Mutations in the N Terminus of the øX174 DNA Pilot Protein H Confer Defects in both Assembly and Host Cell Attachment

Abstract

. 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.

Structural changes of tailless bacteriophage ΦX174 during penetration of bacterial cell walls

“…In time course experiments, most of the infectivity was lost during the first 5 min ( Fig. S1), consistent with the results of previous in vivo studies (17,19,20). Both native and LPS-treated ΦX174 particles…”

“…Proteins G and H are known to interact with the LPS of suitable hosts in a species-specific manner (12,14). Mutations affecting host cell recognition and/or the kinetics of DNA ejection have been isolated and identified in genes F, G, and H (16)(17)(18).…”

Structure-Function Analysis of the ϕX174 DNA-Piloting Protein Using Length-Altering Mutations

“…H protein is involved in multiple aspects of the viral life cycle, from host attachment to intracellular assembly. Previously characterized mutants exhibited both attachment and assembly defects (27,28). Therefore, all aspects of the X174 H ϩ 14 life cycle were analyzed.…”

Mutagenic Analysis of a DNA Translocating Tube’s Interior Surface

“…Gene H mutations can prevent H protein incorporation during morphogenesis or result in H-containing, uninfectious particles [32,44]. To determine whether the H proteins were incorporated at 22 • C, lysis resistant cells expressing the wild-type, T244Q, M251Q, and T244Q/M251Q H genes were infected with am(H)M251.…”