scholarly journals HIV-1 Capsid Protein Forms Spherical (Immature-Like) and Tubular (Mature-Like) Particles in Vitro: Structure Switching by pH-induced Conformational Changes

2001 ◽  
Vol 81 (1) ◽  
pp. 586-594 ◽  
Author(s):  
Lorna S. Ehrlich ◽  
Tianbo Liu ◽  
Suzanne Scarlata ◽  
Benjamin Chu ◽  
Carol A. Carter
Keyword(s):  
Capsid Protein ◽  
Conformational Changes ◽  
Structure Switching ◽  
Hiv 1

Structural determinants of virion assembly and release in the C-terminus of the M-PMV capsid protein

2021 ◽  
Author(s):  
Marlene V. Buckmaster ◽  
Kaneil K. Zadrozny ◽  
Barbie K. Ganser-Pornillos ◽  
Owen Pornillos ◽  
Stephen P. Goff
Keyword(s):  
Capsid Protein ◽  
Conformational Changes ◽  
Structural Determinants ◽  
Particle Assembly ◽  
Gag Protein ◽  
Virion Assembly ◽  
C Terminus ◽  
Hiv 1

The transition from an immature to a fully infectious mature retrovirus particle is associated with molecular switches that trigger dramatic conformational changes in the structure of the Gag proteins. A dominant maturation switch that stabilizes the immature capsid lattice is located downstream of the capsid (CA) protein in many retroviral Gags. The HIV-1 Gag contains a stretch of five amino acid residues termed the ‘clasp motif’, important for the organization of the hexameric subunits that provide stability to the overall immature HIV-1 shell. Sequence alignment of the CA C-terminal domains (CTDs) of the HIV-1 and Mason-Pfizer Monkey Virus (M-PMV) highlighted a spacer-like domain in M-PMV that may provide comparable function. The importance of the sequences spanning the CA-NC cleavage has been demonstrated by mutagenesis, but the specific requirements for the clasp motif in several steps of M-PMV particle assembly and maturation have not been determined in detail. In the present study we report an examination of the role of the clasp motif in the M-PMV life cycle. We generated a series of M-PMV Gag mutants and assayed for assembly of the recombinant protein in vitro , and for the assembly, maturation, release, genomic RNA packaging, and infectivity of the mutant virus in vivo . The mutants revealed major defects in virion assembly and release in 293T and HeLa cells, and even larger defects in infectivity. Our data identifies the clasp motif as a fundamental contributor to CA-CTD interactions necessary for efficient viral infection. Importance The C-terminal domain of the capsid protein of many retroviruses has been shown to be critical for virion assembly and maturation, but the functions of this region of M-PMV are uncertain. We show that a short ‘clasp’ motif in the capsid domain of the M-PMV Gag protein plays a key role in M-PMV virion assembly, genome packaging, and infectivity.

scholarly journals Discovery and Characterization of a Novel CD4-Binding Adnectin with Potent Anti-HIV Activity

2017 ◽  
Vol 61 (8) ◽  
Author(s):  
David Wensel ◽  
Yongnian Sun ◽  
Zhufang Li ◽  
Sharon Zhang ◽  
Caryn Picarillo ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Viral Entry ◽  
High Affinity ◽  
Glycosylation Sites ◽  
Spectrum Activity ◽  
Anti Hiv ◽  
Hiv 1 ◽  
Broad Spectrum Activity

ABSTRACT A novel fibronectin-based protein (Adnectin) HIV-1 inhibitor was generated using in vitro selection. This inhibitor binds to human CD4 with a high affinity (3.9 nM) and inhibits viral entry at a step after CD4 engagement and preceding membrane fusion. The progenitor sequence of this novel inhibitor was selected from a library of trillions of Adnectin variants using mRNA display and then further optimized for improved antiviral and physical properties. The final optimized inhibitor exhibited full potency against a panel of 124 envelope (gp160) proteins spanning 11 subtypes, indicating broad-spectrum activity. Resistance profiling studies showed that this inhibitor required 30 passages (151 days) in culture to acquire sufficient resistance to result in viral titer breakthrough. Resistance mapped to the loss of multiple potential N-linked glycosylation sites in gp120, suggesting that inhibition is due to steric hindrance of CD4-binding-induced conformational changes.

scholarly journals Binding of the C-terminal domain of the HIV-1 capsid protein to lipid membranes: a biophysical characterization

2006 ◽  
Vol 394 (1) ◽  
pp. 345-353 ◽  
Author(s):  
Francisco N. Barrera ◽  
Estefanía Hurtado-Gómez ◽  
María C. Lidón-Moya ◽  
José L. Neira
Keyword(s):  
Capsid Protein ◽  
Lipid Membranes ◽  
Three Dimensional ◽  
Lipid Vesicles ◽  
Lipid Binding ◽  
Scanning Calorimetry ◽  
Form Part ◽  
Virion Envelope ◽  
Hiv 1

The capsid protein, CA, of HIV-1 forms a capsid that surrounds the viral genome. However, recent studies have shown that an important proportion of the CA molecule does not form part of this capsid, and its location and function are still unknown. In the present work we show, by using fluorescence, differential scanning calorimetry and Fourier-transform infrared spectroscopy, that the C-terminal region of CA, CA-C, is able to bind lipid vesicles in vitro in a peripheral fashion. CA-C had a greater affinity for negatively charged lipids (phosphatidic acid and phosphatidylserine) than for zwitterionic lipids [PC/Cho/SM (equimolar mixture of phosphatidylcholine, cholesterol and sphingomyelin) and phosphatidylcholine]. The interaction of CA-C with lipid membranes was supported by theoretical studies, which predicted that different regions, occurring close in the three-dimensional CA-C structure, were responsible for the binding. These results show the flexibility of CA-C to undergo conformational rearrangements in the presence of different binding partners. We hypothesize that the CA molecules that do not form part of the mature capsid might be involved in lipid-binding interactions in the inner leaflet of the virion envelope.

Characterization of a reactive groove in HIV-1 capsid protein defined by an in vitro assembly inhibitor

2007 ◽  
Vol 2007 (Spring) ◽  
Author(s):  
Vanda Bartonova ◽  
Jana Sticht ◽  
Peter Sehr ◽  
Joe Lewis ◽  
Hans-Georg Kraeusslich
Keyword(s):  
Capsid Protein ◽  
In Vitro Assembly ◽  
Hiv 1

scholarly journals Activity of the Small Modified Amino Acid α-Hydroxy Glycineamide on In Vitro and In Vivo Human Immunodeficiency Virus Type 1 Capsid Assembly and Infectivity

2008 ◽  
Vol 52 (10) ◽  
pp. 3737-3744 ◽  
Author(s):  
Samir Abdurahman ◽  
Ákos Végvári ◽  
Masoud Youssefi ◽  
Michael Levi ◽  
Stefan Höglund ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Capsid Protein ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Antiviral Compound ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACT Upon maturation of the human immunodeficiency virus type 1 (HIV-1) virion, proteolytic cleavage of the Gag precursor protein by the viral protease is followed by morphological changes of the capsid protein p24, which will ultimately transform the virus core from an immature spherical to a mature conical structure. Virion infectivity is critically dependent on the optimal semistability of the capsid cone structure. We have reported earlier that glycineamide (G-NH2), when added to the culture medium of infected cells, inhibits HIV-1 replication and that HIV-1 particles with aberrant core structures were formed. Here we show that it is not G-NH2 itself but a metabolite thereof, α-hydroxy-glycineamide (α-HGA), that is responsible for the antiviral activity. We show that α-HGA inhibits the replication of clinical HIV-1 isolates with acquired resistance to reverse transcriptase and protease inhibitors but has no effect on the replication of any of 10 different RNA and DNA viruses. α-HGA affected the ability of the HIV-1 capsid protein to assemble into tubular or core structures in vitro and in vivo, probably by binding to the hinge region between the N- and C-terminal domains of the HIV-1 capsid protein as indicated by matrix-assisted laser desorption ionization-mass spectrometry results. As an antiviral compound, α-HGA has an unusually simple structure, a pronounced antiviral specificity, and a novel mechanism of antiviral action. As such, it might prove to be a lead compound for a new class of anti-HIV substances.

scholarly journals Conserved Surface Residues on the Feline Calicivirus Capsid Are Essential for Interaction with Its Receptor Feline Junctional Adhesion Molecule A (fJAM-A)

2018 ◽  
Vol 92 (8) ◽  
Author(s):  
Zhengchun Lu ◽  
Emily D. Ledgerwood ◽  
Meleana M. Hinchman ◽  
Robert Dick ◽  
John S. L. Parker
Keyword(s):  
Capsid Protein ◽  
Receptor Binding ◽  
Adhesion Molecule ◽  
Conformational Changes ◽  
Growth Kinetics ◽  
Feline Calicivirus ◽  
Viral Capsid ◽  
Virus Receptor ◽  
Receptor Interactions

ABSTRACTHost cell surface receptors are required for attachment, binding, entry, and infection by nonenveloped viruses. Receptor binding can induce conformational changes in the viral capsid and/or the receptor that couple binding with downstream events in the virus life cycle (intracellular signaling, endocytosis and trafficking, and membrane penetration). Virus-receptor interactions also influence viral spread and pathogenicity. The interaction between feline calicivirus (FCV) and its receptor, feline junctional adhesion molecule A (fJAM-A), on host cells is required for infection and induces irreversible, inactivating conformational changes in the capsid of some viral strains. Cryoelectron microscopy (cryo-EM) structures of FCV bound to fJAM-A showed several possible virus-receptor interactions. However, the specific residues on the viral capsid required for binding are not known. Capsid residues that may be involved in postbinding events have been implicated by isolation of soluble receptor-resistant (srr) mutants in which changes in the capsid protein sequence change the capacity of such srr mutants to be inactivated upon incubation with soluble fJAM-A. To clarify which residues on the surface of FCV are required for its interaction with fJAM-A and to potentially identify residues required for postreceptor binding events, we used the existing atomic-resolution structures of FCV and the FCV-fJAM-A cryo-EM structures to select 14 capsid residues for mutation and preparation of recombinant viral capsids. Using this approach, we identified residues on the FCV capsid that are required for fJAM-A binding and other residues that are not required for binding but are required for infection that are likely important for subsequent postbinding events.IMPORTANCEFeline calicivirus (FCV) is a common cause of mild upper respiratory disease in cats. Some FCV isolates can cause virulent systemic disease. The genetic determinants of virulence for FCV are unknown. We previously found that virulent FCV isolates have fasterin vitrogrowth kinetics than less virulent isolates. Differences in viral growthin vitromay correlate with differences in virulence. Here, we investigated the roles of specific FCV capsid residues on the receptor-virus interaction and viral growthin vitro. We show that the capsid protein genes of the virulent FCV-5 isolate determine its fasterin vitrogrowth kinetics compared to those of the nonvirulent FCV-Urbana infectious clone. We also identified residues on the capsid VP1 protein that are important for receptor binding or for steps subsequent to receptor binding. Our data provide further insight into the specific molecular interactions between fJAM-A and the FCV capsid that regulate binding and infectious entry.

scholarly journals Conformational Changes in the 5′ End of the HIV-1 Genome Dependent on the Debranching Enzyme DBR1 during Early Stages of Infection

2017 ◽  
Vol 91 (23) ◽  
Author(s):  
Alvaro E. Galvis ◽  
Hugh E. Fisher ◽  
Hung Fan ◽  
David Camerini
Keyword(s):  
Reverse Transcriptase ◽  
Conformational Changes ◽  
Rnase H ◽  
Genome Replication ◽  
Hiv Replication ◽  
Debranching Enzyme ◽  
Qrt Pcr ◽  
The Difference ◽  
Hiv 1

ABSTRACT Previous studies in our laboratory showed that the RNA debranching enzyme (DBR1) is not required for early steps in HIV cDNA formation but is necessary for synthesis of intermediate and late cDNA products. To further characterize this effect, we evaluated the topology of the 5′ end of the HIV-1 RNA genome during early infection with and without inhibition of DBR1 synthesis. Cells were transfected with DBR1 short hairpin RNA (shRNA) followed 48 h later by infection with an HIV-1-derived vector containing an RNase H-deficient reverse transcriptase (RT). RNA was isolated at several times postinfection and treated with various RNA-modifying enzymes prior to rapid amplification of 5′ cDNA ends (5′ RACE) for HIV-1 RNA and quantitative reverse transcriptase PCR (qRT-PCR). In infected cells, DBR1 knockdown inhibited detection of free HIV-1 RNA 5′ ends at all time points. The difference in detection of free HIV-1 RNA 5′ ends in infected DBR1 knockdown versus control cells was eliminated by in vitro incubation of infected cell RNAs with yeast or human DBR1 enzyme prior to 5′ RACE and qRT-PCR. This was dependent on the 2′-5′ phosphatase activity of DBR1, since it did not occur when we used the catalytically inactive DBR1(N85A) mutant. Finally, HIV-1 RNA from infected DBR1 knockdown cells was resistant to RNase R that degrades linear RNAs but not RNAs in circular or lariat-like conformations. These results provide evidence for formation of a lariat-like structure involving the 5′ end of HIV-1 RNA during an early step in infection and the involvement of DBR1 in resolving it. IMPORTANCE Our findings support a new view of the early steps in HIV genome replication. We show that the HIV genomic RNA is rapidly decapped and forms a lariat-like structure after entering a cell. The lariat-like structure is subsequently resolved by the cellular enzyme DBR1, leaving a 5′ phosphate. This pathway is similar to the formation and resolution of pre-mRNA intron lariats and therefore suggests that similar mechanisms may be used by HIV. Our work therefore opens a new area of investigation in HIV replication and may ultimately uncover new targets for inhibiting HIV replication and for preventing the development of AIDS.

scholarly journals Effect of Macromolecular Crowding Agents on Human Immunodeficiency Virus Type 1 Capsid Protein Assembly In Vitro

2005 ◽  
Vol 79 (22) ◽  
pp. 14271-14281 ◽  
Author(s):  
Marta del Álamo ◽  
Germán Rivas ◽  
Mauricio G. Mateu
Keyword(s):  
Human Immunodeficiency Virus ◽  
Ionic Strength ◽  
Capsid Protein ◽  
High Ionic Strength ◽  
Immunodeficiency Virus ◽  
Low Ionic Strength ◽  
Very High ◽  
Hiv 1

ABSTRACT Previous studies on the self-assembly of capsid protein CA of human immunodeficiency virus type 1 (HIV-1) in vitro have provided important insights on the structure and assembly of the mature HIV-1 capsid. However, CA polymerization in vitro was previously observed to occur only at very high ionic strength. Here, we have analyzed the effects on CA assembly in vitro of adding unrelated, inert macromolecules (crowding agents), aimed at mimicking the crowded (very high macromolecular effective concentration) environment within the HIV-1 virion. Crowding agents induced fast and efficient polymerization of CA even at low (close to physiological) ionic strength. The hollow cylinders thus assembled were indistinguishable in shape and dimensions from those formed in dilute protein solutions at high ionic strength. However, two important differences were noted: (i) disassembly by dilution of the capsid-like particles was undetectable at very high ionic strength, but occurred rapidly at low ionic strength in the presence of a crowding agent, and (ii) a variant CA from a presumed infectious HIV-1 with mutations at the CA dimerization interface was unable to assemble at any ionic strength in the absence of a crowding agent; in contrast, this mutation allowed efficient assembly, even at low ionic strength, when a crowding agent was used. The use of a low ionic strength and inert macromolecules to mimic the crowded environment inside the HIV-1 virion may lead to a better in vitro evaluation of the effects of conditions, mutations or/and other molecules, including potential antiviral compounds, on HIV-1 capsid assembly, stability and disassembly.

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