Structural basis for bacteriophage phi X174 assembly and eclipse as defined by temperature-sensitive mutations.

Abstract

We present a screening procedure for classifying bacteriophage phi X174 mutants as defective in either "early" (genome delivery and RF DNA replication) or "late" (progeny ssDNA replication and morphogenesis) functions. When applied to 108 ts mutants, only five were classified as "early" mutants. Quantitative one-step growth curves confirmed the classification for 22 "late" mutants, while only one "early" mutant was correctly classified by the procedure. The other four "early" mutants gave burst sizes that required a third, or "intermediate," class of functional defects. The mutation in the "early" mutant mapped in gene A, which encodes an endonuclease required for RF replication. However, most of the mutations in the "late" and "intermediate" mutants mapped in genes for F, G, and H, three structural proteins found in mature virions. The amino acid substitutions for each mutation in F capsid and G "spike" genes were located in the atomic structure of wt virions. All but two replacements are on the outer surface of the virion or at interfaces between neighboring polypeptide chains, suggesting defects in protein-protein interactions involved in quaternary rearrangements during maturation. The kinetics of viral eclipse are also altered in most gene F "late" mutants, but only in one gene G "intermediate" mutant. Thus, the F capsid protein appears to have a central role in DNA ejection from the capsid.