High resolution localization of replicating viral genome in adenovirus-infected HeLa cells.
Previous autoradiographical and in situ hybridization experiments have revealed that the replication of viral genomes in adenovirus type 5 infected HeLa cells induces changes in nuclear structure of which one of the more striking is the formation of distinctive replicative foci. The latter consist of a viral ssDNA accumulation site and a surrounding fibrillogranular network. We have reexamined these structures and processes by a more direct and higher resolution approach, that is, incorporation of bromodeoxyuridine (BrdU) by the infected cells and subsequent immunogold detection of the BrdU incorporated into DNA. Short pulses with BrdU in pulse-chase experiments confirmed that viral DNA replication at the early stage of nuclear transformation was confined within small virus-induced structures, the so-called early replicative sites, and revealed the persistence of the newly synthesized viral DNA at those sites for at least 2 h. At intermediate and late stages of nuclear transformation, the intensity of viral DNA replication, which varies from one type of replicative focus to another, was most intense in that layer of the fibrillogranular network which was in closest proximity to the viral single-stranded DNA (ssDNA) accumulation sites, whereas replication was weakest over the latter. Subsequently, BrdU-containing viral DNA molecules became more widely distributed in the viral ssDNA accumulation sites and the fibrillogranular network, the two constituents of the viral replicative machinery. Two hours later, some labeled molecules attained the viral genome storage site and/or became encapsulated. The most striking observation is the presence of a limited region in the replicative focus which is the preferential site for viral genome replication. The data also indicated that viral DNA molecules which were labeled during the short pulses remained in the replicative foci themselves, to be replicated and transcribed prior to attaining the pool of inactive genomes and/or becoming encapsulated.
Besse
S
S
Laboratoire de Biologie et Ultrastructure du Noyau de l'UPR 272 CNRS, Villejuif/France.
Puvion-Dutilleul
F
F
D000260:Adenoviruses, Human / Q000235:genetics / Q000302:isolation & purification / Q000502:physiology*; D001973:Bromodeoxyuridine; D002467:Cell Nucleus / Q000648:ultrastructure; D004247:DNA / Q000032:analysis; D004277:DNA, Single-Stranded / Q000032:analysis; D004279:DNA, Viral / Q000032:analysis* / Q000096:biosynthesis; D016679:Genome, Viral; D006367:HeLa Cells / Q000382:microbiology* / Q000648:ultrastructure; D006801:Humans; D007150:Immunohistochemistry; D014779:Virus Replication
D004277:DNA, Single-Stranded; D004279:DNA, Viral; D004247:DNA; D001973:Bromodeoxyuridine