Rollie J. Clem - US grants

The process of apoptosis, or programmed cell death, is of vital importance in human health and disease. Improved understanding of the regulation of apoptosis promises to lead to new therapeutic strategies for a number of diseases, including cancer, neurological degenerative conditions, and autoimmune dysfunction. Much of our current understanding of the regulation of apoptosis rests on a foundation established by studies in lower animals, including nematodes and insects. An example of the importance of studying cell death in lower animals is provided by the anti-apoptotic gene family called iap. The first iap genes were identified by the PI in baculoviruses, which are viruses of lepidopteran insects. Subsequently, iap homologs have been identified by sequence homology in the genomes of insects and higher animals, and there is evidence linking at least two iap genes to human diseases, namely cancer and spinal muscular atrophy. A novel sequence motif found only in IAP proteins, the BIR motif, is required for anti- death function, presumably through the ability of different BIR motifs to bind to a number of distinct pro-apoptotic proteins. However, the BIR motifs from different IAP proteins do not appear to be inter- changeable, and no detailed information is currently available on the sequence requirements for BIR function. The first two Specific Aims of this proposal address the molecular basis for IAP function, through detailed structure-function analyses and characterization of protein- protein interactions. The third Specific Aim utilizes an improved version of the functional assay used to identify the first iap genes. This improved assay will be used to screen both viral and cellular cDNA libraries for novel anti-apoptotic genes. The identification and characterization of new genes involved in the regulation of cell death will lead to improved understanding of apoptotic pathways in both insects and higher animals, and may also aid in understanding the function of IAP proteins.

gastrointestinal epithelium; gastrointestinal infection; host organism interaction; Arthropoda; clinical research;

[unreadable] DESCRIPTION (provided by applicant): Mosquitoes are important vectors for a number of infectious diseases caused by arboviruses including West Nile virus, dengue virus, yellow fever virus, LaCrosse virus, and Venezuelan, eastern and western equine encephalitis viruses. Mosquitoes acquire arboviruses from vertebrate hosts during blood feeding, and after entry into the mosquito vector, the virus must first infect, replicate in, and escape from the epithelial cells of the midgut before it can be disseminated to other organs. Dissemination to the salivary glands is necessary for transmission to another host. Thus, the midgut epithelium constitutes a potential barrier to infection and subsequent transmission, and is therefore a target for interrupting the transmission cycle. It is well documented that only certain vector-virus combinations result in efficient transmission of disease, and data exist indicating that arboviruses must overcome the immune defenses of the insect vector in order to be transmitted successfully. However, little is known about anti-viral defenses in insects. For several years the Clem laboratory has been studying the role of apoptosis in defense against baculovirus infection in lepidopteran insects. This application seeks to explore the question of whether apoptosis can serve as a defense against arbovirus invasion in the mosquito midgut. In order to accomplish this goal a strong and diverse team has been assembled that includes expertise in apoptosis, mosquito vector biology, and arboviruses. In Aim 1, midguts and other tissues from arbovirus-infected mosquitoes will be examined for apoptosis, including a collection of Aedes aegypti strains that vary in their ability to vector dengue virus. In Aim 2, the hypothesis that apoptosis can limit virus replication and spread will be directly tested by manipulating apoptotic pathways in the mosquito and examining the effects on the ability of arboviruses to successfully cause disseminated infections. [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Arboviruses cause a significant world-wide health burden, with well over 100 million people becoming infected each year with viruses such as dengue, West Nile, yellow fever, and chikungunya, among others. Arboviruses are transmitted by arthropod vectors such as mosquitoes that become infected after ingestion of a viremic blood meal from a vertebrate host. Transmission to a new host requires that the arbovirus replicate in the midgut cells of the vector and then spread to secondary tissues eventually reaching the salivary glands. Once the latter are infected, the arthropod is able to transmit the virus to a new host. A long standing question in the field of vector biology is how arboviruses escape from the midgut, bypassing barriers such as basal laminae as well as host immune mechanisms. In some cases, arboviruses are able to infect and replicate in midgut epithelium but are not able to disseminate to other organs. The existence of this so-called midgut escape barrier implies that virus midgut escape is an active process. However, the mechanisms involved in midgut escape by arboviruses are almost completely unknown. This proposal addresses the signaling mechanisms used by the mosquito-borne viruses dengue, chikungunya, and Sindbis viruses to escape the midgut. Previous work by the investigators has defined a novel mechanism used by baculovirus to escape the midgut of their insect host. Baculoviruses use an elegant mechanism that signals a stepwise cascade of protease activation, wherein matrix metalloproteases become activated and in turn activate effector caspases, which directly cleave components of the basal lamina. This leads to remodeling of the basal lamina which lines tracheal cells associated with the midgut and culminates in the establishment of efficient systemic infections. The hypothesis underlying this proposal is that mosquito-borne arboviruses utilize this same pathway for midgut escape, and preliminary results support this hypothesis. Specifically, (1) it will be determined whether the mechanisms used by baculoviruses to remodel the midgut barrier are also utilized by arboviruses, including activation of matrix metalloproteases and caspases; (2) the contribution of candidate genes involved in midgut escape will be evaluated by RNA interference, arbovirus transducing systems, and transgenic Aedes aegypti mosquitoes by silencing or overexpressing target genes; and (3) the contribution of apoptosis and key apoptotic regulatory genes to arbovirus midgut escape will be tested. The results of these studies will contribute important new information to the understanding of arbovirus-vector interactions and potentially lead to new strategies for control of arbovirus transmission in the field.

This EAGER will advance the national prosperity and add to national bioeconomy by creating a new bee cell line that will help identify how bees succumb to microbial infections. Populations of bees and other pollinators are in decline worldwide, which has major implications for ecosystem health and agricultural production interests. Colony collapse disorder, involving colonies of the European honey bee, Apis mellifera, is just one dramatic example of rapid loss of pollinators. Although a large number of viruses infect bees, it is not known how these viruses impact bee decline. A bee cell line would benefit science because it would allow scientists to identify how bee viruses infect and kill bees. The goal of this research is to establish and characterize the first immortalized cell lines from Apis mellifera. The resulting immortalized cell line(s) will provide an invaluable resources for the virology research field. In addition to helping the honey bee scientific community, this research may help understand disease dynamics in other pollinator species, many of which are important for US agriculture. This cell line may be the basis for commercially-available testing kits to identify or track bee diseases. The research uses funding to train graduate and undergraduate students, including under-represented students. As such this funding is training the next generation of leaders in science. Researchers will share their findings to the public through the use of beekeeping workshops, and honey bee health and research awareness activities through farmer?s markets and video documentaries.

The lack of immortalized bee cell lines has greatly hampered research on bee viruses. Thus far, only primary cell cultures have been established from Apis mellifera, the European honey bee. These primary cell cultures are difficult to establish and maintain, and suffer from several major drawbacks including the presence of multiple resident viruses, extremely slow cell division rates, and limitations on the number of passages before reaching senescence. This research will examine the ability of candidate honey bee genes to immortalize A. mellifera embryonic cells. This will be accomplished by stably expressing candidate genes in primary embryonic cells isolated from embryos and monitoring growth and survival of the resulting cells. Primary cells isolated from different developmental stages of the embryo will be used, in order to attempt to obtain immortalized cell lines that represent various cell types. Cell clones with optimum growth characteristics that are able to be stably passaged will be selected. The resulting immortalized cell lines will be screened for resident viruses by deep sequencing, and any viruses present will be cleared by using a combination of RNA interference and single cell cloning. The resulting virus-free, immortalized honey bee cell lines will be made available to the research community. If successful, this approach will also provide a template for researchers to isolate immortalized cell lines from other bee species. This research was supported by the Symbiosis, Defense and Self-Recognition program of the National Science Foundation.

This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.