My research in the Stedman Lab at PSU: Functions of Cryptic Viral ORFs in Fuselloviridae
The archaeon genus Sulfolobus is one of the largest and most ubiquitous taxa in the Crenarchaea. This extremely thermophilic and acidophilic genus has been found around the world in Japan, Russia, Iceland, and in the Americas. The SSV1 virus in the family Fuselloviridae infects Sulfolobales and is among the best-characterized viruses in the Crenarchaea. Despite this, little is known about their replication, genome integration, or even their method of entry into the host. Because most of the Open Reading Frames (ORFs) show no similarity to known genes in the molecular database, we are developing a system to produce knockouts and mutants for functional analysis of all of the 34 ORFs in the genome. Due to the lack of selectable markers in Sulfolobales, we are using a shuttle vector containing a bacterial plasmid in the SSV1genome. This allows us to replicate the genome as a plasmid in E.coli or as a functional virus in its host. We are amplifying the18.5 kbp shuttle vector using primers that will introduce specific point mutations or remove individual ORFs. This long PCR was accomplished using a high fidelity, highly processive PhusionTM polymerase from MJ Research. This process began with the removal of one of the only genes that shows similarity to anything in the nucleotide database, the integrase gene. Belonging to a large family of tyrosine recombinases, the integrase gene is thought to facilitate insertion and removal of the viral genome in a specific site in the host. Constructs are being used to test specificity of the recognition of the integrase. Integrase genes from other viruses in the family Fuselloviridae that have different integration sites are also being tested in the recombinant construct. This research will provide us with a better understanding of the role of the integrase in the replication of thermophilic viruses and will serve as a model to elucidate the function of other ORFs in the SSV1 genome.
Papers and books related to my research:
B. Wiedenheft, K. M. Stedman, F. Roberto, D. Willits, A. Gleske, L. Zoeller,
J. Snyder, T. Douglas, and M. Young. Comparative Genomic Analysis of Hyperthermophilic
Archaeal Fuselloviridae Viruses. (2004) J. Virology, 78/4, 1954-1961.
K. M. Stedman, Q. She, H. Phan, H. P. Arnold, I. Holz, R. A. Garrett, and W.
Zillig. Relationships between fuselloviruses infecting the extremely thermophilic
archaeon Sulfolobus: SSV1 and SSV2. (2003) Research in Microbiology 154/4, 295-302.
M. Jonuscheit, E. Martusewitsch, K. M. Stedman, and C. Schleper. A Reporter
Gene System for the Hyperthermophilic Archaeon Sulfolobus solfataricus based
on a selectable and integrative shuttle-vector. (2003) Molecular Microbiology
48/5. 1241-1252.
These include links to some other researchers in the field
Sulfolobus
P2 site
The Phage Ecology
Website
Nasa's Astrobiology Website