HIV-1 infection of non-dividing cells: evidence that the amino-terminal basic region of the viral matrix protein is important for Gag processing but not for post-entry nuclear import

ABSTRACTThe HIV-1 matrix (MA) protein is the amino-terminal domain of the HIV-1 precursor Gag (Pr55Gag) protein. MA binds to membranes and RNAs, helps transport Pr55Gag proteins to virus assembly sites at the plasma membranes of infected cells, and facilitates the incorporation of HIV-1 envelope (Env) proteins into virions by virtue of an interaction with the Env protein cytoplasmic tails (CTs). MA has been shown to crystallize as a trimer and to organize on membranes in hexamer lattices. MA mutations that localize to residues near the ends of trimer spokes have been observed to impair Env protein assembly into virus particles, and several of these are suppressed by the 62QR mutation at the hubs of trimer interfaces. We have examined the binding activities of wild-type (WT) MA and 62QR MA variants and found that the 62QR mutation stabilized MA trimers but did not alter the way MA proteins organized on membranes. Relative to WT MA, the 62QR protein showed small effects on membrane and RNA binding. However, 62QR proteins bound significantly better to Env CTs than their WT counterparts, and CT binding efficiencies correlated with trimerization efficiencies. Our data suggest a model in which multivalent binding of trimeric HIV-1 Env proteins to MA trimers contributes to the process of Env virion incorporation.IMPORTANCEThe HIV-1 Env proteins assemble as trimers, and incorporation of the proteins into virus particles requires an interaction of Env CT domains with the MA domains of the viral precursor Gag proteins. Despite this knowledge, little is known about the mechanisms by which MA facilitates the virion incorporation of Env proteins. To help elucidate this process, we examined the binding activities of an MA mutant that stabilizes MA trimers. We found that the mutant proteins organized similarly to WT proteins on membranes, and that mutant and WT proteins revealed only slight differences in their binding to RNAs or lipids. However, the mutant proteins showed better binding to Env CTs than the WT proteins, and CT binding correlated with MA trimerization. Our results suggest that multivalent binding of trimeric HIV-1 Env proteins to MA trimers contributes to the process of Env virion incorporation.

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HIV-1 Nuclear Import: Matrix Protein Is Back on Center Stage, This Time Together with Vpr

Molecular Medicine ◽

10.1007/bf03401911 ◽

1998 ◽

Vol 4 (3) ◽

pp. 138-143 ◽

Cited By ~ 22

Author(s):

Michael I. Bukrinsky ◽

Omar K. Haffar

Keyword(s):

Nuclear Import ◽

Matrix Protein ◽

Center Stage ◽

Hiv 1

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Two nuclear localization signals in the HIV-1 matrix protein regulate nuclear import of the HIV-1 pre-integration complex 1 1Edited by M. Gottesman

Journal of Molecular Biology ◽

10.1006/jmbi.2000.3768 ◽

2000 ◽

Vol 299 (2) ◽

pp. 359-368 ◽

Cited By ~ 95

Author(s):

Omar K Haffar ◽

Serguei Popov ◽

Larisa Dubrovsky ◽

Isabelle Agostini ◽

Hao Tang ◽

...

Keyword(s):

Nuclear Localization ◽

Nuclear Import ◽

Matrix Protein ◽

Nuclear Localization Signals ◽

Hiv 1

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Nuclear Import of HIV-1

Viruses ◽

10.3390/v13112242 ◽

2021 ◽

Vol 13 (11) ◽

pp. 2242

Author(s):

Qi Shen ◽

Chunxiang Wu ◽

Christian Freniere ◽

Therese N. Tripler ◽

Yong Xiong

Keyword(s):

Reverse Transcription ◽

Nuclear Import ◽

Integration Site ◽

Nuclear Pore ◽

Nuclear Pore Complexes ◽

New Paradigm ◽

Retroviral Infection ◽

Dividing Cells ◽

Pore Complexes ◽

Hiv 1

The delivery of the HIV-1 genome into the nucleus is an indispensable step in retroviral infection of non-dividing cells, but the mechanism of HIV-1 nuclear import has been a longstanding debate due to controversial experimental evidence. It was commonly believed that the HIV-1 capsid would need to disassemble (uncoat) in the cytosol before nuclear import because the capsid is larger than the central channel of nuclear pore complexes (NPCs); however, increasing evidence demonstrates that intact, or nearly intact, HIV-1 capsid passes through the NPC to enter the nucleus. With the protection of the capsid, the HIV-1 core completes reverse transcription in the nucleus and is translocated to the integration site. Uncoating occurs while, or after, the viral genome is released near the integration site. These independent discoveries reveal a compelling new paradigm of this important step of the HIV-1 life cycle. In this review, we summarize the recent studies related to HIV-1 nuclear import, highlighting the spatial–temporal relationship between the nuclear entry of the virus core, reverse transcription, and capsid uncoating.

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Oxadiazols: a New Class of Rationally Designed Anti-Human Immunodeficiency Virus Compounds Targeting the Nuclear Localization Signal of the Viral Matrix Protein

Journal of Virology ◽

10.1128/jvi.79.20.13028-13036.2005 ◽

2005 ◽

Vol 79 (20) ◽

pp. 13028-13036 ◽

Cited By ~ 17

Author(s):

Omar Haffar ◽

Larisa Dubrovsky ◽

Richard Lowe ◽

Reem Berro ◽

Fatah Kashanchi ◽

...

Keyword(s):

Human Immunodeficiency Virus ◽

Nuclear Localization ◽

Nuclear Import ◽

Nuclear Localization Signal ◽

Matrix Protein ◽

Computer Assisted ◽

Viral Dna ◽

Immunodeficiency Virus ◽

Localization Signal ◽

Hiv 1

ABSTRACT Despite recent progress in anti-human immunodeficiency virus (HIV) therapy, drug toxicity and emergence of drug-resistant isolates during long-term treatment of HIV-infected patients necessitate the search for new targets that can be used to develop novel antiviral agents. One such target is the process of nuclear translocation of the HIV preintegration complex. Previously we described a class of arylene bis(methylketone) compounds that inhibit HIV-1 nuclear import by targeting the nuclear localization signal (NLS) in the matrix protein (MA). Here we report a different class of MA NLS-targeting compounds that was selected using computer-assisted drug design. The leading compound from this group, ITI-367, showed potent anti-HIV activity in cultures of T lymphocytes and macrophages and also inhibited HIV-1 replication in ex vivo cultured lymphoid tissue. The virus carrying inactivating mutations in MA NLS was resistant to ITI-367. Analysis by real-time PCR demonstrated that the compound specifically inhibited nuclear import of viral DNA, measured by two-long terminal repeat circle formation. Evidence of the existence of this mechanism was provided by immunofluorescent microscopy, using fluorescently labeled HIV-1, which demonstrated retention of the viral DNA in the cytoplasm of drug-treated macrophages. Compounds inhibiting HIV-1 nuclear import may be attractive candidates for further development.

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From Entry to Egress: Strategic Exploitation of the Cellular Processes by HIV-1

Frontiers in Microbiology ◽

10.3389/fmicb.2020.559792 ◽

2020 ◽

Vol 11 ◽

Author(s):

Pavitra Ramdas ◽

Amit Kumar Sahu ◽

Tarun Mishra ◽

Vipin Bhardwaj ◽

Ajit Chande

Keyword(s):

Life Cycle ◽

Nuclear Import ◽

Arms Race ◽

Surface Proteins ◽

Restriction Factors ◽

Host Restriction ◽

Cellular Processes ◽

Host Restriction Factors ◽

Post Entry ◽

Hiv 1

HIV-1 employs a rich arsenal of viral factors throughout its life cycle and co-opts intracellular trafficking pathways. This exquisitely coordinated process requires precise manipulation of the host microenvironment, most often within defined subcellular compartments. The virus capitalizes on the host by modulating cell-surface proteins and cleverly exploiting nuclear import pathways for post entry events, among other key processes. Successful virus–cell interactions are indeed crucial in determining the extent of infection. By evolving defenses against host restriction factors, while simultaneously exploiting host dependency factors, the life cycle of HIV-1 presents a fascinating montage of an ongoing host–virus arms race. Herein, we provide an overview of how HIV-1 exploits native functions of the host cell and discuss recent findings that fundamentally change our understanding of the post-entry replication events.

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Analysis of the Viral Elements Required in the Nuclear Import of HIV-1 DNA

Journal of Virology ◽

10.1128/jvi.01952-09 ◽

2009 ◽

Vol 84 (2) ◽

pp. 729-739 ◽

Cited By ~ 48

Author(s):

Lise Rivière ◽

Jean-Luc Darlix ◽

Andrea Cimarelli

Keyword(s):

Nuclear Import ◽

Matrix Protein ◽

Active Phase ◽

Cell Types ◽

Target Cells ◽

Nuclear Pore ◽

Viral Dna ◽

The Matrix ◽

Central Polypurine Tract ◽

Hiv 1

ABSTRACT HIV-1 possesses an exquisite ability to infect cells independently from their cycling status by undergoing an active phase of nuclear import through the nuclear pore. This property has been ascribed to the presence of karyophilic elements present in viral nucleoprotein complexes, such as the matrix protein (MA); Vpr; the integrase (IN); and a cis-acting structure present in the newly synthesized DNA, the DNA flap. However, their role in nuclear import remains controversial at best. In the present study, we carried out a comprehensive analysis of the role of these elements in nuclear import in a comparison between several primary cell types, including stimulated lymphocytes, macrophages, and dendritic cells. We show that despite the fact that none of these elements is absolutely required for nuclear import, disruption of the central polypurine tract-central termination sequence (cPPT-CTS) clearly affects the kinetics of viral DNA entry into the nucleus. This effect is independent of the cell cycle status of the target cells and is observed in cycling as well as in nondividing primary cells, suggesting that nuclear import of viral DNA may occur similarly under both conditions. Nonetheless, this study indicates that other components are utilized along with the cPPT-CTS for an efficient entry of viral DNA into the nucleus.

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Quantitative microscopy of functional HIV post-entry complexes reveals association of replication with the viral capsid

eLife ◽

10.7554/elife.04114 ◽

2014 ◽

Vol 3 ◽

Cited By ~ 107

Author(s):

Ke Peng ◽

Walter Muranyi ◽

Bärbel Glass ◽

Vibor Laketa ◽

Stephen R Yant ◽

...

Keyword(s):

Reverse Transcription ◽

Nuclear Import ◽

Viral Proteins ◽

Host Factors ◽

Quantitative Detection ◽

Quantitative Microscopy ◽

Nuclear Entry ◽

High Concentrations ◽

Post Entry ◽

Hiv 1

The steps from HIV-1 cytoplasmic entry until integration of the reverse transcribed genome are currently enigmatic. They occur in ill-defined reverse-transcription- and pre-integration-complexes (RTC, PIC) with various host and viral proteins implicated. In this study, we report quantitative detection of functional RTC/PIC by labeling nascent DNA combined with detection of viral integrase. We show that the viral CA (capsid) protein remains associated with cytoplasmic RTC/PIC but is lost on nuclear PIC in a HeLa-derived cell line. In contrast, nuclear PIC were almost always CA-positive in primary human macrophages, indicating nuclear import of capsids or capsid-like structures. We further show that the CA-targeted inhibitor PF74 exhibits a bimodal mechanism, blocking RTC/PIC association with the host factor CPSF6 and nuclear entry at low, and abrogating reverse transcription at high concentrations. The newly developed system is ideally suited for studying retroviral post-entry events and the roles of host factors including DNA sensors and signaling molecules.

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