Richard J. Roller - US grants

Herpes simplex viruses (HSVs) are widely distributed human pathogens. Serological studies suggest that at least 50 percent of the american adult population have antibodies to at least one of the two herpes simplex viruses, indicating infection at some time. The severity of disease ranges from asymptomatic to lethal, depending upon the age and immune competence of the infected individual. Control of herpes simplex viruses and related herpesviruses can be improved by understanding the functions of the virus-encoded proteins, and the mechanisms by which those products are regulated in the infected cell. The research proposed here will identify the function of the product of the essential UL34 gene. Preliminary studies have shown that the UL34 protein is a membrane associated protein whose timing of synthesis and localization in the cell suggest a function in envelopment or egress. These studies will entail the construction and characterization of UL34-deficient viruses, and identification of cellular proteins that interact with, and are regulated by the UL34 protein. These results should, in turn, illuminate the function of the regulatory events mediated by the US11 and US3 proteins, which regulate these products of the UL34 gene. The proposed research is novel and likely to be highly medically significant from several points of view. (i) The UL34 gene product is conserved among alphaherpesviruses including varicella zoster virus (VZV) and is very likely an essential virus gene product for replication in cell culture and in the human host. Its function, therefore, represents a potential therapeutic target. (ii) The function of the UL34 gene product is apparently regulated by phosphorylation, and UL34, in turn appears to regulate the phosphorylation of cellular proteins. Regulation of phosphorylation is a ubiquitous strategy for controlling a wide range of cellular processes, and these studies are likely to expose a novel example of viral regulation of cellular phosphorylation. (iii) The UL34 locus is regulated by the US11 RNA binding protein. Specific RNA-protein interactions like that involved in US11-mediated regulation of transcripts from the UL34 gene are critical in regulating many processes important to the control of cell proliferation (including regulation of oncogene activity), and in regulating the infection of lymphotropic retroviruses. These studies will contribute to the understanding of the ways in which RNA-protein interactions give rise to regulatory effects.

Herpesviruses replicate and packagetheir genomes in the cell nucleus. In order to begin its processof escape from the cell, the virus must pass through the nuclear membrane by first budding into the inner nuclear membrane in a process called primary envelopment. The mechanism of herpesvirus envelopment is significant to human health from two points of view. First, this process represents an attractive target for therapy in that it is dissimilar in many waysto any normal cellular process, essential to the virus, and common to all herpesviruses. Second, the envelopment machinery alters the organization of the nucler anvelope and provides a usefultool for studying that organization. The research proposed here builds on our studies of herpes simplex virus UL34, an essential component of the envelopment apparatus. Results from our initialperiod of funding suggest that this protein plays critical roles inat least three aspects of envelopment including (i) Recruitment of viral proteins to the nuclear envelope, (ii)dispersal of nuclear envelope components that prevent access of the capsid to the nuclear membrane, and (iii)wrapping of the nucleocapsid in the nuclear membrane. The specific goals of the proposed research are threefold 1. We will define essential functions of the UL34 protein by characterizing a set of seven non-functional mutant UL34 proteins already in hand, and others that we will generate by mutagenesis of conserved residues, for their~ability1o perform various functions required for envelopment. 2. We will characterize critical interactions between the UL34 protein and other viral proteins involved in envelopment. This will include experiments to test the hypothesis that UL34 mediates membrane wrapping of the capsid, and a screen for interactions of UL34 with other viral proteins that may participate in envelopment. 3. We will fully characterize changes in the host cell nuclear lamina associated with infection and determine the mechanism by which UL34 and other viral proteins accomplish those changes.

DESCRIPTION (provided by applicant): There is a continuing and urgent need for greater understanding of virus infections and, consequently, a need for a robust virology research community in the U.S and the world. The purpose of the Virology Training Program at the University of Iowa is to train young scientists to be productive members of that research community. The Virology Training Grant at the University of Iowa helps in two ways. First, it fosters the rigorous training of PhD students in the study of virology. Second, it promotes interaction among students and faculty interested in virology across the University of Iowa. By providing stipend support and travel funds for graduate students, the Training Grant will facilitate the recruitment of students who are interested in virology. By establishing curriculum requirements that include research and literature seminars for the virology community and the collaborative teaching of virology training courses the Training Grant will promote interaction in the larger virology community at the University of Iowa and beyond. There are ten Virology Training Grant faculty members who represent a wide variety of research interests from study of very basic processes in the molecular and cellular biology of virus replication, to study of host animal responses to virus infection, to study of the most efficient mechanisms for gene delivery by viruses. We seek support for five predoctoral graduate students for two years between their second and fourth years of study. RELEVANCE (See instructions): Virus infections have critical public health relevance because: (i) They are a major cause of human disease, (ii) Their intimate relationship with host cells makes them useful tools for studying normal cellular functions, (iii) They are vehicles for gene delivery in remedial gene therapy and vaccine development. There is a need to train scientists who can meet the public health threat and exploit opportunities presented by viruses.

DESCRIPTION (provided by applicant): Nuclear egress of herpesviruses is an essential and conserved process in virus replication. Over the past decade, viral proteins required for this process have been identified and functionally characterized. Progress in identification of cellular factors that participate in nuclear egress has been slower. Identification of cellular factors that participate in nuclear egress is highly significant from two points of view. On one hand, identification of essential factors might yield targets for antiviral therapy. On the other, characterization of the function of these factors will certainly provide insight into their normal functions in the uninfected cell. This is an important problem in human disease biology because many interesting inherited diseases are caused by mutations in genes that encode proteins of the nuclear envelope and its underlying lamina. These diseases include muscular dystrophies, cardiac, and bone and connective tissue disorders and torsion dystonias. In no case is the relationship between the mutant protein and disease pathogenesis completely clear. Here, we propose to explore the function of the torsinA gene product in HSV infection. Mutation of the gene encoding torsinA results in a neuromuscular disease called early-onset torsion dystonia, and the mechanism of disease is unclear. We have found preliminary evidence for a functional interaction between torsinA and herpes simplex type 1, and evidence specifically for a role for torsinA in regulation of membrane fusion at the nuclear envelope. We propose to fully characterize the interaction between torsinA and HSV pursuing two specific aims. Aim 1. Phenotypic characterization of the function of TorsinA in HSV-1 infection. We will use complementary approaches of TorsinA over-expression and knock-down/knock-out to test the hypothesis that normal Torsin expression is required for efficient HSV infection and to test the hypothesis that Torsin A is specifically required for efficient nuclear egress of HSV. Aim 2. Identification of the mechanism of TorsinA function in HSV infection. TorsinA function is thought to be mediated by interaction with, and regulation of, the activity of other proteins. Our preliminary data suggest that Torsin A might regulate HSV nuclear egress either by regulating the activity of previously identified cellular proteins or by directly regulating the function of HV glycoproteins in the nuclear envelope or the primary virion envelope. We will test both of these hypotheses using a combination of genetic and biochemical approaches.

DESCRIPTION (provided by applicant): All of the manifestations of alphaherpesvirus disease (herpes simplex, varicella zoster) result from the ability of the virus to spread from the initial infected cell or cells at mucosal surfaces to other cells in the area and to nerve cells that innervate the site of primary replication. Recurrence of symptoms and consequent spread of the virus to new hosts similarly requires the ability to spread from the sensory ganglion cells to cell at the periphery and among the cells on the mucosal surface. Amazingly, spread of the virus in recurrent infection occurs in the face of an adaptive immune response, including an antibody response that should neutralize virus released from the cell. The disease-causing properties of these viruses therefore depend on the mechanisms used for spread from cell to cell that protect the virus from exposure to effectors of the adaptive immune response. Spread of the human alphaherpesviruses within the host requires trafficking of newly assembled virus particles from their assembly site at the Golgi to exposed cell surfaces for release to extracellular medium or to cell junctions for cell-to-cell spread (CCS). Neither trafficking pathway is well understood. In part this is because, other than viral proteins required for entry of virus into host cells, no virl gene functions have been identified that are required for the process in most cell types. We have discovered that two viral gene products, pUL34 and pUL51, play critical roles in efficient virus release and/or CCS. Both proteins are apparently multifunctional. pUL34 is required for nuclear egress of herpesvirus capsids, and pUL51 has been shown to be required for efficient cytoplasmic assembly of the virus. We have discovered, however, that both proteins play critical roles in release and CCS that can be genetically uncoupled from their roles in virion assembly. Our overall goal is to test the hypothesis that HSV release and CCS are accomplished by viral hijacking of cellular pathways that sort host cell membrane proteins to appropriate surfaces and junctions. We will use two general approaches to this overall goal. The first approach (contained in the first two specific aims) is to characterize the functions and interactions of pUL34 and pUL51 that are required for virus release and CCS. These viral proteins can thus be used as tools to identify critical viral and cellular proteins that also participate. The second approach (contained in the third specific aim is to take advantage of recently developed information about the host pathways that deliver cellular membrane proteins to basolateral and junctional surfaces of cells and to probe those pathways using dominant negative inhibitors of critical molecules.

Nuclear egress of herpesviruses is an essential and conserved process in virus replication, making it an attractive target for therapeutic interventions to target the broad array of human and animal herpesviruses. The egress process is carried out by a nuclear egress complex (NEC), the core of which is composed of two conserved virus proteins called, in HSV, pUL31 and pUL34. These two proteins may recruit various other virus and host cell proteins to the NEC, the identity and function of those other proteins depending upon the species of herpesvirus. Nuclear egress requires DNA containing capsids to bud into the inner nuclear membrane, and this budding is driven by multimerization of the pUL31/pUL34 heterodimer. Several independent lines of evidence show that the pUL31/pUL34 complex is sufficient to drive this budding. Nevertheless, formation of buds without capsids is not commonly observed in TEM analysis of infected cells, suggesting that capsids must somehow trigger budding, and that capsid-less budding is inhibited in the infected cell. The mechanism of this negative regulation is not understood, and a similar knowledge gap exists for many matrix-driven virus budding processes. Herpesvirus nuclear egress can therefore provide an attractive model for understanding general principles of regulation of viral envelopment. We have previously characterized a pUL34 mutant that is deficient in nuclear egress and shows extensive formation of empty vesicles that bud into the inner nuclear membrane. This adds further evidence that the budding function of the NEC is subject to negative regulation and that this regulation can be disrupted by specific mutations in the NEC proteins. We have selected for extragenic suppressor mutants that suppress the replication defect. Interestingly, we have found two types. In one type, we see one of several mutations in the other known component of the NEC, pUL31. In the other, there are no mutations in the UL31 gene. The existence of this second class of mutants strongly suggests the participation of a third, unidentified viral gene product that is a negative regulator of budding (NRB), and provides an avenue for identification of that gene product. We will identify and characterize the function of the NRB and its interaction with the pUL31/pUL34 heterodimer by pursuing two aims: (i) Our promiscuous budding mutant contains two discrete point mutations in pUL34, and we will determine which of these is responsible for its growth and promiscuous budding phenotypes. (ii) By isolating several non-UL31 suppressors and use of whole-genome sequencing, we will identify the NRB locus. We will confirm its function using recombinant mutant viruses that carry mutant UL34 and the putative NRB suppressor mutant. We will confirm the presence of the NRB in the NEC, and then confirm that it acts as a negative regulator of budding by co-expression with pUL31 and pUL34 in the absence of other viral proteins.

Recently, we have observed that, in at least one mouse and one human neuronal cell line, a putative cytoplasmic viral assembly center (cVAC) is formed around the microtubule organization center (MTOC). It is well-established that human cytomegalovirus (HCMV), a non-neuro-invasive human herpesvirus, forms a visually distinct cVAC in epithelial cells and fibroblasts. This feature of HCMV assembly has made it a rich source for discovery of functions of viral tegument proteins in viral assembly as well as host factors that are required for viral morphogenesis and egress. We believe that the study of an a HSV cVAC would allow similar rapid advances in the field by establishing a neuronal cell-based model that 1) would provide highly interpretable results for revealing the nature of the HSV assembly compartment, such as the dynamics of host membrane rearrangement during infection and essential viral or host factors required, 2) would be a rich source of discovery of mechanisms of HSV assembly protein function, 3) will allow us to compare cytoplasmic assembly between HCMV and HSV, which does not only provide insights regarding the functions of HSV homologs, but also has implications in the assembly of other herpesviruses. We propose here to explore the composition of the putative HSV cVAC and test specific hypotheses about the importance of microtubule networks, endocytosis, and the viral tegument protein pUL51 in its formation.

Alphaherpesviruses encode two proteins kinases that are critical regulators of pathogenesis, pUS3 and pUL13. pUS3 has multiple functions in infection including facilitation of nuclear egress, protection from apoptosis, promotion of viral protein synthesis, and inhibition of antigen presentation to the immune system. The regulation of the activity of pUS3 is, therefore, of great interest, both as a tool for understanding the molecular biology of herpesvirus infections, and as an avenue for exploring antiviral therapies based on inhibition of its various activities. pUS3 kinase activity has been reported to be regulated both by autophosphorylation and by phosphorylation by another herpesvirus-encoded kinase, pUL13. Intriguingly, regulation of pUS3 activity by pUL13 appears to affect some pUS3 functions, but not others. Specifically, it appears to be necessary for at least some of the activities of pUS3 in egress of virus capsids from the nucleus, but not for protection of infected cells from some pro-apoptotic stimuli (). These observations highlight two very significant gaps in our understanding of the interaction between pUL13 and pUS3. First, it is unclear which pUS3 functions are regulated by pUL13, and what distinguished UL13-dependent from UL13-independent functions. We propose the simple hypothesis that phosphorylation of pUS3 by pUL13 specifically regulates its nuclear functions by regulating its access to the nucleus. Second, the mechanism and functional significance of pUL13 regulation of pUS3 is unclear. It is known that pUL13 phosphorylates pUS3, but it is not clear whether this phosphorylation is necessary for pUL13 regulation of pUS3. A recently published proteomic analysis of HSV infected cells suggests that pUL13 phosphorylates pUS3 at S139. We will use a genetic approach to test the hypothesis that S139 is the critical residue for regulation of pUS3 by pUL13, and then to test the significance of S139 phosphorylation for viral growth and spread.