Advisory Committee Chair
Colleen Jonsson
Advisory Committee Members
Elizabeth Sztul
William Britt
Peter Prevelige
Louise Chow
Document Type
Dissertation
Date of Award
2007
Degree Name by School
Doctor of Philosophy (PhD) Heersink School of Medicine
Abstract
Hantaviruses are enveloped, negative-sense RNA viruses belonging to genus Hantavirus, within the family Bunyaviridae. Hantaviruses include many serious human pathogens and are classified as Old and New World based on the geographic distribution of rodent reservoirs and their ability to in induce distinct illnesses in humans. Currently there are no reservoirs known for hantaviruses other than the rodents. Human transmission of hantaviruses occurs via inhalation of aerosolized rodent excreta leading to hemorrhagic fever with renal syndrome (HFRS) or hantavirus pulmonary syndrome (HPS). Hantavirus genome consists of small (S), medium (M) and large (L) segments that encode nucleocapsid (N) protein, two surface glycoproteins (Gn and Gc) and L protein (L), the viral polymerase respectively. In contrast to most negative-stranded RNA viruses, hantaviruses and other viruses in the family Bunyaviridae mature intracellularly and derive the virion envelope from the Golgi or pre-Golgi compartments. Overall, the steps leading to virion assembly remain largely unanswered for hantaviruses. The pathways of trafficking and assembly for the Old and New World hantaviruses are under active investigation; Also, the cellular factors facilitating post-entry viral replication events are not well understood. Most of the reported information comes from the studies of Gn and Gc. Because hantaviruses do not have matrix proteins, the N protein has been proposed to play a key role in assembly. iii This study focused on the intracellular trafficking and morphogenesis of the hantaviruses using the N protein as a probe. The hantaviral N protein (approximately 50kDa) is the most abundant viral component in both virions and infected cells. The N protein is synthesized very early during the infection and hence can facilitate examination of early events in viral trafficking. Further, we looked at the association of N protein with cellular factors. In chapter 2, using a combination of drugs that inhibit cellular trafficking and confocal microscopy, we show that N trafficked to and colocalized with the endoplasmic reticulum-Golgi intermediate compartment (ERGIC) in HTNV-infected Vero E6 cells in a microtubule-dependent manner, and did not colocalize with endoplasmic reticulum (ER), Golgi, or early endosomes. Overexpression of dynamitin which, dominant negatively regulates dynein mediated transport, reduced N accumulation in the perinuclear region further supporting the role of microtubule components in N trafficking. Finally, we demonstrated the requirement of an intact ERGIC for viral replication, suggesting the possibility of virus factories in this region. In chapter 3, I designed experiments to probe for differences in trafficking pathways and utilization of host cytoskeleton between representatives of Old and New World hantaviruses. Such a difference could help identify the differences in the pathogenesis of Old and New World hantaviruses. I observed that all viruses targeted the ERGIC and required an intact microtubule network for replication. Additionally, I noted the unique requirement of actin for replication of ANDV. Furthermore during early events in the life cycle, I noted distinct pathways for Andes (AND) virus a New World hantavirus and Hantaan (HTN) virus an Old World hantavirus in entry and post-entry processes. iv In chapter 4, I designed in vitro pull down experiments to look for direct interaction between recombinant HTNV N protein and dynein light chains as well as intermediate chain. Amino acid sequence alignment confirmed the presence of a putative dynein light chain-8 (LC8) binding sequence DRIEQWL in at region 234-240 in HTNV N protein. Though our initial experiments did not reveal interactions between the N protein and dynein subunits, further experiments to confirm or deny such direct interactions are being undertaken. In summary, the hantaviral N protein participates in many interactions involving viral and cellular factors during viral replication. Future work to develop an understanding of the molecular mechanisms of N protein interaction with cytoskeletal components along with other viral components will provide clues to study trafficking as well as assembly and pathogenesis. Further it will provide with a good model system to study functions in other members of Bunyaviridae.
Recommended Citation
Ramanathan, Harish N., "Trafficking Of Hantaviral Nucleocapsid Proteins" (2007). All ETDs from UAB. 3785.
https://digitalcommons.library.uab.edu/etd-collection/3785