Development of a genetic system for the archaeal virus Sulfolobus turreted icosahedral virus (STIV)

Abstract

Our understanding of archaeal viruses has been limited by the lack of genetic systems for examining viral function. We describe the construction of an infectious clone for the archaeal virus Sulfolobus turreted icosahedral virus (STIV). STIV was isolated from a high temperature (82°C) acidic (pH 2.2) hot spring in Yellowstone National Park and replicates in the archaeal model organism Sulfolobus solfataricus (Rice et al., 2004). While STIV is one of most studied archaeal viruses, little is known about its replication cycle. The development of an STIV infectious clone allows for directed gene disruptions and detailed genetic analysis of the virus. The utility of the STIV infectious clone was demonstrated by gene disruption of STIV open reading frame (ORF) B116 which resulted in crippled virus replication, while disruption of ORFs A197, C381 and B345 was lethal for virus replication.

Genomics of Bacterial and Archaeal Viruses: Dynamics within the Prokaryotic Virosphere

“…Importantly, a genetic system has recently been established for STIV (290). This development will now allow a detailed genetic analysis of the virus and promises that in the near future, Sulfolobus-STIV will become one of the most VOL.…”

Insights into a Viral Lytic Pathway from an Archaeal Virus-Host System

“…The resulting STIV (⌬C92) DNA was prepared for transfection into S. solfataricus P2 3 by digestion with BamHI (NEB) to remove the pCRII-TOPO-TA vector, followed by ligation with T4 DNA ligase (NEB) to generate a full-length STIV genome containing an AgeI site in place of the C92 gene. This construct was transformed into S. solfataricus P2 3 cells as described previously (7,9). After transformation, samples were collected every 24 h and analyzed for viral DNA replication via quantitative PCR (qPCR).…”

“…Standard qPCR protocols were followed, using the manufacturer's recommended protocols (SsoFast EvaGreen supermix; Bio-Rad, Hercules, CA) and 7.5 pmol STIV-5F and mid-r primers (Table 1) to amplify a portion of the STIV major capsid protein (MCP). If STIV DNA was detected in qPCR, a Western blot analysis using the anti-B345 polyclonal antibody raised against the STIV MCP was performed using standard procedures as previously described (7,9). If viral DNA and the viral MCP were detected, Sulfolobus P2 3 cultures were then infected with virus particles to determine whether the genetically modified virus was infectious.…”

“…Much of the limited knowledge that we do have comes from examining crenarchaeal viruses that infect members of the Sulfolobales species (1-9). Since its isolation from an acidic hot spring in Yellowstone National Park (YNP), Sulfolobus turreted icosahedral virus (STIV) has emerged as a model system for examining archaeal viruses due to its relative ease of propagation in the laboratory (4), the availability of multiple host and viral genome sequences, and the development of both host and viral genetic systems (9). The 72-nm icosahedral virion is built upon a Tϭ31 lattice that contains an internal lipid membrane and packages a 17.6-kb circular double-stranded (dsDNA) genome (10)(11)(12)(13)(14).…”

“…The 72-nm icosahedral virion is built upon a Tϭ31 lattice that contains an internal lipid membrane and packages a 17.6-kb circular double-stranded (dsDNA) genome (10)(11)(12)(13)(14). STIV has been the subject of transcriptomic, proteomic, genetic, and structural studies (2,4,7,(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20).…”