scholarly journals Conjugation of Native-Like HIV-1 Envelope Trimers onto Liposomes Using EDC/Sulfo-NHS Chemistry: Requirements and Limitations

Pharmaceutics ◽  
2020 ◽  
Vol 12 (10) ◽  
pp. 979
Author(s):  
Ehsan Suleiman ◽  
Julia Mayer ◽  
Elisabeth Lehner ◽  
Bianca Kohlhauser ◽  
Alexandra Katholnig ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Ionic Strength ◽  
Functional Group ◽  
Positive Charge ◽  
Immunodeficiency Virus ◽  
Close Proximity ◽  
Ph Dependent ◽  
Hiv 1 ◽  
Positively Charged

The display of native-like human immunodeficiency virus type 1 envelope (HIV-1 Env) trimers on liposomes has gained wide attention over the last few years. Currently, available methods have enabled the preparation of Env-liposome conjugates of unprecedented quality. However, these protocols require the Env trimer to be tagged and/or to carry a specific functional group. For this reason, we have investigated N-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide/N-Hydroxysulfosuccinimide (EDC/Sulfo-NHS) chemistry for its potential to covalently conjugate tag-free, non-functionalized native-like Env trimers onto the surface of carboxyl-functionalized liposomes. The preservation of the liposome’s physical integrity and the immunogen’s conformation required a fine-tuned two-step approach based on the controlled use of β-mercaptoethanol. The display of Env trimers was strictly limited to activated liposomes of positive charge, i.e., liposomes with a positive zeta potential that carry amine-reactive Sulfo-NHS esters on their surface. In agreement with that, conjugation was found to be highly ionic strength- and pH-dependent. Overall, we have identified electrostatic pre-concentration (i.e., close proximity between negatively charged Env trimers and positively charged liposomes established through electrostatic attraction) to be crucial for conjugation reactions to proceed. The present study highlights the requirements and limitations of potentially scalable EDC/Sulfo-NHS-based approaches and represents a solid basis for further research into the controlled conjugation of tag-free, non-functionalized native-like Env trimers on the surface of liposomes, and other nanoparticles.

scholarly journals Human immunodeficiency virus type 1 gp120 envelope characteristics associated with disease progression differ in family members infected with genetically similar viruses

2013 ◽  
Vol 94 (1) ◽  
pp. 20-29 ◽  
Author(s):  
Elly Baan ◽  
Renée M. van der Sluis ◽  
Margreet E. Bakker ◽  
Vincent Bekker ◽  
Dasja Pajkrt ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Disease Progression ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Positive Charge ◽  
Gp120 Envelope ◽  
Immunodeficiency Virus ◽  
Virus Strains ◽  
Hiv 1

The human immunodeficiency virus type 1 (HIV-1) envelope protein provides the primary contact between the virus and host, and is the main target of the adaptive humoral immune response. The length of gp120 variable loops and the number of N-linked glycosylation events are key determinants for virus infectivity and immune escape, while the V3 loop overall positive charge is known to affect co-receptor tropism. We selected two families in which both parents and two children had been infected with HIV-1 for nearly 10 years, but who demonstrated variable parameters of disease progression. We analysed the gp120 envelope sequence and compared individuals that progressed to those that did not in order to decipher evolutionary alterations that are associated with disease progression when individuals are infected with genetically related virus strains. The analysis of the V3-positive charge demonstrated an association between higher V3-positive charges with disease progression. The ratio between the amino acid length and the number of potential N-linked glycosylation sites was also shown to be associated with disease progression with the healthier family members having a lower ratio. In conclusion in individuals initially infected with genetically linked virus strains the V3-positive charges and N-linked glycosylation are associated with HIV-1 disease progression and follow varied evolutionary paths for individuals with varied disease progression.

scholarly journals Nef Can Enhance the Infectivity of Receptor-Pseudotyped Human Immunodeficiency Virus Type 1 Particles

2008 ◽  
Vol 82 (21) ◽  
pp. 10811-10819 ◽  
Author(s):  
Massimo Pizzato ◽  
Elena Popova ◽  
Heinrich G. Göttlinger
Keyword(s):  
Human Immunodeficiency Virus ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Viral Infectivity ◽  
Endosomal Acidification ◽  
Immunodeficiency Virus ◽  
Ph Dependent ◽  
Env Protein ◽  
Hiv 1

ABSTRACT Nef is an accessory protein of human immunodeficiency virus type 1 (HIV-1) that enhances the infectivity of progeny virions when expressed in virus-producing cells. The requirement for Nef for optimal infectivity is, at least in part, determined by the envelope (Env) glycoprotein, because it can be eliminated by pseudotyping HIV-1 particles with pH-dependent Env proteins. To investigate the role of Env in the function of Nef, we have examined the effect of Nef on the infectivity of Env-deficient HIV-1 particles pseudotyped with viral receptors for cells expressing cognate Env proteins. We found that Nef significantly enhances the infectivity of CD4-chemokine receptor pseudotypes for cells expressing HIV-1 Env. Nef also increased the infectivity of HIV-1 particles pseudotyped with Tva, the receptor for subgroup A Rous sarcoma virus (RSV-A), even though Nef had no effect if the pH-dependent Env protein of RSV-A was used for pseudotyping. However, Nef does not always enhance viral infectivity if the normal orientation of the Env-receptor interaction is reversed, because the entry of Env-deficient HIV-1 into cells expressing the vesicular stomatitis virus G protein was unaffected by Nef. Together, our results demonstrate that the presence of a viral Env protein during virus production is not required for the ability of Nef to increase viral infectivity. Furthermore, since the infectivity of Tva pseudotypes was blocked by inhibitors of endosomal acidification, we conclude that low-pH-dependent entry does not always bypass the requirement for Nef.

scholarly journals Effects of Mutations in Residues near the Active Site of Human Immunodeficiency Virus Type 1 Integrase on Specific Enzyme-Substrate Interactions

1998 ◽  
Vol 72 (6) ◽  
pp. 5046-5055 ◽  
Author(s):  
Jennifer L. Gerton ◽  
Sharron Ohgi ◽  
Mari Olsen ◽  
Joseph DeRisi ◽  
Patrick O. Brown
Keyword(s):  
Human Immunodeficiency Virus ◽  
Active Site ◽  
Catalytic Core Domain ◽  
Viral Dna ◽  
Catalytic Core ◽  
Core Domain ◽  
Immunodeficiency Virus ◽  
Close Proximity ◽  
Hiv 1

ABSTRACT The phylogenetically conserved catalytic core domain of human immunodeficiency virus type 1 (HIV-1) integrase contains elements necessary for specific recognition of viral and target DNA features. In order to identify specific amino acids that determine substrate specificity, we mutagenized phylogenetically conserved residues that were located in close proximity to the active-site residues in the crystal structure of the isolated catalytic core domain of HIV-1 integrase. Residues composing the phylogenetically conserved DD(35)E active-site motif were also mutagenized. Purified mutant proteins were evaluated for their ability to recognize the phylogenetically conserved CA/TG base pairs near the viral DNA ends and the unpaired dinucleotide at the 5′ end of the viral DNA, using disintegration substrates. Our findings suggest that specificity for the conserved A/T base pair depends on the active-site residue E152. The phenotype of IN(Q148L) suggested that Q148 may be involved in interactions with the 5′ dinucleotide of the viral DNA end. The activities of some of the proteins with mutations in residues in close proximity to the active-site aspartic and glutamic acids were salt sensitive, suggesting that these mutations disrupted interactions with DNA.

scholarly journals A Patch of Positively Charged Amino Acids Surrounding the Human Immunodeficiency Virus Type 1 Vif SLVx4Yx9Y Motif Influences Its Interaction with APOBEC3G

2009 ◽  
Vol 83 (17) ◽  
pp. 8674-8682 ◽  
Author(s):  
Gongying Chen ◽  
Zhiwen He ◽  
Tao Wang ◽  
Rongzhen Xu ◽  
Xiao-Fang Yu
Keyword(s):  
Amino Acids ◽  
Human Immunodeficiency Virus ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Amino Terminal ◽  
Critical Residue ◽  
Immunodeficiency Virus ◽  
Hiv 1 ◽  
Positively Charged

ABSTRACT The amino-terminal region of the Vif molecule in human immunodeficiency virus type 1 (HIV-1), HIV-2, and simian immunodeficiency virus (SIV) contains a conserved SLV/Ix4Yx9Y motif that was first described in 1992, but the importance of this motif for Vif function has not yet been examined. Our characterization of the amino acids surrounding this motif in HIV-1 Vif indicated that the region is critical for APOBEC3 suppression. In particular, amino acids K22, K26, Y30, and Y40 were found to be important for the Vif-induced degradation and suppression of cellular APOBEC3G (A3G). However, mutation of these residues had little effect on the Vif-mediated suppression of A3F, A3C, or A3DE, suggesting that these four residues are not important for Vif assembly with the Cul5 E3 ubiquitin ligase or protein folding in general. The LV portion of the Vif SLV/Ix4Yx9Y motif was found to be required for optimal suppression of A3F, A3C, or A3DE. Thus, the SLV/Ix4Yx9Y motif and surrounding amino acids represent an important functional domain in the Vif-mediated defense against APOBEC3. In particular, the positively charged K26 of HIV-1 Vif is invariably conserved within the SLV/Ix4Yx9Y motif of HIV/SIV Vif molecules and was the most critical residue for A3G inactivation. A patch of positively charged and hydrophilic residues (K22x3K26x3Y30x9YRHHY44) and a cluster of hydrophobic residues (V55xIPLx4-5LxΦx2YWxL72) were both involved in A3G binding and inactivation. These structural motifs in HIV-1 Vif represent attractive targets for the development of lead inhibitors to combat HIV infection.

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.

scholarly journals ADS-J1 Inhibits Human Immunodeficiency Virus Type 1 Entry by Interacting with the gp41 Pocket Region and Blocking Fusion-Active gp41 Core Formation

2009 ◽  
Vol 53 (12) ◽  
pp. 4987-4998 ◽  
Author(s):  
Hongtao Wang ◽  
Zhi Qi ◽  
Angi Guo ◽  
Qinchao Mao ◽  
Hong Lu ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Heptad Repeat ◽  
Enzyme Linked Immunosorbent Assay ◽  
Core Formation ◽  
Immunodeficiency Virus ◽  
Hiv 1 ◽  
Positively Charged

ABSTRACT We previously identified a small-molecule anti-human immunodeficiency virus type 1 (anti-HIV-1) compound, ADS-J1, using a computer-aided molecular docking technique for primary screening and a sandwich enzyme-linked immunosorbent assay (ELISA) as a secondary screening method. In the present study, we demonstrated that ADS-J1 is an HIV-1 entry inhibitor, as determined by a time-of-addition assay and an HIV-1-mediated cell fusion assay. Further mechanism studies confirmed that ADS-J1 does not block gp120-CD4 binding and exhibits a marginal interaction with the HIV-1 coreceptor CXCR4. However, ADS-J1 inhibited the fusion-active gp41 core formation mimicked by peptides derived from the viral gp41 N-terminal heptad repeat (NHR) and C-terminal heptad repeat (CHR), as determined by ELISA, native polyacrylamide gel electrophoresis, and circular dichroism analysis. Moreover, using a surface plasmon resonance assay, we found that ADS-J1 could bind directly to IQN17, a trimeric peptide containing the gp41 pocket region, resulting in the conformational change of IQN17 and the blockage of its interaction with a short D peptide, PIE7. The positively charged residue (K574) located in the gp41 pocket region is critical for the binding of ADS-J1 to NHR. These results suggest that ADS-J1 may bind to the viral gp41 NHR region through its hydrophobic and ionic interactions with the hydrophobic and positively charged resides located in the pocket region, subsequently blocking the association between the gp41 NHR and CHR regions to form the fusion-active gp41 core, thereby inhibiting HIV-1-mediated membrane fusion and virus entry.

scholarly journals Removal of Arginine 332 Allows Human TRIM5α To Bind Human Immunodeficiency Virus Capsids and To Restrict Infection

2006 ◽  
Vol 80 (14) ◽  
pp. 6738-6744 ◽  
Author(s):  
Yuan Li ◽  
Xing Li ◽  
Matthew Stremlau ◽  
Mark Lee ◽  
Joseph Sodroski
Keyword(s):  
Human Immunodeficiency Virus ◽  
Therapeutic Strategies ◽  
Charged Residue ◽  
Virus Capsids ◽  
Immunodeficiency Virus ◽  
Binding Loop ◽  
Hiv 1 ◽  
Positively Charged ◽  
B30.2 Domain

ABSTRACT Human TRIM5α (TRIM5αhu) only modestly inhibits human immunodeficiency virus type 1 (HIV-1) and does not inhibit simian immunodeficiency virus (SIVmac). Alteration of arginine 332 in the TRIM5αhu B30.2 domain to proline, the residue found in rhesus monkey TRIM5α, has been shown to create a potent restricting factor for both HIV-1 and SIVmac. Here we demonstrate that the potentiation of HIV-1 inhibition results from the removal of a positively charged residue at position 332 of TRIM5αhu. The increase in restricting activity correlated with an increase in the ability of TRIM5αhu mutants lacking arginine 332 to bind HIV-1 capsid complexes. A change in the cyclophilin A-binding loop of the HIV-1 capsid decreased TRIM5αhu R332P binding and allowed escape from restriction. The ability of TRIM5αhu to restrict SIVmac could be disrupted by the presence of any charged residue at position 332. Thus, charged residues in the v1 region of the TRIM5αhu B30.2 domain can modulate capsid binding and restriction potency. Therapeutic strategies designed to neutralize arginine 332 of TRIM5αhu might potentiate the innate resistance of human cells to HIV-1 infection.

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