The minute virus of mice capsid specifically recognizes the 3' hairpin structure of the viral replicative-form DNA: mapping of the binding site by hydroxyl radical footprinting

Abstract

The terminal hairpin structures of the DNA of minute virus of mice (MVM) are essential for viral replication. Here we show that the hairpin 3' terminus of MVM replicative-form DNA binds specifically to empty MVM capsids. Binding of the same terminal DNA sequence in its linear double-stranded (extended) conformation was not observed. After heat denaturation and quick cooling of 3'-terminal extended-form fragments, not only the virion strand but also the complementary strand was found to bind to the capsid, presumably because each strand reformed a similar hairpin structure. No binding affinity for the capsid was found to be associated with hairpin or extended 5' termini or with any other region of the viral DNA. Hydroxyl radical footprinting analyses revealed three protected nucleotide stretches forming a binding site at the branch point of the two 3'-terminal hairpin arms looping out from the DNA stem (T structure). Single base changes within this site did not affect the binding. In band shift experiments, specific binding to the T structure was demonstrated for VP1 but not for VP2.

The minor capsid protein VP1 of the autonomous parvovirus minute virus of mice is dispensable for encapsidation of progeny single-stranded DNA but is required for infectivity

“…Although the capsid proteins are not essential for the amplification of double-stranded RF (Fig. 2), previous results had suggested that the capsid proteins might participate in proper maturation of the viral left-hand (3') end: (i) empty MVM capsids, as well as reconstituted VP1 (but not VP2), bind specifically to the 3' end of MVM DNA (65), and (ii) the expression of the capsid proteins precedes the appearance of left-end turnaround forms in a highly synchronized infection (64). Therefore, we chose to examine specific viral replicative intermediates generated by these capsid mutants by using a combination of two-dimensional agarose gel electrophoresis and restriction enzyme analysis.…”

Evidence of Pervasive Biologically Functional Secondary Structures within the Genomes of Eukaryotic Single-Stranded DNA Viruses

“…Although the sequence of this structure is divergent in all five mastrevirus data sets, it is plausible that this structure has some function during splicing of the movement protein intron. (Table S2) (42), and the 3= complementary strand T-shaped structure that binds to the viral capsid in some parvoviruses (Par-voMPV; rank 3) (Table S2) (96). Besides these well-known structures, we sought to identify other uncharacterized, but likely biologically functional, structural elements within some of these genomes.…”

Adeno-Associated Virus Type 2 Protein Interactions: Formation of Pre-Encapsidation Complexes

“…Current concepts for the solution of this problem postulate a specific interaction of a part of the genome, i.e., the packaging sequence, with components of the packaging apparatus and capsid. For the autonomous parvoviruses, a specific interaction of the 3Ј hairpin structure of MVM with the capsid (43) and an interaction of a 3Ј-terminal fragment of the Aleutian mink disease virus with VP1 (44) have been demonstrated. In contrast, a direct interaction of AAV-2 ITRs, which are necessary and sufficient for AAV-2 genome encapsidation, with free capsid proteins or capsids produced in the absence of Rep proteins could not be demonstrated.…”