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....
After interaction of human immunodeficiency virus type 1 (HIV-1) virions with cell surface receptors, a series of poorly characterized events results in establishment of a viral reverse transcription complex in the host cell cytoplasm. This process is coordinated in such a way that reverse transcription is initiated shortly after formation of the viral reverse transcription complex. However, the mechanism through which virus entry and initiation of reverse transcription are coordinated and how these events are compartmentalized in the infected cell are not known. In this study, we demonstrate that viral reverse transcription complexes associate rapidly with the host cell cytoskeleton during HIV-1 infection and that reverse transcription occurs almost entirely in the cytoskeletal compartment. Interruption of actin polymerization before virus infection reduced association of viral reverse transcription complexes with the cytoskeleton. In addition, efficient reverse transcription was dependent on intact actin microfilaments. The localization of reverse transcription to actin microfilaments was mediated by the interaction of a reverse transcription complex component (gag MA) with actin but not vimentin (intermediate filaments) or tubulin (microtubules). In addition, fusion, but not endocytosis-mediated HIV-1 infectivity, was impaired when actin depolymerizing agents were added to target cells before infection but not when added after infection. These results point to a previously unsuspected role for the host cell cytoskeleton in HIV-1 entry and suggest that components of the cytoskeleton promote establishment of the reverse transcription complex in the host cell and also the process of reverse transcription within this complex.
In the replication of human immunodeficiency virus type 1 (HIV-1), gag MA (matrix), a major structural protein of the virus, carries out opposing targeting functions. During virus assembly, gag MA is cotranslationally myristoylated, a modification required for membrane targeting of gag polyproteins. During virus infection, however, gag MA, by virtue of a nuclear targeting signal at its N terminus, facilitates the nuclear localization of viral DNA and establishment of the provirus. We now show that phosphorylation of gag MA on tyrosine and serine prior to and during virus infection facilitates its dissociation from the membrane, thus allowing it to translocate to the nucleus. Inhibition of gag MA phosphorylation either on tyrosine or on serine prevents gag MA-mediated nuclear targeting of viral nucleic acids and impairs virus infectivity. The requirement for gag MA phosphorylation in virus infection is underscored by our finding that a serine/threonine kinase is associated with virions of HIV-1. These results reveal a novel level of regulation of primate lentivirus infectivity.
An important aspect of the pathophysiology of human immunodeficiency virus type-1 (HIV-1) infection is the ability of the virus to replicate in non-dividing cells. HIV-1 matrix (MA), the amino-terminal domain of the Pr55 gag polyprotein (Pr55), bears a nuclear localization signal that promotes localization of the viral preintegration complex to the nucleus of non-dividing cells following virus entry. However, late during infection, MA, as part of Pr55, directs unspliced viral RNA to the plasma membrane, the site of virus assembly. How MA can mediate these two opposing targeting functions is not understood. Here we demonstrate that MA has a previously undescribed nuclear export activity. Although MA lacks the canonical leucine-rich nuclear export signal, nuclear export is mediated through the conserved Crm1p pathway and functions in both mammalian cells and yeast. A mutation that disrupts the MA nuclear export signal (MA-M4) mislocalizes Pr55 and genomic viral RNA to the nucleus, thereby severely impairing viral replication. Furthermore, we show that MA-M4 can act in a dominant-negative fashion to mislocalize genomic viral RNA even in the presence of wild-type MA. We conclude that the MA nuclear export signal is required to counteract the MA nuclear localization signal, thus ensuring the cytoplasmic availability of the components required for virion assembly.
SUMMARYUncoating of influenza virus (strain WSN) in MDCK cells was studied by following the fate of the virus labelled with radioactive precursors. The accumulation of subviral components of input virus was observed in nuclear-associated cytoplasm (NAC) obtained by treatment of the nuclei with citric acid. Two types of subviral components were found there, ribonucleoproteins (RNPs) and larger subviral particles (SVP) containing RNPs in association with M protein. SVP, with different relative amounts of M protein, were revealed in NAC, suggesting that M protein was gradually released from RNPs. The released RNPs entered the nuclei while M protein accumulated within perinuclear membranes. Thus, SVP could be regarded as probable intermediates in virus uncoating. Rimantadine prevented the release of M protein from RNPs and their penetration into the nuclei provoking the accumulation of subviral components in NAC.
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