Mutation V111I in HIV-2 Reverse Transcriptase Increases the Fitness of the Nucleoside Analogue-Resistant K65R and Q151M Viruses

International audienceInfection with HIV-2 can ultimately lead to AIDS, although disease progression is much slower than with HIV-1. HIV-2 patients are mostly treated with a combination of nucleoside reverse transcriptase (RT) inhibitors (NRTIs) and protease inhibitors designed for HIV-1. Many studies have described the development of HIV-1 resistance to NRTIs and identified mutations in the polymerase domain of RT. Recent studies have shown that mutations in the connection and RNase H domains of HIV-1 RT may also contribute to resistance. However, only limited information exists regarding the resistance of HIV-2 to NRTIs. In this study, therefore, we analyzed the polymerase, connection, and RNase H domains of RT in HIV-2 patients failing NRTI-containing therapies. Besides the key resistance mutations K65R, Q151M, and M184V, we identified a novel mutation, V111I, in the polymerase domain. This mutation was significantly associated with mutations K65R and Q151M. Sequencing of the connection and RNase H domains of the HIV-2 patients did not reveal any of the mutations that were reported to contribute to NRTI resistance in HIV-1. We show that V111I does not strongly affect drug susceptibility but increases the replication capacity of the K65R and Q151M viruses. Biochemical assays demonstrate that V111I restores the polymerization defects of the K65R and Q151M viruses but negatively affects the fidelity of the HIV-2 RT enzyme. Molecular dynamics simulations were performed to analyze the structural changes mediated by V111I. This showed that V111I changed the flexibility of the 110-to-115 loop region, which may affect deoxynucleoside triphosphate (dNTP) binding and polymerase activity.IMPORTANCEMutation V111I in the HIV-2 reverse transcriptase enzyme was identified in patients failing therapies containing nucleoside analogues. We show that the V111I change does not strongly affect the sensitivity of HIV-2 to nucleoside analogues but increases the fitness of viruses with drug resistance mutations K65R and Q151M

Differentiation of primary adult microglia alters their response to TLR8-mediated activation but not their capacity as APC

Activated microglia are found in a variety of neuroinflammatory disorders where they have attributed roles as effector as well as antigen-presenting cells (APC). Critical determinants for the multifaceted role of microglia are the differentiation potential of microglia and their mode of activation. In this study, we have investigated the effects of M-CSF and GM-CSF-mediated differentiation of adult primate microglia on their cellular phenotype, antigen presentation, and phagocytic function as well as on Toll-like receptor (TLR)-mediated responses. We show that although cell morphology and expression levels of activation markers were markedly different, differentiation with either factor yielded microglia that phenotypically and functionally resemble macrophages. Both M-CSF and GM-CSF-differentiated microglia were responsive to TLR1/2, 2, 3, 4, 5, 6/2, and 8-mediated activation, but not to TLR7 or 9-mediated activation. Intriguingly, M-CSF-differentiated microglia expressed higher levels of TLR8-encoding mRNA and protein, and produced larger amounts of proinflammatory cytokines in response to TLR8-mediated activation as compared to GM-CSF-differentiated microglia. While differentiation of adult microglia by growth factors that can be produced endogenously in the central nervous system is thus unlikely to change their APC function, it can alter their innate responses to infectious stimuli such as ssRNA viruses. Resident primate microglia may thereby help shape rather than initiate adaptive immune responses

Structure of the HIV-1 reverse transcriptase (RT) enzyme.

<p>(A) Shows RT bound to a DNA/DNA template/primer complex (shown in surface representation). The enzyme is shown in cartoon representation and coloured by subdomain; fingers are blue, palm red, thumb green, connection yellow and the RNaseH orange. The p66 subunit is shown in the foreground and in darker shades. A black rectangle indicates the location of the p51 RNaseH primer grip. (B) and (C) show detailed views of this region, including chemical representations of the residues K395 and E396 in the wild-type and A400T RT enzymes, respectively. A green line is used to illustrate the hydrogen bond formed between T400 and E396.</p

Changes in the RT sequence of HIV-1 subtype B viruses that are found with increased frequency in patients failing therapy.

a<p>Correction based on the False Discovery Rate (FDR) approach.</p>b<p>p-values <0.05 are considered significant.</p

The average angle between RNaseH primer grip residues in the p51 (residues 395 and 396) and p66 (359 and 360) subunits observed in simulations of HIV-1 RT with alanine and threonine at position 400.

<p>Standard deviations are shown in parentheses.</p

Drug susceptibility and replication of A400T viruses.

<p>(A) Susceptibility of the A400T virus to NNRTI (light grey) and NRTI (dark grey) was measured using single-cycle drug susceptibility assays. The average of three independent experiments is plotted, and the standard deviation is shown (error bars). The susceptibility of the A400T virus is shown as the relative difference in IC₅₀ compared to the wild-type pNL4.3-XN virus. (B) Replication of viruses containing A400T alone, or in combination with M184V or TAMs. SupT1 cells were infected with an equal amount of virus (50 ng). CA-p24 production was measured in the culture medium at several days post infection. A representative experiment is shown. Three independent experiments were performed with similar results.</p

Distance between the scissile phosphate (−19) and the centre of mass of the RNaseH active site (p66 residues 443, 478, 498 and 549) over the course of the simulations of the HIV-1 RT bound to an RNA/DNA substrate.

<p>The simulation for the wild-type A400 is shown in red, and for substitution T400 in blue. Frequency distributions of the χ₁ dihedral of p51 residue E396 in simulations of (B) RNA/DNA hybrid substrate, (C) double stranded DNA substrate and (D) NVP bound HIV-1 RT (results for the unbound RT are shown in Fig S16 in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0074078#pone.0074078.s001" target="_blank">File S1</a>). The simulation for A400 is shown in red, T400 in blue.</p

Association between position 400 (alanine vs threonine) and polymerase domain mutations in subtype B viruses from patients failing therapy.

*<p>p-values <0.05 are considered significant.</p

DNA polymerase activity of virus-derived RT enzymes containing substitution A400T.

<p>(A) DNA polymerase activity was measured using a 51-nt RNA template, representing the HIV-1 primer binding site (PBS) with flanking sequences. A 21-nt DNA primer complementary to the PBS was 5′-end-labeled (asterisk), and heat-annealed onto the RNA template. Extension of the primer by RT will result in a 38-nt labeled cDNA product. (B) The RNA template/DNA primer complex was incubated with the different virion-derived RTs in the presence of dNTPs. Formation of the cDNA product (cDNA) was monitored after 3, 7 and 15 min incubation. A control reaction was performed in the absence of RT (lane 19) showing only the labeled primer (P). (C) The amount of cDNA production was quantified in three independent experiments. Plotted is the average cDNA production with standard deviation (error bars). The polymerase activity of the wild-type virus-derived RT was set at 100%.</p

RNaseH activity of virus-derived RT enzymes containing substitution A400T.

<p>(A) RNaseH cleavage was measured using a 5′-end labeled (asterisk) 51-nt HIV-1 RNA template representing the PBS region. A 21-nt DNA primer complementary to the PBS was heat-annealed onto the RNA template. Primary (−19) and secondary (−12/−9) cleavages were monitored. (B) Representative gel showing RNaseH cleavage mediated by the virion-derived RTs with substitution A400T in the wild-type and M184V background. The reactions were initiated by the addition of Mg²⁺, and template (T) cleavage was monitored after 3, 7, 20 and 40 min of incubation. Control reactions were performed in the absence of Mg²⁺ (lane 18) or RT (lane 19), showing no template degradation. (C) The formation of secondary cleavage products over time was quantified, and is plotted for this representative gel. (D) RNaseH cleavage mediated by the virion-derived RTs with substitution A400T in the wild-type and TAM background was analyzed, and formation of secondary cleavage products is plotted in (E). Representative experiments are shown, and three independent experiments were performed with similar results.</p