Essential and dispensable virus-encoded replication elements revealed by efforts to develop hypoviruses as gene expression vectors

Nobuhiro Suzuki, L. M. Geletka, D. L. Nuss

Research output: Contribution to journalArticle

46 Citations (Scopus)

Abstract

We have investigated whether hypoviruses, viral agents responsible for virulence attenuation (hypovirulence) of the chestnut blight fungus Cryphonectria parasitica, could serve as gene expression vectors. The infectious cDNA clone of the prototypic hypovirus CHV1-EP713 was modified to generate 20 different vector candidates. Although transient expression was achieved for a subset of vectors that contained the green fluorescent protein gene from Aequorea victoria, long-term expression (past day 8) was not observed for any vector construct. Analysis of viral RNAs recovered from transfected fungal colonies revealed that the foreign genes were readily deleted from the replicating virus, although small portions of foreign sequences were retained by some vectors after months of replication. However, the results of vector viability and progeny characterization provided unexpected new insights into essential and dispensable elements of hypovirus replication. The N-terminal portion (codons 1 to 24) of the 5'-proximal open reading frame (ORF), ORF A, was found to be required for virus replication, while the remaining 598 codons of this ORF were completely dispensable. Substantial alterations were tolerated in the pentanucleotide UAAUG that contains the ORF A termination codon and the overlapping putative initiation codon of the second of the two hypovirus ORFs, ORF B. Replication competence was maintained following either a frameshift mutation that caused a two-codon extension of ORF A or a modification that produced a single-ORF genomic organization. These results are discussed in terms of determinants of hypovirus replication, the potential utility of hypoviruses as gene expression vectors, and possible mechanisms by which hypoviruses recognize and delete foreign sequences.

Original languageEnglish
Pages (from-to)7568-7577
Number of pages10
JournalJournal of Virology
Volume74
Issue number16
DOIs
Publication statusPublished - 2000
Externally publishedYes

Fingerprint

Hypoviridae
Virus Replication
Open Reading Frames
open reading frames
Hypovirus
Gene Expression
gene expression
viruses
codons
Codon
Cryphonectria parasitica
virulence
Aequorea victoria
frameshift mutation
Frameshift Mutation
start codon
Initiator Codon
Terminator Codon
stop codon
Viral RNA

ASJC Scopus subject areas

  • Immunology

Cite this

Essential and dispensable virus-encoded replication elements revealed by efforts to develop hypoviruses as gene expression vectors. / Suzuki, Nobuhiro; Geletka, L. M.; Nuss, D. L.

In: Journal of Virology, Vol. 74, No. 16, 2000, p. 7568-7577.

Research output: Contribution to journalArticle

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