Keisuke Yusa

HIV-1 acquires resistance to two classes of antiviral drugs through homologous recombination

Genetic recombination contributes to the genomic heterogeneity of human immunodeficiency virus type 1 (HIV-1). In the present study, we demonstrate that HIV-1 readily develops resistance to two classes of anti-HIV-1 drugs through in vitro genetic recombination involving large segments of the viral genome. Co-transfection of COS-7 cells with an HIV-1 plasmid (pSUM13) carrying five mutations in the reverse transcriptase (RT)-encoding region (A62V, V75I, F77L, F116Y, Q151M), conferring resistance to multiple dideoxynucleoside analogs (ddNs), and another HIV-1 plasmid (pSUM431) carrying five mutations in the protease-encoding region (V321, L33F, K451, 184V, L89M), conferring resistance to protease inhibitors such as KNI-272, readily produced HIV-1 carrying both sets of mutations when propagated in MT-2 cells in the presence of azidothymidine (AZT) and KNI-272. The resultant HIV-1 variant was highly resistant to both ddNs and KNI-272. Co-infection of MT-2 cells with HIV-1SUM13 carrying the RT mutations and HIV-1SUM431 carrying the mutations in the protease also generated HIV-1 with both sets of mutations when cultured with AZT and KNI-272. We also report here that the problematic artifactual recombination occurring during genetic analyses of heterogeneous nucleic acid sequences using polymerase chain reaction can be successfully obviated.

Isolation of TAK-779-resistant HIV-1 from an R5 HIV-1 GP120 V3 Loop Library

The human immunodeficiency virus (HIV-1) envelope glycoprotein (GP) 120 interacts with CD4 and the CCR5 coreceptor for viral entry. The V3 loop in GP120 is a crucial region for determining coreceptor usage during viral entry, and a variety of amino acid substitutions has been observed in clinical isolates. To construct an HIV-1 V3 loop library, we chose 10 amino acid positions in the V3 loop and incorporated random combinations (27,648 possibilities) of the amino acid substitutions derived from 31 R5 viruses into the V3 loop of HIV-1JR-FL proviral DNA. The constructed HIV-1 library contained 6.6 × 106 independent clones containing a set of 0–10 amino acid substitutions in the V3 loop. To address whether restricted steric alteration in the V3 loop could confer resistance to an entry inhibitor, TAK-779, we selected entry inhibitor-resistant HIV-1 by increasing the concentration of TAK-779 from 0.10 to 0.30 μm in PM1-CCR5 cells with high expression of CCR5. The selected viruses at passage 8 contained five amino acid substitutions in the V3 loop without any other mutations in GP120 and showed 15-fold resistance compared with the parental virus. These results indicated that a certain structure of the V3 loop containing amino acid substitutions derived from 31 R5 viruses can contribute to the acquisition of resistance to entry inhibitors binding to CCR5. Taken together, this type of HIV-1 V3 loop library is useful for isolating and analyzing the specific biological features of HIV-1 with respect to alterations of the V3 loop structure.

A combination of polymorphic mutations in V3 loop of HIV-1 gp120 can confer noncompetitive resistance to maraviroc

Maraviroc binds to the pocket of extracellular loops of the cell surface CCR5 and prevents R5 HIV-1 from using CCR5 as a coreceptor for entry into CD4-positive cells. To evaluate the contribution of the V3 loop structure in gp120 to maraviroc resistance, we isolated maraviroc-resistant variants from the V3 loop library virus (HIV-1(V3Lib)) containing a set of random combinations of 0-10 polymorphic mutations in vitro. HIV-1(V3Lib) at passage 17 could not be suppressed even at 10 μM (>1400-fold resistance), while HIV-1(JR-FL) at passage 17 revealed an 8-fold resistance to maraviroc. HIV-1(V3Lib-P17) contained T199K and T275M plus 5 mutations in the V3 loop, I304V/F312W/T314A/E317D/I318V. The profile of pseudotyped virus containing I304V/F312W/T314A/E317D/I318V in V3 loop alone revealed a typical noncompetitive resistance, although T199K and/or T275M could not confer noncompetitive resistance. This type of library virus is useful for isolation of escape viruses from effective entry inhibitors.

Acquisition of multi-PI (protease inhibitor) resistance in HIV-1 in vivo and in vitro.

Protease inhibitors are effective antiviral agents which can lead to a severe decrease in HIV RNA copies in plasma of naive patients, however even successful suppression of the virus with antiretroviral agents including protease inhibitor(s) (PI(s)) generates PI-resistant HIV-1 after long term treatment. Occasionally HIV-1 acquires cross-resistance to other PIs with which the patients have not been treated. Cross-resistance to multiple PIs (multi-PI resistance) leads to a restricted salvage strategy; therefore multi-PI resistance is one of the serious obstacles to efficient antiretroviral chemotherapy. The most common PI-resistance mechanism in HIV-1 is the emergence and accumulation of multiple amino acid substitutions within the viral protease. As well, additional substitutions in protease cleavage sites or substitutions in the Gag protein at non-cleavage sites are involved in recovery of the reduced replication fitness of HIV-1 caused by these mutations in the viral protease. To address or predict the resistance mechanisms of PIs, resistant HIV-1 variants have been intensively studied in vitro. However, the profiles of the amino acid substitutions obtained in PI resistant variants are more diverse and complex than that found in vitro. More elaborate in vitro systems for further analysis of acquisition of PI resistance mechanisms are needed.

Structure and Dynamics of the gp120 V3 Loop That Confers Noncompetitive Resistance in R5 HIV-1JR-FL to Maraviroc

Maraviroc, an (HIV-1) entry inhibitor, binds to CCR5 and efficiently prevents R5 human immunodeficiency virus type 1 (HIV-1) from using CCR5 as a coreceptor for entry into CD4+ cells. However, HIV-1 can elude maraviroc by using the drug-bound form of CCR5 as a coreceptor. This property is known as noncompetitive resistance. HIV-1V3-M5 derived from HIV-1JR-FLan is a noncompetitive-resistant virus that contains five mutations (I304V/F312W/T314A/E317D/I318V) in the gp120 V3 loop alone. To obtain genetic and structural insights into maraviroc resistance in HIV-1, we performed here mutagenesis and computer-assisted structural study. A series of site-directed mutagenesis experiments demonstrated that combinations of V3 mutations are required for HIV-1JR-FLan to replicate in the presence of 1 µM maraviroc, and that a T199K mutation in the C2 region increases viral fitness in combination with V3 mutations. Molecular dynamic (MD) simulations of the gp120 outer domain V3 loop with or without the five mutations showed that the V3 mutations induced (i) changes in V3 configuration on the gp120 outer domain, (ii) reduction of an anti-parallel β-sheet in the V3 stem region, (iii) reduction in fluctuations of the V3 tip and stem regions, and (iv) a shift of the fluctuation site at the V3 base region. These results suggest that the HIV-1 gp120 V3 mutations that confer maraviroc resistance alter structure and dynamics of the V3 loop on the gp120 outer domain, and enable interactions between gp120 and the drug-bound form of CCR5.

A broad antiviral neutral glycolipid, fattiviracin FV-8, is a membrane fluidity modulator.

To screen for an effective antiviral compound which acts as a membrane fluidity modulator, dichotomous effects on human immunodeficiency virus type 1 (HIV‐1) infection due to different treatments of several glycolipids and lipids were examined. Continuous treatment of infected cells with 40 μg ml−1 fattiviracin FV‐8, a neutral glycolipid isolated from Streptomycetes, inhibited HIV‐1 infection by 96%, whereas pretreatment with 400 μg ml−1 enhanced infectivity 4.7‐fold. The glycolipid showed similar effects as glycyrrhizin; it inhibited infection by broad enveloped viruses, blocked cell–cell fusion, reduced the infectivity of treated virions and enhanced susceptibility to viral infection and cell–cell fusion of cells pretreated with high doses of the compound. Suppression and enhancement was correlated with decreased and increased fluidity of plasma membrane of the fattiviracin FV‐8‐treated cells. Restricted movement of membrane molecules might impede the formation of a wide fusion pore, and therefore be critical to the entry of viruses. Thus, this can be applied as a new strategy to inhibit viral infections.

Adsorption and Infectivity of Human Immunodeficiency Virus Type 1 Are Modified by the Fluidity of the Plasma Membrane for Multiple-Site Binding

Based on the assumption that fluidity of the plasma membrane and viral envelope is necessary for recruiting additional receptors and ligands to the initial attachment site for “multiple‐site binding,” we determined the effect of increased temperature on viral infectivity. Infection of human immunodeficiency virus type 1 (HIV‐1) and a pseudotyped luciferase‐expressing chimeric virus using MAGI and GHOST/CXCR4 cells showed that in 1 hr of viral adsorption the extent of virus infection and the amount of tightly adsorbed viruses depended on temperature; and that membrane fluidity increased according to increased temperature. Augmented infection was observed as post‐attachment enhancement (PAE) when cells were washed and incubated at 40 C for 1 hr after viral adsorption. PAE was completely inhibited by 1 μM of anti‐CXCR4 peptide T140, and addition of T140 at 20 min resulted in a gradual loss of inhibition of PAE, indicating the need for a 30 to 40 min timelag to ensure tight multiple‐site binding. These data suggest that the accumulation of gp120 and receptor complex (multiple‐site binding) was needed to complete the infection. Treatments of cells with 0.05% Tween 20 or 2 μg/ml of anti‐HLA‐II antibody resulted in increases or decreases, respectively, of attached viruses and the infectivity. As well, Tween 20 and anti‐HLA‐II antibody enhanced and suppressed the fluidity of the plasma membrane, respectively. Amounts of adsorbed viruses and degrees of viral infectivity correlated with the intensity of fluidity of the plasma membrane, probably due to the formation of multiple‐site binding.

Preferential recognition of monomeric CCR5 expressed in cultured cells by the HIV-1 envelope glycoprotein gp120 for the entry of R5 HIV-1

Bimolecular fluorescence complementation (BiFC) and western blot analysis demonstrated that CCR5 exists as constitutive homo-oligomers, which was further enhanced by its antagonists such as maraviroc (MVC) and TAK-779. Staining by monoclonal antibodies recognizing different epitopes of CCR5 revealed that CCR5 oligomer was structurally different from the monomer. To determine which forms of CCR5 are well recognized by CCR5-using HIV-1 for the entry, BiFC-positive and -negative cell fractions in CD4-positive 293T cells were collected by fluorescent-activated cell sorter, and infected with luciferase-reporter HIV-1 pseudotyped with CCR5-using Envs including R5 and R5X4. R5 and dual-R5 HIV-1 substantially infected BiFC-negative fraction rather than BiFC-positive fraction, indicating the preferential recognition of monomeric CCR5 by R5 and dual-R5 Envs. Although CCR5 antagonists enhanced oligomerization of CCR5, MVC-resistant HIV-1 was found to still recognize both MVC-bound and -unbound forms of monomeric CCR5, suggesting the constrained use of monomeric CCR5 by R5 HIV-1.

Construction of a Human Immunodeficiency Virus Type 1 (HIV-1) Library Containing Random Combinations of Amino Acid Substitutions in the HIV-1 Protease due to Resistance by Protease Inhibitors

ABSTRACT Human immunodeficiency virus type 1 (HIV-1) heterogeneity contributes to the emergence of drug-resistant virus, escape from host defense systems, and/or conversion of the cellular tropism. To establish an in vitro system to address a heterogeneous virus population, we constructed a library of HIV-1 molecular clones containing a set of random combinations of zero to 11 amino acid substitutions associated with resistance to protease inhibitors by the HIV-1 protease. The complexity (2.1 × 105) of the HIV-1 library pNG-PRL was large enough to cover all of the possible combinations of zero to 11 amino acid substitutions (a total of 4,096 substitutions possible). The T-cell line MT-2 was infected with the HIV-1 library, and resistant viruses were selected after treatment by the protease inhibitor ritonavir (0.03 to 0.30 μM). The viruses that contained three to eight amino acid substitutions could be selected within 2 weeks. These results demonstrate that this HIV-1 library could serve as an alternative in vitro system to analyze the emergence of drug resistance and to evaluate the antiviral activity of novel compounds against multidrug-resistant viruses.

Gp120 V3-dependent Impairment of R5 HIV-1 Infectivity Due to Virion-incorporated CCR5

Entry of R5 human immunodeficiency virus type 1 (HIV-1) into target cells requires sequential interactions of the envelope glycoprotein gp120 with the receptor CD4 and the coreceptor CCR5. We investigated replication of 45 R5 viral clones derived from the HIV-1JR-FLan library carrying 0–10 random amino acid substitutions in the gp120 V3 loop. It was found that 6.7% (3/45) of the viruses revealed ≥10-fold replication suppression in PM1/CCR5 cells expressing high levels of CCR5 compared with PM1 cells expressing low levels of CCR5. In HIV-1V3L#08, suppression of replication was not associated with entry events and viral production but with a marked decrease in infectivity of nascent progeny virus. HIV-1V3L#08, generated from infected PM1/CCR5 cells, was 98% immunoprecipitated by anti-CCR5 monoclonal antibody T21/8, whereas the other infectious viruses were only partially precipitated, suggesting that incorporation of larger amounts of CCR5 into the virions caused impairment of viral infectivity in HIV-1V3L#08. The results demonstrate the implications of an alternative influence of CCR5 on HIV-1 replication.