Mutations in the N terminus of the øX174 DNA pilot protein H confer defects in both assembly and host cell attachment

Lindsey N. Young, Alyson M. Hockenberry, Bentley A. Fane

Research output: Contribution to journalArticle

7 Scopus citations

Abstract

The øX174 DNA pilot protein H forms an oligomeric DNA-translocating tube during penetration. However, monomers are incorporated into 12 pentameric assembly intermediates, which become the capsid's icosahedral vertices. The protein's N terminus, a predicted transmembrane helix, is not represented in the crystal structure. To investigate its functions, a series of absolute and conditional lethal mutations were generated. The absolute lethal proteins, a deletion and a triple substitution, were efficiently incorporated into virus-like particles lacking infectivity. The conditional lethal mutants, bearing cold-sensitive (cs) and temperature-sensitive (ts) point mutations, were more amenable to further analyses. Viable particles containing the mutant protein can be generated at the permissive temperature and subsequently analyzed at the restrictive temperature. The characterized cs defect directly affected host cell attachment. In contrast, ts defects were manifested during morphogenesis. Particles synthesized at permissive temperature were indistinguishable from wild-type particles in their ability to recognize host cells and deliver DNA. One mutation conferred an atypical ts synthesis phenotype. Although the mutant protein was efficiently incorporated into virus-like particles at elevated temperature, the progeny appeared to be kinetically trapped in a temperature-independent, uninfectious state. Thus, substitutions in the N terminus can lead to H protein misincorporation, albeit at wild-type levels, and subsequently affect particle function. All mutants exhibited recessive phenotypes, i.e., rescued by the presence of the wild-type H protein. Thus, mixed H protein oligomers are functional during DNA delivery. Recessive and dominant phenotypes may temporally approximate H protein functions, occurring before or after oligomerization has gone to completion.

Original languageEnglish (US)
Pages (from-to)1787-1794
Number of pages8
JournalJournal of virology
Volume88
Issue number3
DOIs
StatePublished - Feb 1 2014

ASJC Scopus subject areas

  • Microbiology
  • Immunology
  • Insect Science
  • Virology

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