF, and that ICP27-mediated disruption of host mRNA processing is important for HSV replication.  

Project Summary The development of trans-synaptic viral tracers is an important component of the BRAIN Initiative. At present, the lack of viral-based anterograde monosynaptic tracing tools with high signal strength and low toxicity is a gap in neuroscience. Herpes simplex virus (HSV) type 1 strain 129 (H129) is the most promising viral tool for anterograde neuronal tracing. However, current versions of genetically modified H129 viruses are limited by high virulence and toxicity, weak label signals that require immunostaining for detection, and time-dependent spread across multiple synapses. There is also a concern of the directional specificity of anterograde propagation of H129 recombinants, as they may propagate retrogradely. Investigators in the field have been working actively to develop improved versions of anterograde viral tracers, but progress has been limited. We have formed a strong interdisciplinary collaborative team composed of virologists and systems neuroscientists to develop anterograde monosynaptic recombinant H129 tracers with high signal strength and little or no toxicity for multi-species neural circuit analysis. Our published work and preliminary data establish the feasibility and key methodologies for the proposed research. We will capitalize on our established bacterial artificial chromosome (BAC) based system for rapid generation of recombinant H129 vectors and precise control of the H129 payload. We have a sound plan to reduce viral toxicity, enhance label signals and generate variants carrying different functional payloads. Our overall goal is to create a new set of safe, effective and validated anterograde-directed viral vectors that allow efficient labeling in monosynaptic projection targets of specific neuron types. These new tools will have a broad impact by enabling optical imaging, physiological recording, and activity manipulation of defined anterograde projection networks. For rapid resource sharing, we will create a service platform through the UCI Center for Virus Research to disseminate the new molecular tools to the neuroscience community.