Structure-Based Mutagenesis of Sulfolobus Turreted Icosahedral Virus B204 Reveals Essential Residues in the Virion-Associated DNA-Packaging ATPase

Abstract

Sulfolobus turreted icosahedral virus (STIV), an archaeal virus that infects the hyperthermoacidophile Sulfolobus solfataricus, is one of the most well-studied viruses of the domain Archaea. STIV shares structural, morphological, and sequence similarities with viruses from other domains of life, all of which are thought to belong to the same viral lineage. Several of these common features include a conserved coat protein fold, an internal lipid membrane, and a DNA-packaging ATPase. B204 is the ATPase encoded by STIV and is thought to drive packaging of viral DNA during the replication process. Here, we report the crystal structure of B204 along with the biochemical analysis of B204 mutants chosen based on structural information and sequence conservation patterns observed among members of the same viral lineage and the larger FtsK/HerA superfamily to which B204 belongs. Both in vitro ATPase activity assays and transfection assays with mutant forms of B204 confirmed the essentiality of conserved and nonconserved positions. We also have identified two distinct particle morphologies during an STIV infection that differ in the presence or absence of the B204 protein. The biochemical and structural data presented here are not only informative for the STIV replication process but also can be useful in deciphering DNA-packaging mechanisms for other viruses belonging to this lineage. IMPORTANCE STIV is a virus that infects a host from the domainArchaea that replicates in high-temperature, acidic environments. While STIV has many unique features, there exist several striking similarities between this virus and others that replicate in different environments and infect a broad range of hosts from Bacteria and Eukarya. Aside from structural features shared by viruses from this lineage, there exists a significant level of sequence similarity between the ATPase genes carried by these different viruses; this gene encodes an enzyme thought to provide energy that drives DNA packaging into the virion during infection. The experiments described here highlight the elements of this enzyme that are essential for proper function and also provide supporting evidence that B204 is present in the mature STIV virion. In Sulfolobus turreted icosahedral virus (STIV), the open reading frame B204 (previously described as B164) (1, 2) encodes a putative ATPase that is predicted to function as the molecular motor driving viral DNA packaging during infection (3). Interestingly, it has been demonstrated that viruses harboring ATPases homologous to B204 share other common features, such as a spherical morphology, an internal lipid membrane, and a structurally homologous coat protein fold (termed the double ␤-barrel or double jelly roll fold) (4). The structural and functional assessment of viruses belonging to this putative viral lineage can provide insights on viral infection strategies across the three domains of life. In the case of B204, understanding its function has implications for mechanisms by which an ancient molecular motor may h...

Structure and assembly of archaeal viruses

“…3E). Biochemical analysis together with the presence of B204 in purified mature virions, reinforce the fundamental role of this protein in viral DNA packaging and virion assembly (Dellas et al, 2015;Happonen et al, 2013). Notably, B204 assembles at a unique genomepackaging vertex suggesting that the genome packaging occurs specifically at one of the 12 five-fold vertices (Fu et al, 2010;Happonen et al, 2014), as in the case of PRD1 (Hong et al, 2014;Stromsten et al, 2003).…”

“…The mechanism underlying the incorporation of the inner membrane into the virion remains enigmatic. Because partially assembled particles were not detected budding from the cytoplasmatic membrane and based on the fact that viral and cellular membranes have different lipid compositions (Maaty et al, 2006), it has been proposed that the inner viral membrane is formed de novo (Dellas et al, 2015;Fu and Johnson, 2012;Fu et al, 2010). Notably, this process might be facilitated by the cellular Endosomal Sorting Complex Required for Transport (ESCRT) machinery, because perturbation of normal ESCRT function abrogates viral replication (Snyder et al, 2013).…”

Vast diversity of prokaryotic virus genomes encoding double jelly-roll major capsid proteins uncovered by genomic and metagenomic sequence analysis

“…All genomes in this group encode the packaging ATPase highly similar to that of STIV, which has been functionally and structurally characterized [ 57 ], although the juxtaposition of the MCP and ATPase genes is not conserved (Fig. 7 , Additional file 6 ).…”

Isolation and Characterization of Metallosphaera Turreted Icosahedral Virus, a Founding Member of a New Family of Archaeal Viruses

“…Although only 9 nm, they are reminiscent of STIV's 13-nm turrets. In STIV, these are suggested to serve as a tunnel for DNA in the virion-packaging or -unpackaging event (34,35). Additionally, initial cryo-electron tomography analysis suggests a bilayer at the virion's inner surface reminiscent of STIV's internal lipid bilayer.…”