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Permissiveness of the host cell to productive infection by oncoretroviruses is cell-cycle dependent, and nuclear localization of viral nucleoprotein preintegration complexes will occur only after cells have passed through mitosis. In contrast, establishment of an integrated provirus after infection by the lentivirus HIV-1 is independent of host cell proliferation. The ability of HIV-1 to replicate in non-dividing cells is partly accounted for by the karyophilic properties of the viral preintegration complex which, after virus infection, is actively transported to the host cell nucleus. Here we report that the gag matrix protein of HIV-1 contains a nuclear localization sequence which, when conjugated to a heterologous protein, directs its nuclear import. In addition, HIV-1 mutants containing amino-acid substitutions in this nuclear localization signal integrate and replicate within dividing but not growth-arrested cells, and thus display a phenotype more representative of an oncoretrovirus.

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....

A common feature in the life cycle of cytocidal retroviruses, including human immunodeficiency virus type 1 (HIV-1), is the accumulation of large amounts of unintegrated viral DNA. As yet, the role of unintegrated viral DNA in the cytopathogenesis of cytocidal retrovirus infections remains unresolved. HIV-1 mutants which were deleted in the integrase/endonuclease gene and which were unable to establish an integrated form of the virus were constructed. Despite an inability to integrate, these mutants were fully competent templates for HIV-1 core and envelope antigen production. HIV-1 antigen could be detected in the supernatants of lymphocyte cultures infected with HIV-1 integrase mutants. However, an inability to rescue infectious virus from these cultures indicated that HIV-1 integration was required for the production of infectious HIV-1. On the basis of the ability of unintegrated HIV-1 DNA to serve as a template for HIV-1 antigen production, it is plausible that unintegrated viral DNA can contribute to the HIV-1 antigen pool during HIV-1 replication.

A modified polymerase chain reaction protocol was used to amplify the entire envelope-coding region of HIV-1 directly from brain and lymph node tissue obtained at autopsy from three HIV-1-infected individuals. Molecular analysis of amplified DNA by digestion with 18 restriction endonucleases, singly and in combination, revealed different HIV-1 genotypes in the brain and lymph node compartments in each of the three individuals. This anatomic compartmentalization of HIV-1 populations may reflect different viral genomic sequences that determine tropism or differences in host immune selection pressures in the brain and lymphoid compartments that drive the emergence of distinct viral populations.