Sulfolobus spindle shaped viruses

 

The stedman Lab archetype 

One of the main areas of study in the Stedman lab, since it's founding, has been the research of Sulfolobus Spindle-Shaped Viruses, or SSVs. These viruses, categorized as Fuselloviridae, are found throughout the world in high temperature and acid - the environments are typically above 70º C and below pH 4. They exist in these conditions where their hosts, the archaea Saccharolobus (née Sulfolobus), thrive. Saccharolobus species can be found in volcanically heated acid hot springs all over the world - from Japan, to Russia, to Yellowstone National Park in the US, to Iceland, to Italy, and many other places. Consequently, SSVs have been isolated wherever their archaeal hosts are found.

SSV1 Structure from Stedman et al. 2015

SSV1 Structure from Stedman et al. 2015

There are currently 10 known SSVs, and the first discovered and most commonly studied of these viruses is SSV1, which encodes a positively supercoiled, 15.5 kbp circular dsDNA genome (NC_001338.1). This genome is enclosed within a lemon or spindle-shaped capsid, hence the name "spindle-shaped virus". The genome encodes 34 open reading frames, most of which have no recognizable homologues to other viruses or organisms, apart from other Fuselloviridae. Only one SSV1 gene shows clear homology to proteins outside the Fuselloviridae - the viral integrase. Despite being studied for over 40 years, still very little is known about the genomes of the SSVs. 

 

Mutant screening

Much of our progress in understanding the SSV genomes comes from screening mutants created by either targeted deletions, or non-specific transposon insertions. By studying these mutants, we can better understand the function and necessity of the genes that have been altered.

A halo assay testing the infectivity of SSV10 mutants. Notice the difference in border sharpness, and diameter each of the mutants have.

A halo assay testing the infectivity of SSV10 mutants. Notice the difference in border sharpness, and diameter each of the mutants have.

When studying these SSV mutants, the first functional screening that we do is testing for infectivity of one of its natural hosts Saccharolobus solfataricus S441. The technique we use to test this is the halo assay, and involves spotting virus on a lawn of S. solfataricus, with successful infection creating a ring of growth inhibition, or a "halo" around the spot. If a mutant is found to produce (or not produce) a halo on three individual assays, we consider the result repeatable. We can then conclude whether the mutated gene is essential for infection or not, based on the results. 

Halo assays are made possible by the fact that SSV infected cells have their growth slowed compared to uninfected cells. However, SSVs are different from most viruses in that when they emerge from a host, the cell doesn't die. Because of this, in addition to the sharpness and diameter of the halo, the time it takes for infected cells to reach the density of the surrounding lawn can be used as a rough measure of just how infectious and growth retarding the mutant is.

Once halo assays are complete, we use the results to continue piecing together the functions of the mutated open reading frames (ORFs). Because the genomes of SSVs have so little homology to anything else known, we are still far from understanding the function of every ORF. However, a paper published by our lab in 2017 suggests that nearly a third of the SSV1 genome is highly tolerant to mutation, or even complete removal), and our current work suggests this may hold true for some other SSVs as well.

The SSV1 genome showing A) conservation across 11  Fuselloviridae , and B) the infectivity of  host  Saccharolobus solfataricus  by different SSV1 mutants. From Iverson et al., 2017

The SSV1 genome showing A) conservation across 11 Fuselloviridae, and B) the infectivity of  host Saccharolobus solfataricus by different SSV1 mutants. From Iverson et al., 2017

Infection Mechanism

TEM & rendering of SSV1 budding through the host cell membrane and S-layer. From the  2016 Quemin et al. paper   Eukaryotic-Like Virus Budding in Archaea . These beautiful images show how SSV1 fuses with the host cell membrane during egress. 

TEM & rendering of SSV1 budding through the host cell membrane and S-layer. From the 2016 Quemin et al. paper Eukaryotic-Like Virus Budding in Archaea. These beautiful images show how SSV1 fuses with the host cell membrane during egress. 

Even today, after decades of study, little is known about how SSVs initiate infection, but some is known about particle egress from host cells. SSV1 capsid proteins, which are extremely hydrophobic, have been observed localizing at the membrane interface, and have shown to carry out eukaryotic-like budding. This budding is unusual for a virus, because it doesn't actually kill the host cell, only slows it's growth. This is especially important for work with SSVs because not only does the slowed growth make analysis of halo assays possible, but the cells' ability to keep growing after infection makes growing up lots of the viruses as easy as growing up the host cell culture. 

 

the capsid proteins

In addition to doing genome characterization of SSVs, our lab is also working towards unraveling the mysteries behind the structure of SSV capsids. Currently, the ORFs that we know the most about are the genes coding for the virus capsid proteins (vps). The SSV1 capsid is composed of four proteins - vp1, 2, & 3, which make up the main spindle-shaped shell, and vp4, which we have good reason to believe codes for a single, long peptide chain that forms multimeric tail fibers at the base of the spindle. It is believed that vp1 & vp3 dimerize to form the outer capsid shell, while vp2, which is very basic, is thought to reside on the inner surface of the capsid, where it non-specifically binds DNA. In the genome, vp 1, 2, & 3 are all transcribed from a single polycistronic ORF (T 1/2 in the figure above). The vp4 protein is transcribed at the beginning of a larger transcript that also ends with vp2 (T 4/7/8). 

Vp3, one of the major capsid proteins, is important for the correct formation of capsid structure, as a knockout of this gene causes viruses to take on a strangely elongated "cigarillo" shape. A vp2 knockout yields seemingly normal functional virus, while a knockout of vp4 has yet to be made and tested. 

The strangely "cigarillo" shaped viruses that result from the deletion of the vp3 capsid gene (left), as compared to the wild type SSV1 (right).

The strangely "cigarillo" shaped viruses that result from the deletion of the vp3 capsid gene (left), as compared to the wild type SSV1 (right).

A figure from our 2017 paper  Genetic Analysis of the Major Capsid Protein of the Archaeal Fusellovirus SSV1: Mutational Flexibility and Conformational Change,  showing the residue conservation of the vp1 N-terminus across different SSVs.

A figure from our 2017 paper Genetic Analysis of the Major Capsid Protein of the Archaeal Fusellovirus SSV1: Mutational Flexibility and Conformational Change, showing the residue conservation of the vp1 N-terminus across different SSVs.

Vp1, however, has so far proven to be the most important of the capsid genes for capsid formation, as any insertion in or deletion of the gene leads to a non-reproducing virus.

In a 2017 Stedman Lab paper, the importance of vp1 was discussed further, and looked at the role that a post-translational N-terminal cleavage of vp1 plays in virus functionality. The N-terminal region is largely non-conserved, but contains a glutamate towards the middle of the protein that is conserved across all SSVs.  The paper concluded that removal of the five residues N-terminal to this glutamate lead to non-functional virus, indicating cleavage is necessary for virus function. However, point mutations at that glutamate lead to functional, albeit morphologically abnormal virus. Currently, experiments are underway to determine if N-terminal cleavage is still occurring at the same point in these point mutants. 

 

SSV Lassen: the newest SSV

In 2008, the Stedman labs yearly trip to Lassen Volcanic National Park yielded an exciting discovery - a new SSV! Much like SSV1, nearly all of the genome has little homology to anything outside of other SSVs, and we are using the same saturation mutagenesis techniques that we have used to test ORFs for mutation tolerance in SSV1. Many of the core genes that are conserved across other SSVs are also conserved in SSVL (such as the vps), but other less conserved ORFs seem to be okay with mutation. Interestingly, SSVL was found to contain a large stretch of DNA that is completely disposable, and despite not having sequence homology in the region, it is found in the same location as a similarly disposable region in SSV1.

A toroidal multimeric Cas4 protein from  Sulfolobus solfataricus , determined by  Yakunin et al. in 2013 . A monomer of this protein has a similar structure to an uncharacterized ORF in SSV10 that we are investigating. 

A toroidal multimeric Cas4 protein from Sulfolobus solfataricus, determined by Yakunin et al. in 2013. A monomer of this protein has a similar structure to an uncharacterized ORF in SSV10 that we are investigating. 

Much of the rest of the SSVL genome contains homologous ORFs and has a similar arrangement to SSV1, but SSVL also contains five ORFs unique to it and no other SSVs. One of these unique ORFs we have been investigating has a very similar predicted structure to the known structure of a Cas4 protein found in it's Saccharolobus host. This would be the first example of an anti-CRISPR/Cas system in the SSVs, and our lab is working towards expressing and characterizing this protein. 

 

    Mutant Viruses From Hell

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    One of the best ways we screen for SSV mutants is by involving undergraduate students in the research. For the past five years, Dr. Stedman, along with graduate students from the lab, have taught an immersive lab course at Portland State that utilizes this undergrad power. The Advanced Molecular & Cell Biology Research Lab, lovingly called "Mutant Viruses from Hell," gives students the opportunity to screen SSV1 mutants, in hopes of finding a virus with an insertional (transposon) mutant in an ORF that is yet untested for functionality.

     

    This course has been able to not only provide rigorous and engrossing real life research experience to undergraduates, but has also been critical to our understanding of the SSV1 genome. Over the years, many students from this course have gone on to continue doing work in the Stedman lab and elsewhere as undergraduate researchers and graduate students.