Neutralization of X4- and R5-tropic HIV-1 NL4-3 variants by HOCl-modified serum albumins

<p>Abstract</p> <p>Background</p> <p>Myeloperoxidase (MPO), an important element of the microbicidal activity of neutrophils, generates hypochlorous acid (HOCl) from H₂O₂and chloride, which is released into body fluids. Besides its direct microbicidal activity, HOCl can react with amino acid residues and HOCl-modified proteins can be detected <it>in vivo</it>.</p> <p>Findings</p> <p>This report is based on binding studies of HOCl-modified serum albumins to HIV-1 gp120 and three different neutralization assays using infectious virus. The binding studies were carried out by surface plasmon resonance spectroscopy and by standard ELISA techniques. Virus neutralization assays were carried out using HIV-1 NL4-3 virus and recombinant strains with CXCR4 and CCR5 coreceptor usage. Viral infection was monitored by a standard p24 or X-gal staining assay. Our data demonstrate that HOCl-modified mouse-, bovine- and human serum albumins all bind to the HIV-1 NL4-3 gp120 (LAV) glycoprotein in contrast to non-modified albumin. Binding of HOCl-modified albumin to gp120 correlated to the blockade of CD4 as well as that of V3 loop specific monoclonal antibody binding. In neutralization experiments, HOCl-modified serum albumins inhibited replication and syncytium formation of the X4- and R5-tropic NL4-3 isolates in a dose dependent manner.</p> <p>Conclusions</p> <p>Our data indicate that HOCl-modified serum albumin veils the binding site for CD4 and the V3 loop on gp120. Such masking of the viral gp120/gp41 envelope complex might be a simple but promising strategy to inactivate HIV-1 and therefore prevent infection when HOCl-modified serum albumin is applied, for example, as a topical microbicide.</p

Effect of Lysine to Arginine Mutagenesis in the V3 Loop of HIV-1 gp120 on Viral Entry Efficiency and Neutralization

<div><p>HIV-1 infection is characterized by an ongoing replication leading to T-lymphocyte decline which is paralleled by the switch from CCR5 to CXCR4 coreceptor usage. To predict coreceptor usage, several computer algorithms using gp120 V3 loop sequence data have been developed. In these algorithms an occupation of the V3 positions 11 and 25, by one of the amino acids lysine (K) or arginine (R), is an indicator for CXCR4 usage. Amino acids R and K dominate at these two positions, but can also be identified at positions 9 and 10. Generally, CXCR4-viruses possess V3 sequences, with an overall positive charge higher than the V3 sequences of R5-viruses. The net charge is calculated by subtracting the number of negatively charged amino acids (D, aspartic acid and E, glutamic acid) from the number of positively charged ones (K and R). In contrast to D and E, which are very similar in their polar and acidic properties, the characteristics of the R guanidinium group differ significantly from the K ammonium group. However, in coreceptor predictive computer algorithms R and K are both equally rated. The study was conducted to analyze differences in infectivity and coreceptor usage because of R-to-K mutations at the V3 positions 9, 10 and 11. V3 loop mutants with all possible RRR-to-KKK triplets were constructed and analyzed for coreceptor usage, infectivity and neutralization by SDF-1α and RANTES. Virus mutants R₉R₁₀R₁₁ showed the highest infectivity rates, and were inhibited more efficiently in contrast to the K₉K₁₀K₁₁ viruses. They also showed higher efficiency in a virus-gp120 paired infection assay. Especially V3 loop position 9 was relevant for a switch to higher infectivity when occupied by R. Thus, K-to-R exchanges play a role for enhanced viral entry efficiency and should therefore be considered when the viral phenotype is predicted based on V3 sequence data.</p></div

Neutralization of RRR-to-KKK V3 loop mutants by SDF-1α, RANTES.

<p>(a) For each NL-952.1, NL-952.2 and NL-952.3 virus all eight mutants (RRR-to-KKK) were tested for neutralization by SDF-1α. GHOST-X4 cells were infected with an amount of virus representing about 100 ffu. SDF-1α was added to a final concentration of 125, 250, 500 and 1000 ng/ml. (b) Neutralization by RANTES was carried out for X4R5-dualtropic NL-952.1 mutants with the exception of the X4-monotropic mutant RRR, since the RRR mutant does not show any viral growth in GHOST-R5 cells (infection = 0% at RANTES 0 ng/ml). Also all NL-952.2 and NL-952.3 mutants did not replicate in GHOST-R5 cells and therefore could not be tested for neutralization by RANTES.</p

NL-952 virus mutants.

<p>V3 loop regions of the three viruses NL-952.1, NL-952.2 and NL-952.3 which differ by mutations affecting the N-glycosylation sites g13-g17. The three viruses were used to generate mutants with all triple combinations of amino acids R and K at the V3 loop positions 9, 10 and 11 (black symbols). N-linked carbohydrates: C, complex type; HM, high mannose type.</p

Coreceptor prediction for NL-952.1, NL-952.2 and NL-952.3 V3 loop mutants.

<p>* amino acid sequence at V3 loop positions 9-to-11.</p><p>CC = cell culture tested coreceptor usage using GHOST-CXCR4 and GHOST-CCR5 indicator cell lines.</p><p>G2P = prediction algorithm geno2pheno (<a href="http://www.geno2pheno.org" target="_blank">http://www.geno2pheno.org</a>).</p><p>PSSM = position-specific scoring matrices (indra.mullins.microbiol.washington.edu/webpssm).</p><p>HcP = prediction algorithm HIVcoPred (<a href="http://www.imtech.res.in/raghava/hivcopred" target="_blank">http://www.imtech.res.in/raghava/hivcopred</a>).</p><p>R5X4 = prediction algorithm developed using V3 sequences were CCR5 and CXCR4 coreceptor usage was assayed [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0119879#pone.0119879.ref014" target="_blank">14</a>].</p><p>SINSI = prediction algorithm developed using V3 sequences were MT2 cell tropism was assayed [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0119879#pone.0119879.ref014" target="_blank">14</a>].</p><p>Coreceptor prediction for NL-952.1, NL-952.2 and NL-952.3 V3 loop mutants.</p

Infection rates of RRR-to-KKK V3 loop mutants.

<p>(a) Blue stained TZM-bl cells that had been infected by RRR, RKR and KKK mutants of viruses NL-952.1, NL-952.2 and NL-952.3. For each experiment, about 10⁴ cells/96-well were infected with cell culture supernatants containing virus equal to 0,5 ng p24. Staining with X-gal was carried out two days post infection. (b) Viruses containing the amino acid combinations as shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0119879#pone.0119879.t001" target="_blank">Table 1</a> at the V3 loop positions 9-to-11 were tested for infectivity using CD4+, CCR5+ and CXCR4+ TZM-bl cells. Shown are the means and standard deviations based on ten experiments. Virus inocula were standardized based on p24 antigen measurements (0,5 ng p24/10⁴ cells/96-well). Within the set of RRR-to-KKK mutants, all RRR-virus mutants (black bars) showed the highest and the KKK-virus mutants (red bars) the lowest infection rates.</p

Neutralization of RRR-to-KKK V3 loop mutants by mHSA.

<p>The eight RRR-to-KKK mutants were tested for neutralization against the HIV-1 inhibitory protein mHSA [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0119879#pone.0119879.ref030" target="_blank">30</a>]. TZM-bl cells were infected with an amount of virus representing about 100 ffu. The HIV-1 entry inhibitor mHSA was added at various concentrations. Shown are the means and standard deviations based on ten experiments. Black bars, RRR-mutants; red bar, KKK-mutants.</p

Neutralization of NQST- V3 loop mutants by RANTES.

<p>For each of the three viruses NL-952.1, NL-952.2 and NL-952.3, nine mutants containing amino acids N, Q, S and T at the V3 position 9, 10 and/or 11 were tested for neutralization by RANTES. GHOST-R5 cells were infected with an amount of virus representing about 100 ffu. RANTES was added to a final concentration of 125, 250, 500 and 1000 ng/ml. All 27 viruses were R5-monotropic since they showed no growth on GHOST-X4 cells (see also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0119879#pone.0119879.t001" target="_blank">Table 1</a>). Shown are the means and standard deviations based on ten experiments.</p

Generation of Viral Particles with Brain Cell-Specific Tropism by Pseudotyping HIV-1 with the Zika Virus E Protein

Flaviviruses are a family of RNA viruses that includes many known pathogens, such as Zika virus (ZIKV), West Nile virus (WNV), dengue virus (DENV), and yellow fever virus (YFV). A pseudotype is an artificial virus particle created in vitro by incorporating the flavivirus envelope proteins into the structure of, for example, a retrovirus such as human immunodeficiency virus type-1 (HIV-1). They can be a useful tool in virology for understanding the biology of flaviviruses, evaluating immune responses, developing antiviral strategies but can also be used as vectors for gene transfer experiments. This protocol describes the generation of a ZIKV/HIV-1 pseudotype developed as a new tool for infecting cells derived from a highly malignant brain tumor: glioblastoma multiforme grade 4

Zikavirus prME Envelope Pseudotyped Human Immunodeficiency Virus Type-1 as a Novel Tool for Glioblastoma-Directed Virotherapy

Glioblastoma multiforme is the most lethal type of brain tumor that is not yet curable owing to its frequent resurgence after surgery. Resistance is mainly caused by the presence of a subpopulation of tumor cells, the glioma stem cells (GSCs), which are highly resistant to radiation and chemotherapy. In 2015, Zikavirus (ZIKV)-induced microcephaly emerged in newborns, indicating that ZIKV has a specific neurotropism. Accordingly, an oncolytic tropism for infecting GSCs was demonstrated in a murine tumor model. Like other flaviviruses, ZIKV is enveloped by two proteins, prM and E. The pME expression plasmid along with the HIV-1 vector pNL Luc AM generated prME pseudotyped viral particles. Four different prME envelopes, Z1 to Z4, were cloned, and the corresponding pseudotypes, Z1- to Z4-HIVluc, produced by this two-plasmid system, were tested for entry efficiency using Vero-B4 cells. The most efficient pseudotype, Z1-HIVluc, also infected glioma-derived cell lines U87 and 86HG39. The pseudotype system was then extended by using a three-plasmid system including pME-Z1, the HIV-1 packaging plasmid psPAX2, and the lentiviral vector pLenti-luciferase-P2A-Neo. The corresponding pseudotype, designated Z1-LENTIluc, also infected U87 and 86HG39 cells. Altogether, a pseudotyped virus especially targeting glioma-derived cells might be a promising candidate for a prospective glioblastoma-directed virotherapy