scholarly journals Dominant Negative MA-CA Fusion Protein Is Incorporated into HIV-1 Cores and Inhibits Nuclear Entry of Viral Preintegration Complexes

2019 ◽  
Vol 93 (21) ◽  
Author(s):  
Jordan Anderson-Daniels ◽  
Parmit K. Singh ◽  
Gregory A. Sowd ◽  
Wen Li ◽  
Alan N. Engelman ◽  
...  
Keyword(s):  
Antiviral Activity ◽  
Mutational Analysis ◽  
Membrane Binding ◽  
Dominant Negative ◽  
Target Cells ◽  
Virus Infectivity ◽  
Nuclear Entry ◽  
Gag Polyprotein ◽  
Hiv 1 ◽  
Ca Protein

ABSTRACT Particle maturation is a critical step in the HIV-1 replication cycle that requires proteolytic cleavage of the Gag polyprotein into its constitutive proteins: the matrix (MA), capsid (CA), nucleocapsid (NC), and p6 proteins. The accurate and efficient cleavage of Gag is essential for virion infectivity; inhibitors of the viral protease are potent antivirals, and substitutions in Gag that prevent its cleavage result in reduced HIV-1 infectivity. In a previous study, a mutation inhibiting cleavage at the MA-CA junction was observed to potently inhibit virus infection: incorporation of small amounts of uncleaved MA-CA protein into HIV-1 particles inhibited infectivity by ∼95%, and the resulting viral particles exhibited aberrant capsids. Here we report a detailed mechanistic analysis of HIV-1 particles bearing uncleaved MA-CA protein. We show that the particles contain stable cores and can efficiently saturate host restriction by TRIMCyp in target cells. We further show that MA-CA associates with CA in particles without detectably affecting the formation of intermolecular CA interfaces. Incorporation of MA-CA did not markedly affect reverse transcription in infected cells, but nuclear entry was impaired and integration targeting was altered. Additionally, results from mutational analysis of Gag revealed that membrane-binding elements of MA contribute to the antiviral activity of uncleaved MA-CA protein. Our results suggest that small amounts of partially processed Gag subunits coassemble with CA during virion maturation, resulting in impaired capsid functions. IMPORTANCE To become infectious, newly formed HIV-1 particles undergo a process of maturation in which the viral polyproteins are cleaved into smaller components. A previous study demonstrated that inclusion of even small quantities of an uncleavable mutant Gag polyprotein results in a strong reduction in virus infectivity. Here we show that the mechanism of transdominant inhibition by uncleavable Gag involves inhibition of nuclear entry and alteration of viral integration sites. Additionally, the results of mutational analysis suggest that the membrane-binding activity of Gag is a major requirement for the antiviral activity. These results further define the antiviral mechanism of uncleavable Gag, which may be useful for exploiting this effect to develop new antivirals.

scholarly journals HIV-1 CA inhibitors are antagonized by inositol phosphate stabilization of the viral capsid in cells

2021 ◽  
Author(s):  
Gregory A Sowd ◽  
Jiong Shi ◽  
Christopher Aiken
Keyword(s):  
Antiretroviral Therapy ◽  
Antiviral Activity ◽  
Binding Site ◽  
Target Cells ◽  
Nuclear Entry ◽  
Inositol Hexakisphosphate ◽  
Capsid Stability ◽  
Hiv 1 ◽  
In Cells

The HIV-1 capsid, composed of the CA protein, is the target of the novel antiretroviral drug lenacapavir (LCV). CA inhibitors block host factor binding and alter capsid stability to prevent nuclear entry and reverse transcription (RTN), respectively. Capsid stability is mediated in vitro by binding to the host cell metabolite inositol hexakisphosphate (IP6). IP6 depletion in target cells has little effect on HIV-1 infection. We hypothesized that capsid-altering concentrations of CA inhibitors might reveal an effect of IP6 depletion on HIV-1 infection in target cells. To test this, we studied the effects of IP6 depletion on inhibition of infection by the CA inhibitors PF74 and LCV. At low doses of either compound that affect HIV-1 nuclear entry, no effect of IP6 depletion on antiviral activity was observed. Increased antiviral activity was observed in IP6-depleted cells at inhibitor concentrations that affect capsid stability, correlating with increased RTN inhibition. Assays of uncoating and endogenous RTN of purified cores in vitro provided additional support. Our results show that inositol phosphates stabilize the HIV-1 capsid in target cells, thereby dampening the antiviral effects of capsid-targeting antiviral compounds. We propose that targeting of the IP6-binding site in conjunction with CA inhibitors will lead to robust antiretroviral therapy. Importance HIV-1 infection and subsequent depletion of CD4 + T cells results in AIDS. Antiretroviral therapy (ART) treatment of infected individuals prevents progression to AIDS. The HIV-1 capsid has recently become an ART target. Capsid inhibitors block HIV-1 infection at multiple steps, offering advantages over current ART. The cellular metabolite inositol hexakisphosphate (IP6) binds the HIV-1 capsid, stabilizing it in vitro . However, the function of this interaction in target cells is unclear. Our results imply that IP6 stabilizes the incoming HIV-1 capsid in cells, thus limiting the antiviral efficiency of capsid-destabilizing antivirals. We present a model of capsid inhibitor function and propose that targeting of the IP6-binding site in conjunction with capsid inhibitors currently in development will lead to more robust ART.

scholarly journals Conserved Residue Asn-145 in the C-Terminal Heptad Repeat Region of HIV-1 gp41 is Critical for Viral Fusion and Regulates the Antiviral Activity of Fusion Inhibitors

Viruses ◽  
2019 ◽  
Vol 11 (7) ◽  
pp. 609 ◽  
Author(s):  
Xiuzhu Geng ◽  
Zixuan Liu ◽  
Danwei Yu ◽  
Bo Qin ◽  
Yuanmei Zhu ◽  
...  
Keyword(s):  
Antiviral Activity ◽  
Mutational Analysis ◽  
Heptad Repeat ◽  
Titration Calorimetry ◽  
Target Cells ◽  
Viral Fusion ◽  
Fusion Inhibitors ◽  
Structural And Functional Properties ◽  
Relationship Of ◽  
Hiv 1

Entry of HIV-1 into target cells is mediated by its envelope (Env) glycoprotein composed of the receptor binding subunit gp120 and the fusion protein gp41. Refolding of the gp41 N- and C-terminal heptad repeats (NHR and CHR) into a six-helix bundle (6-HB) conformation drives the viral and cellular membranes in close apposition and generates huge amounts of energy to overcome the kinetic barrier leading to membrane fusion. In this study, we focused on characterizing the structural and functional properties of a single Asn-145 residue, which locates at the middle CHR site of gp41 and is extremely conserved among all the HIV-1, HIV-2, and simian immunodeficiency virus (SIV) isolates. By mutational analysis, we found that Asn-145 plays critical roles for Env-mediated cell-cell fusion and HIV-1 entry. As determined by circular dichroism (CD) spectroscopy and isothermal titration calorimetry (ITC), the substitution of Asn-145 with alanine (N145A) severely impaired the interactions between the NHR and CHR helices. Asn-145 was also verified to be important for the antiviral activity of CHR-derived peptide fusion inhibitors and served as a turn-point for the inhibitory potency. Intriguingly, Asn-145 could regulate the functionality of the M-T hook structure at the N-terminus of the inhibitors and displayed comparable activities with the C-terminal IDL anchor. Crystallographic studies further demonstrated the importance of Asn-145-mediated interhelical and intrahelical interactions in the 6-HB structure. Combined, the present results have provided valuable information for the structure-function relationship of HIV-1 gp41 and the structure-activity relationship of gp41-dependent fusion inhibitors.

scholarly journals Antiviral activity of recombinant ankyrin targeted to the capsid domain of HIV-1 Gag polyprotein

Retrovirology ◽  
2012 ◽  
Vol 9 (1) ◽  
pp. 17 ◽  
Author(s):  
Sawitree Nangola ◽  
Agathe Urvoas ◽  
Marie Valerio-Lepiniec ◽  
Wannisa Khamaikawin ◽  
Supachai Sakkhachornphop ◽  
...  
Keyword(s):  
Antiviral Activity ◽  
Gag Polyprotein ◽  
Hiv 1

scholarly journals The Structure-Activity Relationships of 2,4(1H,3H)-pyrimidinedione Derivatives as potent HIV type 1 and type 2 inhibitors

2007 ◽  
Vol 18 (5) ◽  
pp. 259-275 ◽  
Author(s):  
Robert W Buckheit ◽  
Tracy L Hartman ◽  
Karen M Watson ◽  
Ho Seok Kwon ◽  
Sun Hwan Lee ◽  
...  
Keyword(s):  
Antiviral Activity ◽  
Binding Pocket ◽  
Target Cells ◽  
Significant Activity ◽  
In Vitro Test ◽  
Structure Activity Relationships ◽  
Structure Activity ◽  
Test Concentration ◽  
Hiv 1

Since the discovery of the 2,4 (1 H,3 H)-pyrimidinediones as potent non-nucleoside inhibitors of the HIV-1 reverse transcriptase (RT) this class of compounds has yielded a number of N-1 acyclic substituted pyrimidinediones with substantial antiviral activity, which is highly dependent upon their molecular fit into the binding pocket common to this inhibitory class. We have specifically examined the structure activity relationships of compounds with chemical modification made by substituting homocyclic rather than acyclic moieties at N-1 of the pyrimidinedione. Seventy-four compounds were synthesized and evaluated for antiviral activity against HIV-1 and HIV-2. The homocyclic modifications resulted in compounds with significant activity against both HIV-1 and HIV-2, suggesting these compounds represent a new class of non-nucleoside RT inhibitors. The structure-activity relationship (SAR) evaluations indicated that cyclopropyl, phenyl and 1- or 3-cyclopenten-1-yl substitutions at the N-1 of the pyrimidinedione, the addition of a methyl linker between the cyclic moiety and the N-1 and the addition of a benzoyl group at the C-6 of the pyrimidinedione had the greatest contribution to antiviral activity. Five pyrimidinedione analogues with therapeutic indexes (TIs)>450,000 and a specific analogue (1-cyclopropylmethyl-5-isopropyl-6-(3,5-dimethylbenzoyl)-2,4(1 H,3 H)-pyrimidinedione), which exhibited a TI of >2,000,000, were identified. None of the analogues were cytotoxic to target cells at the highest in vitro test concentration, which is the upper limit of compound solubility of the analogues in aqueous solution. Thus, we have identified a series of pyrimidinediones with substantially improved antiviral efficacy and range of action and with significantly reduced cellular cytotoxicity.

scholarly journals HIV-1 requires capsid remodelling at the nuclear pore for nuclear entry and integration

2021 ◽  
Author(s):  
Anabel Guedán ◽  
Callum D Donaldson ◽  
Ophélie Cosnefroy ◽  
Ian A Taylor ◽  
Kate N. Bishop
Keyword(s):  
Reverse Transcription ◽  
Integration Site ◽  
Productive Infection ◽  
Nuclear Pore ◽  
Virus Infectivity ◽  
Nuclear Speckles ◽  
Nuclear Entry ◽  
The Core ◽  
Fold Reduction ◽  
Hiv 1

The capsid (CA) lattice of the HIV-1 core plays a key role during infection. From the moment the core is released into the cytoplasm, it interacts with a range of cellular factors that, ultimately, direct the pre-integration complex to the integration site. For integration to occur, the CA lattice must disassemble. Early uncoating or a failure to do so has detrimental effects on virus infectivity, indicating that an optimal stability of the viral core is crucial for infection. Here, we introduced cysteine residues into HIV-1 CA in order to induce disulphide bond formation and engineer hyper-stable mutants that are slower or unable to uncoat, and then followed their replication. From a panel of mutants, we identified three with increased capsid stability in cells and found that, whilst the M68C/E212C mutant had a 5-fold reduction in reverse transcription, two mutants, A14C/E45C and E180C, were able to reverse transcribe to approximately WT levels. Moreover, these mutants only had a 5-fold reduction in 2-LTR circle production, suggesting that not only could reverse transcription complete in hyper-stable cores, but that the nascent viral cDNA could enter the nuclear compartment. Furthermore, we observed significant levels of A14C/E45C mutant capsid in nuclear and chromatin-associated fractions implying that the hyper-stable cores themselves entered the nucleus. Immunofluorescence studies revealed that although the A14C/E45C mutant capsid reached the nuclear pore with the same kinetics as wild type capsid, it was then retained at the pore in association with Nup153. Crucially, infection with the hyper-stable mutants did not promote CPSF6 re-localisation to nuclear speckles, despite the mutant capsids being competent for CPSF6 binding. These observations suggest that hyper-stable cores are not able to uncoat, or remodel, enough to pass through or dissociate from the nuclear pore and integrate successfully. This, is turn, highlights the importance of capsid lattice flexibility for nuclear entry. In conclusion, we hypothesise that during a productive infection, a capsid remodelling step takes place at the nuclear pore that releases the core complex from Nup153, and relays it to CPSF6, which then localises it to chromatin ready for integration.

scholarly journals HIV-1 Gag release from yeast reveals ESCRT interaction with the Gag N-terminal protein region

2020 ◽  
Vol 295 (52) ◽  
pp. 17950-17972
Author(s):  
Birgit Meusser ◽  
Bettina Purfuerst ◽  
Friedrich C. Luft
Keyword(s):  
Plasma Membrane ◽  
Mutational Analysis ◽  
Genetic Manipulation ◽  
Membrane Binding ◽  
Hek293 Cells ◽  
Binding Studies ◽  
Site Mutation ◽  
Terminal Protein ◽  
Hiv 1 ◽  
Plasma Membrane Binding

The HIV-1 protein Gag assembles at the plasma membrane and drives virion budding, assisted by the cellular endosomal complex required for transport (ESCRT) proteins. Two ESCRT proteins, TSG101 and ALIX, bind to the Gag C-terminal p6 peptide. TSG101 binding is important for efficient HIV-1 release, but how ESCRTs contribute to the budding process and how their activity is coordinated with Gag assembly is poorly understood. Yeast, allowing genetic manipulation that is not easily available in human cells, has been used to characterize the cellular ESCRT function. Previous work reported Gag budding from yeast spheroplasts, but Gag release was ESCRT-independent. We developed a yeast model for ESCRT-dependent Gag release. We combined yeast genetics and Gag mutational analysis with Gag-ESCRT binding studies and the characterization of Gag-plasma membrane binding and Gag release. With our system, we identified a previously unknown interaction between ESCRT proteins and the Gag N-terminal protein region. Mutations in the Gag-plasma membrane–binding matrix domain that reduced Gag-ESCRT binding increased Gag-plasma membrane binding and Gag release. ESCRT knockout mutants showed that the release enhancement was an ESCRT-dependent effect. Similarly, matrix mutation enhanced Gag release from human HEK293 cells. Release enhancement partly depended on ALIX binding to p6, although binding site mutation did not impair WT Gag release. Accordingly, the relative affinity for matrix compared with p6 in GST-pulldown experiments was higher for ALIX than for TSG101. We suggest that a transient matrix-ESCRT interaction is replaced when Gag binds to the plasma membrane. This step may activate ESCRT proteins and thereby coordinate ESCRT function with virion assembly.

scholarly journals Antiviral properties of palinavir, a potent inhibitor of the human immunodeficiency virus type 1 protease.

1997 ◽  
Vol 41 (5) ◽  
pp. 965-971 ◽  
Author(s):  
D Lamarre ◽  
G Croteau ◽  
E Wardrop ◽  
L Bourgon ◽  
D Thibeault ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Antiviral Activity ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Potent Inhibitor ◽  
Clinical Isolates ◽  
Target Cells ◽  
Immunodeficiency Virus ◽  
Hiv 1

Palinavir is a potent inhibitor of the human immunodeficiency virus type 1 (HIV-1) and type 2 (HIV-2) proteases. Replication of laboratory strains (HIV-1, HIV-2, and simian immunodeficiency virus) and HIV-1 clinical isolates is inhibited by palinavir with 50% effective concentrations ranging from 0.5 to 30 nM. The average cytotoxic concentration of palinavir (35 microM) in the various target cells indicates a favorable therapeutic index. Potent antiviral activity is retained with increased doses of virus and with clinical isolates resistant to zidovudine (AZT), didanosine (ddI), or nevirapine. Combinations of palinavir with either AZT, ddI, or nevirapine demonstrate synergy or additivity in the inhibition of HIV-1 replication. Palinavir retains anti-HIV-1 activity when administered postinfection until times subsequent to the reverse transcription step. In chronically infected CR-10 cells, palinavir blocks Gag precursor polyprotein processing completely, reducing greater than 99% of infectious particle production. The results indicate that the antiviral activity of palinavir is specific to inhibition of the viral protease and occurs at a late stage in the replicative cycle of HIV-1. On the basis of the potent in vitro activity, low-level cytotoxicity, and other data, palinavir was selected for in-depth preclinical evaluation.

scholarly journals Bicaudal D2 facilitates the cytoplasmic trafficking and nuclear import of HIV-1 genomes during infection

2017 ◽  
Vol 114 (50) ◽  
pp. E10707-E10716 ◽  
Author(s):  
Adarsh Dharan ◽  
Silvana Opp ◽  
Omar Abdel-Rahim ◽  
Sevnur Komurlu Keceli ◽  
Sabrina Imam ◽  
...  
Keyword(s):  
Viral Genome ◽  
Target Cells ◽  
Adapter Protein ◽  
Nuclear Entry ◽  
Microtubule Network ◽  
Monocytic Cell ◽  
Monocytic Cell Line ◽  
Microtubule Motor ◽  
Hiv 1

Numerous viruses, including HIV-1, exploit the microtubule network to traffic toward the nucleus during infection. Although numerous studies have observed a role for the minus-end microtubule motor dynein in HIV-1 infection, the mechanism by which the viral core containing the viral genome associates with dynein and induces its perinuclear trafficking has remained unclear. Here, we report that the dynein adapter protein bicaudal D2 (BICD2) is able to interact with HIV-1 viral cores in target cells. We also observe that BICD2 can bind in vitro-assembled capsid tubes through its CC3 domain. We observe that BICD2 facilitates infection by promoting the trafficking of viral cores to the nucleus, thereby promoting nuclear entry of the viral genome and infection. Finally, we observe that depletion of BICD2 in the monocytic cell line THP-1 results in an induction of IFN-stimulated genes in these cells. Collectively, these results identify a microtubule adapter protein critical for trafficking of HIV-1 in the cytoplasm of target cells and evasion of innate sensing mechanisms in macrophages.

scholarly journals Nuclear Import Defect of Human Immunodeficiency Virus Type 1 DNA Flap Mutants Is Not Dependent on the Viral Strain or Target Cell Type

2006 ◽  
Vol 80 (20) ◽  
pp. 10262-10269 ◽  
Author(s):  
Nathalie Arhel ◽  
Sandie Munier ◽  
Philippe Souque ◽  
Karine Mollier ◽  
Pierre Charneau
Keyword(s):  
Human Immunodeficiency Virus ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Target Cell ◽  
Nuclear Import ◽  
Target Cells ◽  
Virus Infectivity ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACT We have previously established, using human immunodeficiency virus type 1 (HIV-1) strain LAI, that the HIV-1 central DNA Flap acts as a cis determinant of viral genome nuclear import. Although the impact of the DNA Flap on nuclear import has already found numerous independent confirmations in the context of lentivirus vectors, it has been claimed that it may be nonessential for infectious virus strains LAI, YU-2 (J. D. Dvorin et al., J. Virol. 76:12087-12096, 2002), HXB2, and NL4-3 (A. Limon et al., J. Virol. 76:12078-12086, 2002). We conducted a detailed analysis of virus infectivity using the provirus clones provided by the authors and analogous target cells. In contrast to published data, our results show that all cPPT mutant viruses exhibit reduced infectivity corresponding to a nuclear import defect irrespective of the viral genetic background or target cell.

scholarly journals Capsid Proteins from Human Immunodeficiency Virus Type 1 and Simian Immunodeficiency Virus SIVmac Can Coassemble into Mature Cores of Infectious Viruses

2008 ◽  
Vol 82 (17) ◽  
pp. 8253-8261 ◽  
Author(s):  
Jianbo Chen ◽  
Vinay K. Pathak ◽  
Weiqun Peng ◽  
Wei-Shau Hu
Keyword(s):  
Human Immunodeficiency Virus ◽  
Simian Immunodeficiency Virus ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Infectious Virus ◽  
Virus Infectivity ◽  
Immunodeficiency Virus ◽  
Hiv 1 ◽  
Ca Protein

ABSTRACT We have recently shown that the Gag polyproteins from human immunodeficiency virus type 1 (HIV-1) and HIV-2 can coassemble and functionally complement each other. During virion maturation, the Gag polyproteins undergo proteolytic cleavage to release mature proteins including capsid (CA), which refolds and forms the outer shell of a cone-shaped mature core. Less than one-half of the CA proteins present within the HIV-1 virion are required to form the mature core. Therefore, it is unclear whether the mature core in virions containing both HIV-1 and HIV-2 Gag consists of CA proteins from a single virus or from both viruses. To determine whether CA proteins from two different viruses can coassemble into mature cores of infectious viruses, we exploited the specificity of the tripartite motif 5α protein from the rhesus monkey (rhTRIM5α) for cores containing HIV-1 CA (hCA) but not the simian immunodeficiency virus SIVmac CA protein (sCA). If hCA and sCA cannot coassemble into the same core when equal amounts of sCA and hCA are coexpressed, the infectivities of such virus preparations in cells should be inhibited less than twofold by rhTRIM5α. However, if hCA and sCA can coassemble into the same core structure to form a mixed core, rhTRIM5α would be able to recognize such cores and significantly restrict virus infectivity. We examined the restriction phenotypes of viruses containing both hCA and sCA. Our results indicate that hCA and sCA can coassemble into the same mature core to produce infectious virus. To our knowledge, this is the first demonstration of functional coassembly of heterologous CA protein into the retroviral core.

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