THE MISMATCHED NUCLEOTIDES IN THE 5'-TERMINAL HAIRPIN OF MINUTE VIRUSOF MICE ARE REQUIRED FOR EFFICIENT VIRAL-DNA REPLICATION

The 5'-terminal sequence in the DNA of the parvovirus minute virus of mice (MVM) is a palindrome, It can form a hairpin, the stem of which i s entirely base-paired except for three consecutive unpaired nucleotid es which form a bubble. Since this structure is well conserved among d ifferent parvoviruses, we examined its importance for viral replicatio n by generating MVM mutants with alterations in this region, A clone o f MVMp DNA which contained the entire 3' end and more than half of the 5' palindrome was made. Although it lacked the sequence information t o form a wild-type bubble, this DNA was infectious, On transfection in to A9 fibroblasts, it gave rise to a virus (MVMs) which had a bubble i n its 5' palindrome, The bubble consisted of four mismatched nucleotid es in the same location as the unpaired nucleotides of the wild-type p alindrome, Apparently, neighboring plasmid sequences were incorporated into the viral DNA, enabling formation of the mismatch. This observat ion suggested that a bubble is critical for growth of MVM but that its sequence is not, To find out whether MVM lacking a bubble in the 5' p alindrome is viable, we made a second clone in which the plasmid seque nces incorporated in MVMs were removed, Transfection of this DNA gave rise to a virus (MVMx) in which the nucleotides unpaired in the wild-t ype hairpin are now fully base-paired, Although MVMx can be propagated , it is defective in comparison with wild-type MVMp; it exhibited abou t a 50-fold-lower ratio of plaque-forming units to DNA content, In mix ed infections, MVMp consistently outgrew the bubbleless MVMx, The rate of accumulation of DNA replication intermediates was lower for MVMx t han for the wild-type virus, Quantitative analysis of the 5' termini o f replicative form DNA suggested that the ability of MVMx to convert h airpin 5' termini to extended termini is impaired. In contrast, the vi rus with the altered bubble, MVMs, behaved like the wild-type MVMp in all the assays, We conclude that MVM lacking a bubble in its 5'-termin al DNA hairpin is less infectious than and has a selective disadvantag e compared with wild-type MVM. The nucleotide sequence of the bubble i s not critical, We provide evidence that the presence of a bubble is n ecessary for efficient viral DNA replication.