Active nuclear import of human immunodeficiency virus type 1 preintegration complexes.

Abstract

After cell infection by the human immuno deficiency virus type 1 (HIV-1), nascent viral DNA in the form of a high molecular weight nucleoprotein preintegration comin plex must be transported to the nucleus of the host cell prior to integration of viral DNA with the host genome. The mechm used by retroviruses for nuclear targeting of preintegration complexes and dependence on cell division has not been established. Our studies show that, after infection, the preintegration complex of HIV-1 was rapidly transported to the nucleus of the host cell by a process that required ATP but was independent of cell division. Functional HIV-1 integrase, an essential component of the preintegration complex, was not required for nuclear import of these complexes. The ability of iHV-1 to use host cell active transport processes for nuclear import of the viral preintegration complex obviates the requireenlt for host cell division in establishment of the integrated provires. These findgs are pertinent to our underding of early events in the life cycle of HIV-1 and to the mode of HIVE1 replication in terminally differentiated nondividing cells such as macrophages (monocytes, tissue macrophages, follicular dendritic cells, and microglial cells).Integration of the retroviral genome with cellular DNA and establishment of the provirus is an essential step in retrovirus replication (1). The integration reaction is catalyzed by a virus-encoded integrase, which is derived from the virus particle and which, after reverse transcription of genomic viral RNA, remains associated with the viral cDNA in a high molecular weight nucleoprotein preintegration complex (2). Targeting of the viral preintegration complex to host cell DNA is therefore dependent on transport of this complex to the nucleus of the host cell. The process that directs nuclear localization of retroviral preintegration complexes after infection and the dependence ofthis process on cell division are unknown. Oncogenic HIV-1-infected cells were lysed in hypotonic medium (6) using multiple strokes of a Dounce homogenizer, and nuclear integrity during cell lysis was monitored by phase-contrast microscopy. Nuclei were extracted with a hypertonic buffer (6) and both nuclear and cytoplasmic extracts were fractionated on nonionic density gradients as described (2). Integration activity in each gradient fraction was analyzed in vitro by a modification of a previous protocol (7). Briefly, 100 ul of each gradient fraction was mixed with 1 pug of ir AN7 target DNA (8) in a reaction volume of 150 Al and was incubated 60 min at 220C. Samples were treated with DNA polymerase I and deproteinated; in vitro integration products were identified by two rounds of PCR with nested HIV-1 U5 and R long terminal repeat (LTR) primers. PCR products were visualized by Southern blot hybridization with 32P-end-labeled oligonucleotide probes as described elsewhere (4).PCR Analysis of HIV-1 DNA in Nuclear and Cytoplasmic Cell Extracts. Cells were washed once in ice-cold phosphatebuffered saline (pH 7....

Cell-specific nuclear import of plasmid DNA in smooth muscle requires tissue-specific transcription factors and DNA sequences

“…10,27,28 Similar NLS-mediated nuclear import models have been proposed for viral genomes, including HIV and adenovirus. 29,30 In addition, several groups have shown that by complexing plasmids with proteins, such as histone proteins and nuclear factor kB, or with peptide nucleic acids (PNAs) that contain nuclear localization signals, nuclear import and expression are enhanced. [31][32][33][34] As SRF and NK3 factors are coexpressed specifically in SMCs, other cell types are unable to form an import complex on the pDNA, making the SMGA promoter a tissuespecific DTS.…”

Macrophages and HIV-1: dangerous liaisons

“…PIC is transported through nuclear pores via an active, energydependent mechanism (Bukrinsky et al, 1992;Depienne et al, 2001;Gallay et al, 1997).…”

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