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A genetic study of SSV1, the prototypical fusellovirus.

Iverson E, Stedman K - Front Microbiol (2012)

Bottom Line: Recently we have developed genetic tools to analyze these genes.In this study, we have deleted three SSV1 open reading frames (ORFs) ranging from completely conserved to poorly conserved: VP2, d244, and b129.Deletion of the universally conserved ORF b129, which encodes a predicted transcriptional regulator, results in loss of infectivity.

View Article: PubMed Central - PubMed

Affiliation: Biology Department, Center for Life in Extreme Environments, Portland State University, Portland, OR, USA.

ABSTRACT
Viruses of thermophilic Archaea are unique in both their structures and genomic sequences. The most widespread and arguably best studied are the lemon-shaped fuselloviruses. The spindle-shaped virus morphology is unique to Archaea but widespread therein. The best studied fusellovirus is SSV1 from Beppu, Japan, which infects Sulfolobus solfataricus. Very little is known about the function of the genes in the SSV1 genome. Recently we have developed genetic tools to analyze these genes. In this study, we have deleted three SSV1 open reading frames (ORFs) ranging from completely conserved to poorly conserved: VP2, d244, and b129. Deletion of the universally conserved ORF b129, which encodes a predicted transcriptional regulator, results in loss of infectivity. Deletion of the poorly conserved predicted DNA-binding protein gene VP2 yields viable virus that is indistinguishable from wild-type. Deletion of the well-conserved ORF d244 that encodes a predicted nuclease yields viable virus. However, infection of S. solfataricus with virus lacking ORF d244 dramatically retards host growth, compared to the wild-type virus.

No MeSH data available.


Related in: MedlinePlus

Typical growth inhibition of S. solfataricus on plates due to infectious virus. Lawns of S. solfataricus strain GΘ were prepared as in Stedman et al. (2003). Two microliters of supernatant from cultures transformed with either (A) SSV-ΔVP2 or (B) SSV-Δd244 were placed on the lawns where indicated. Δ indicates where SSV-ΔVP2 was spotted, ΔD where SSV-Δd244 was spotted. P indicates SSV-WT spotted as a positive control. T or Tx indicates 2 μL of 0.01% Triton X-100 spotted as a control for lawn growth.
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Figure 2: Typical growth inhibition of S. solfataricus on plates due to infectious virus. Lawns of S. solfataricus strain GΘ were prepared as in Stedman et al. (2003). Two microliters of supernatant from cultures transformed with either (A) SSV-ΔVP2 or (B) SSV-Δd244 were placed on the lawns where indicated. Δ indicates where SSV-ΔVP2 was spotted, ΔD where SSV-Δd244 was spotted. P indicates SSV-WT spotted as a positive control. T or Tx indicates 2 μL of 0.01% Triton X-100 spotted as a control for lawn growth.

Mentions: To confirm the presence of infectious virus, halo assays were performed in duplicate 48 and 72 h post-electroporation (Stedman et al., 2003). Uninfected Sulfolobus GΘ cells were diluted to an OD600 nm = ~0.3 and allowed to grow until the OD600 nm reached ~0.35 (about 2.5 h). Half of a milliliter of this uninfected culture was added to 5 mL YS media containing 0.2% wt/vol Gelrite® as a softlayer and poured onto prewarmed YS plates. Two microliters of supernatant from electroporated cultures was spotted onto the lawns and plates were incubated at 75°C for up to 3 days. A halo of host growth inhibition, typically observed 48–72 h after incubation, indicated the presence of an infectious virus (Figure 2).


A genetic study of SSV1, the prototypical fusellovirus.

Iverson E, Stedman K - Front Microbiol (2012)

Typical growth inhibition of S. solfataricus on plates due to infectious virus. Lawns of S. solfataricus strain GΘ were prepared as in Stedman et al. (2003). Two microliters of supernatant from cultures transformed with either (A) SSV-ΔVP2 or (B) SSV-Δd244 were placed on the lawns where indicated. Δ indicates where SSV-ΔVP2 was spotted, ΔD where SSV-Δd244 was spotted. P indicates SSV-WT spotted as a positive control. T or Tx indicates 2 μL of 0.01% Triton X-100 spotted as a control for lawn growth.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC3367457&req=5

Figure 2: Typical growth inhibition of S. solfataricus on plates due to infectious virus. Lawns of S. solfataricus strain GΘ were prepared as in Stedman et al. (2003). Two microliters of supernatant from cultures transformed with either (A) SSV-ΔVP2 or (B) SSV-Δd244 were placed on the lawns where indicated. Δ indicates where SSV-ΔVP2 was spotted, ΔD where SSV-Δd244 was spotted. P indicates SSV-WT spotted as a positive control. T or Tx indicates 2 μL of 0.01% Triton X-100 spotted as a control for lawn growth.
Mentions: To confirm the presence of infectious virus, halo assays were performed in duplicate 48 and 72 h post-electroporation (Stedman et al., 2003). Uninfected Sulfolobus GΘ cells were diluted to an OD600 nm = ~0.3 and allowed to grow until the OD600 nm reached ~0.35 (about 2.5 h). Half of a milliliter of this uninfected culture was added to 5 mL YS media containing 0.2% wt/vol Gelrite® as a softlayer and poured onto prewarmed YS plates. Two microliters of supernatant from electroporated cultures was spotted onto the lawns and plates were incubated at 75°C for up to 3 days. A halo of host growth inhibition, typically observed 48–72 h after incubation, indicated the presence of an infectious virus (Figure 2).

Bottom Line: Recently we have developed genetic tools to analyze these genes.In this study, we have deleted three SSV1 open reading frames (ORFs) ranging from completely conserved to poorly conserved: VP2, d244, and b129.Deletion of the universally conserved ORF b129, which encodes a predicted transcriptional regulator, results in loss of infectivity.

View Article: PubMed Central - PubMed

Affiliation: Biology Department, Center for Life in Extreme Environments, Portland State University, Portland, OR, USA.

ABSTRACT
Viruses of thermophilic Archaea are unique in both their structures and genomic sequences. The most widespread and arguably best studied are the lemon-shaped fuselloviruses. The spindle-shaped virus morphology is unique to Archaea but widespread therein. The best studied fusellovirus is SSV1 from Beppu, Japan, which infects Sulfolobus solfataricus. Very little is known about the function of the genes in the SSV1 genome. Recently we have developed genetic tools to analyze these genes. In this study, we have deleted three SSV1 open reading frames (ORFs) ranging from completely conserved to poorly conserved: VP2, d244, and b129. Deletion of the universally conserved ORF b129, which encodes a predicted transcriptional regulator, results in loss of infectivity. Deletion of the poorly conserved predicted DNA-binding protein gene VP2 yields viable virus that is indistinguishable from wild-type. Deletion of the well-conserved ORF d244 that encodes a predicted nuclease yields viable virus. However, infection of S. solfataricus with virus lacking ORF d244 dramatically retards host growth, compared to the wild-type virus.

No MeSH data available.


Related in: MedlinePlus