Sulfolobus islandicus rod-shaped virus 2

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By Andrew Van Horn

Introduction

Sulfolobus islandicus rod-shaped virus 2 (SIRV2) is a lytic double-stranded DNA archaeal virus that infects Sulfolobus archaea . SIRV2 falls into the greater taxonomy of the Rudiviridae family. Microbiologists have taken interest in SIRV2 due to its thermophilic and acidophilic properties needed to infect Sulfolobus. SIRV2 displays unique properties from many other viruses . These include a unique viral release mechanism of which there are very few similar structures found in the natural world. Furthermore, SIRV2 is a lytic virus that acts by severely degrading host chromosomes. Additionally, SIRV2 is commonly at the forefront of the generally limited field of archaeal viral research .

Sulfolobus islandicus Archaeon

Sulfolobus islandicus is an archaeon commonly found in Icelandic sulfur hot springs . S. islandicus is a member of the Sulfolobaceae family and the Sulfolobales order. Once more, S. islandicus is a member of the Crenaracheota kingdom of the phylum Archaea. S. islandicus thrives in extreme environments of acidic pH 3 and high temperatures around 80°C. Notably, the intracellular pH of S. islandicus is only slightly acidic, approximately pH 6. Additionally, S. islandicus metabolizes sulfur, though the methods can vary between strains and species . Sulfur metabolism can be done through sulfur oxygenase/reductase and sulfur reductase, among others. However, the extensive methods of sulfur metabolism in S. islandicus are poorly understood.

Pathogenicity

While believed until very recently to be a host-carrier virus that transmitted vertically without lysing host cells, this has been revealed to not be the case. In 2009, Bize et al. used flow cytometry to document large-scale chromosomal degradations and DNA damage that led to them recharacterizing SIRV2 as a cytocidal lytic virus. In S. islandicus cells infected with SIRV2, reduction in genome size were documented as early as half an hour after infection. After 12 hours, the majority of their cells populations did not contain any flow cytometry-detectable DNA. Interestingly, a slight increase in intracellular DNA of 1.3 Mb was detected around 3 hours after infection. Following the spike, decreases in intracellular DNA occurred. This spike was attributed to viral DNA replication that occurs quickly after infection.

The latent period of SIRV2 was determined by Bize et al. to be 8-10 hours. After 8-10 hours, almost no host DNA was detectable. Additionally, virions are then released through Virus-Associated Pyramids. Estimates of burst size for SIRV2 in S. islandicus is generally estimated around 30-50 virions. Bize et al. also showed that no virions were released before the 8-10 hours after infections. Furthermore, cell death was associated with virion release at 8-10 hours by using a membrane potential sensitive probe.

Additionally, it was demonstrated that SIRV2 is able to superinfect S. islandicus. This means that a cell infected by SIRV2 is unable to be infected by other viruses while presently infected by SIRV2.

Viral Life Cycle

Infection

SIRV2 binds to filaments protruding from the surface of S. islandicus to reach the cell for infection . SIRV2 virions use their tail fibers to bind the tips of long filaments on the cell surface. Virions then migrate down the length of the filament until reaching the cell surface. Upon contact with the cell surface, the virions begin to dissemble and release their genome into the new host cell. Approximately three virions can be bound to an individual filament at a given time. Virions show an initial binding preference for the tips of the filaments for initial attachment. While only two virions can be bound at a given time to the tip of the filament, other virions can simultaneously be migrating down the length of the filament towards the cell surface. Fortunately, filaments are highly abundant on the S. islandicus cell surface. Once virions are bond to the filaments or cell surface, they are irreversibly bound.

Moreover, the rate of infection by SIRV2 of S. islandicus is extremely high. In Quemin et al. 2013, they demonstrated that approximately 80% of virions injected to a culture of s. islandicus were absorbed within thirty seconds, and almost all virions were absorbed after approximately 25 minutes. This rapid absorption is likely facilitated at least in part by the great abundance of filaments for virions to bind to. Furthermore, the ability of multiple virions to bind to each filament allows for a rapid infection process. This rapid infection process combined with lengthy latent period of 8-10 hours has led scientists to suggest that this cycle timing is highly evolved for the extreme conditions in which S. islandicus, and thus SIRV2, live. If living in an unfavorable environment of Icelandic hot springs in which the temperatures are greater than 80°C and as acidic as pH 3, it is favorable for SIRV2 to reduce exposure to the extreme environment by minimizing infection time and increasing the time spent within the safety of S. islandicus.

Genome Replication

SIRV2 replicates its genome by a unique mechanism that involves localizing replication and using host cell proteins . SIRV2 creates a replication focus near the terminus of the host cell. Viral DNA localizes to one end of the cell for replication. Furthermore, three proteins have been identified to localize in the replication focus: viral ssDNA binding protein gp17, host cell proliferating nuclear antigen (PCNA), and DNA polymerase I (Dpo1). Interestingly, Sulfolobus encodes four DNA polymerases. However, Dpo2, Dpo3, and Dpo4 do not play any consequential role in the viral replication of SIRV2.As such, Dpo1 is accepted to be the polymerase involved in viral DNA replication. Simultaneously, host PCNA is also utilized by SIRV2. It is believed that SIRV2 uses PCNA because PCNA recruits and organizes the DNA replication and repair processes. In this sense, by recruiting PCNA, SIRV2 unlocks a variety of tools that it can use to replicate and protect its genome. During DNA replication, PCNA and Dpo1 are almost completely confined to the replication focus. Interestingly, Dpo1 and gp17 expression increase throughout infection. This suggests mechanism by which SIRV2 is able to not increase its own gene expression but alter the gene expression of the host cell.

The SIRV2 replication cycle is also unique in that in undergoes multiple diverse stages. Replication begins with the binding of a replication initiating protein (Rep). Asymmetric strand-displacement replication then proceeds to make circular dimer intermediates. Displacement replication is a process of continuous DNA replication on both stands that results in a hairpin loop on the end of the slower (light) strand when the faster (heavy) strand reaches the origin of light strand replication. This hairpin loop is then replicated by additional DNA polymerases in lagging strand replication. Once circular dimer intermediates have been synthesized, both strand displacement and strand-coupled replication occur together. Rolling circle replication then occurs once then occurs once to produce multimers of ssDNA. Then multiple re-initiation evens of DNA replication occur that cause the DNA to form brush-like structures. These brush-like structures result from multiple strands of associated ssDNA that fan out. The brush-like structures then undergo coupled-replication to produce multiple new progeny dsDNA genomes.

Virion Release

SIRV2 employs one of the most unique viral release mechanisms seen in nature. Encoded in SIRV2-ORF98 is the gene for SIRV2-P98, the protein constituent of the SIRV2 Virus-Associated Pyramid (VAP) that releases virions. After P98 synthesis, the proteins self-assemble in the cytoplasm into a seven-sided open-base pyramidal structure. This shape of VAP is exceptionally rare in nature, with only two other known proteins with a seven-fold structure in nature: the archaeal 20S proteasome and scallop muscle myosin filaments. The continued growth and synthesis of the VAP occurs along the base of each triangular face. As a result, there is a constant presence of growing VAPs of various sizes in the host cell’s cytoplasm.

To release virions, the VAP embeds itself in the membrane with its base facing inwards. Virions can then assemble in the opening forged by the VAP. Once enough virions have assembled near the VAP, the VAP undergoes a conformational shift releases the virions. The seven triangular open away from the center the create an open channel for virion release. Interestingly, the faces are curved in the open state. Additionally, the VAPs must grow to certain size before they are capable of opening and releasing virions. A suggested size for functionality has been a minimum diameter of 250 nm.

When SIRV2-ORF98 has been placed into other organisms such as Escherichia coli the bacterial cells still produced the VAP. This demonstrated to researchers that the P98 proteins were able to self-assemble into the VAP. Moreover, the VAP inserted itself into the inner membrane of the bacterial cells. This finding was curious given biochemical differences between bacterial and archaeal membranes that often lead to protein insertion specificity. Notably, the Sulfolobus turreted icosahedral virus (STIV) shows a similar viral release mechanism, though the VAP geometry maintains some key differences and is less stable.

References

Authored for BIOL 238 Microbiology, taught by Joan Slonczewski, 2022, Kenyon College