Dataset Information

Complete Genome Sequence of a UL96 Mutant Cytomegalovirus Towne-BAC (Bacterial Artificial Chromosome) Isolate Passaged in Fibroblasts To Allow Accumulation of Compensatory Mutations.

ABSTRACT: Here, we present the complete genome sequence of a cytomegalovirus Towne-BAC (bacterial artificial chromosome) isolate that we first genetically engineered to mutate the UL96 gene and then serially passaged in human fibroblasts to allow for the accumulation of compensatory mutations. A total of 17 single-base substitutions were discovered in the passaged genome compared to the reference sequence (KF493877).

SUBMITTER: Brechtel TM

PROVIDER: S-EPMC3813188 | biostudies-literature | 2013 Oct

REPOSITORIES: biostudies-literature

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Publications

Complete Genome Sequence of a UL96 Mutant Cytomegalovirus Towne-BAC (Bacterial Artificial Chromosome) Isolate Passaged in Fibroblasts To Allow Accumulation of Compensatory Mutations.

Brechtel Teal M TM Tyner Molly M Tandon Ritesh R

Genome announcements 20131024 5

Here, we present the complete genome sequence of a cytomegalovirus Towne-BAC (bacterial artificial chromosome) isolate that we first genetically engineered to mutate the UL96 gene and then serially passaged in human fibroblasts to allow for the accumulation of compensatory mutations. A total of 17 single-base substitutions were discovered in the passaged genome compared to the reference sequence (KF493877). ...[more]

PMID: 24158558

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Project description:Human cytomegalovirus (CMV) has evolved to replicate while causing minimal damage, maintain life-long latency, reactivate sub-clinically, and, in spite of robust host immunity, produce and shed infectious virus in order to transmit to new hosts. The CMV temperance factor RL13 may contribute to this strategy of coexistence with the host by actively restricting viral replication and spread. Viruses with an intact RL13 gene grow slowly in cell culture, release little extracellular virus, and form small foci. By contrast, viruses carrying disruptive mutations in the RL13 gene form larger foci and release higher amounts of cell-free infectious virions. Such mutations invariably arise during cell culture passage of clinical isolates and are consistently found in highly adapted strains. The potential existence in such strains of other mutations with roles in mitigating RL13's restrictive effects, however, has not been explored. To this end, a mutation that frame shifts the RL13 gene in the highly cell culture-adapted laboratory strain Towne was repaired, and a C-terminal FLAG epitope was added. Compared to the frame-shifted parental virus, viruses encoding wild-type or FLAG-tagged wild-type RL13 produced small foci and replicated poorly. Within six to ten cell culture passages, mutations emerged in RL13 that restored replication and focus size to those of the RL13-frame-shifted parental virus, implying that none of the numerous adaptive mutations acquired by strain Towne during more than 125 cell culture passages mitigate the temperance activity of RL13. Whilst RL13-FLAG expressed by passage zero stocks was localized exclusively within the virion assembly compartment, RL13-FLAG with a E208K substitution that emerged in one lineage was mostly dispersed into the cytoplasm, suggesting that localization to the virion assembly compartment is likely required for RL13 to exert its growth-restricting activities. Changes in localization also provided a convenient way to assess the emergence of RL13 mutations during serial passage, highlighting the usefulness of RL13-FLAG Towne variants for elucidating the mechanisms underlying RL13's temperance functions.

Dataset Information

Complete Genome Sequence of a UL96 Mutant Cytomegalovirus Towne-BAC (Bacterial Artificial Chromosome) Isolate Passaged in Fibroblasts To Allow Accumulation of Compensatory Mutations.

Publications

Complete Genome Sequence of a UL96 Mutant Cytomegalovirus Towne-BAC (Bacterial Artificial Chromosome) Isolate Passaged in Fibroblasts To Allow Accumulation of Compensatory Mutations.

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