Effect of lysine to arginine mutagenesis in the V3 loop of HIV-1 gp120 on viral entry efficiency and neutralization

Abstract

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.

Fig 1. NL-952 virus mutants.

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.

Fig 2. Infection rates of RRR-to-KKK V3 loop mutants.

(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 Table 1 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.

Fig 3. Neutralization of RRR-to-KKK V3 loop mutants by SDF-1α, RANTES.

(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.

Fig 4. Neutralization of RRR-to-KKK V3 loop mutants by mHSA.

The eight RRR-to-KKK mutants were tested for neutralization against the HIV-1 inhibitory protein mHSA [30]. 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.

Fig 5. Neutralization of NQST- V3 loop mutants by RANTES.

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 Table 1). Shown are the means and standard deviations based on ten experiments.

Fig 6. Impact of soluble gp120 on viral entry.

For each experiment, TZM-bl cells were infected with cell culture supernatants containing virus equal to 0,5 ng p24 antigen in a volume of 100 μl and in the presence of 7,5 ng sgp120/ml. Soluble gp120 was produced in HeLa-P4 cells using pSVATGrev expression vectors for sgp120-RRR and sgp120-KKK. White bars, experiments with KKK-viruses and RRR-sgp120. Black bars, experiments with RRR-viruses and KKK-sgp120. +, indicate the component parts of each virus/sgp120 experiment. Shown are the means and standard deviations based on three experiments.