Extreme Mutation Tolerance: Nearly Half of the Archaeal Fusellovirus Sulfolobus Spindle-Shaped Virus 1 Genes Are Not Required for Virus Function, Including the Minor Capsid Protein Gene vp3

Abstract

Viruses infecting the Archaea harbor a tremendous amount of genetic diversity. This is especially true for the spindle-shaped viruses of the family Fuselloviridae, where Ͼ90% of the viral genes do not have detectable homologs in public databases. This significantly limits our ability to elucidate the role of viral proteins in the infection cycle. To address this, we have developed genetic techniques to study the well-characterized fusellovirus Sulfolobus spindle-shaped virus 1 (SSV1), which infects Sulfolobus solfataricus in volcanic hot springs at 80°C and pH 3. Here, we present a new comparative genome analysis and a thorough genetic analysis of SSV1 using both specific and random mutagenesis and thereby generate mutations in all open reading frames. We demonstrate that almost half of the SSV1 genes are not essential for infectivity, and the requirement for a particular gene correlates well with its degree of conservation within the Fuselloviridae. The major capsid gene vp1 is essential for SSV1 infectivity. However, the universally conserved minor capsid gene vp3 could be deleted without a loss in infectivity and results in virions with abnormal morphology. IMPORTANCE Most of the putative genes in the spindle-shaped archaeal hyperthermophile fuselloviruses have no sequences that are clearly similar to characterized genes. In order to determine which of these SSV genes are important for function, we disrupted all of the putative genes in the prototypical fusellovirus, SSV1. Surprisingly, about half of the genes could be disrupted without destroying virus function. Even deletions of one of the known structural protein genes that is present in all known fuselloviruses, vp3, allows the production of infectious viruses. However, viruses lacking vp3 have abnormal shapes, indicating that the vp3 gene is important for virus structure. Identification of essential genes will allow focused research on minimal SSV genomes and further understanding of the structure of these unique, ubiquitous, and extremely stable archaeal viruses.

Genetic Analysis of the Major Capsid Protein of the Archaeal Fusellovirus SSV1: Mutational Flexibility and Conformational Change<strong> </strong>

“…Sulfolobus cells were grown in YS media [20] and E.coli were grown in Lysogeny Broth (LB) with appropriate antibiotics [21,22]. Chemically competent E.coli and electrocompetent Sulfolobus were prepared and transformed as in Iverson et al [15].…”

“…LIPCR was used to delete the vp1 gene, portions of the vp1 gene, or to introduce single base pair mutations into the SSV1 genome [15,18,19]. All primers used in this study are listed in Table 2.…”

“…ORFs in the SSV1 genome are not essential for virus function, including the minor capsid protein VP3 [15]. Interestingly, virions lacking the vp3 gene have aberrant shapes yet still fit the fused fullerene cone model.…”

“…Proteolytic cleavage of virus structural proteins is very well-known; from the polyproteins of picornaviruses, flaviviruses and retroviruses [16] to the maturation of receptorbinding proteins in orthomyxoviruses [17]. There are now 10 described SSVs from hot springs throughout the world [2,15]. All known SSVs contain vp1 genes with N-terminal extensions and conserved C-termini, including the universally conserved glutamate residue [2,15].…”

“…Frames in the SSV1 genome [15,18,19] to make partial deletions, point mutations and complement mutants in the SSV1 genome. Both complete and partial deletions of the N-terminal region of the vp1 gene of SSV1 led to nonfunctional virus.…”