Development of Human Immunodeficiency Virus Type 1 Resistance to 4'-Ethynyl-2-Fluoro-2'-Deoxyadenosine Starting with Wild-Type or Nucleoside Reverse Transcriptase Inhibitor-Resistant Strains.

4'-Ethynyl-2-fluoro-2'-deoxyadenosine (EFdA, MK-8591, islatravir) is a nucleoside reverse transcriptase translocation inhibitor (NRTTI) with exceptional potency against wild-type (WT) and drug-resistant HIV-1 in phase III clinical trials. EFdA resistance is not well characterized. To study EFdA resistance patterns that may emerge in naive or tenofovir (TFV)-, emtricitabine/lamivudine (FTC/3TC)-, or zidovudine (AZT)-treated patients, we performed viral passaging experiments starting with WT, K65R, M184V, or D67N/K70R/T215F/K219Q HIV-1. Regardless of the starting viral sequence, all selected EFdA-resistant variants included the M184V reverse transcriptase (RT) mutation. Using recombinant viruses, we validated the role for M184V as the primary determinant of EFdA resistance; none of the observed connection subdomain (R358K and E399K) or RNase H domain (A502V) mutations significantly contributed to EFdA resistance. A novel EFdA resistance mutational pattern that included A114S was identified in the background of M184V. A114S/M184V exhibited higher EFdA resistance (∼24-fold) than either M184V (∼8-fold) or A114S alone (∼2-fold). Remarkably, A114S/M184V and A114S/M184V/A502V resistance mutations were up to 50-fold more sensitive to tenofovir than was WT HIV-1. These mutants also had significantly lower specific infectivities than did WT. Biochemical experiments confirmed decreases in the enzymatic efficiency (kcat/Km) of WT versus A114S (2.1-fold) and A114S/M184V/A502V (6.5-fold) RTs, with no effect of A502V on enzymatic efficiency or specific infectivity. The rather modest EFdA resistance of M184V or A114S/M184V (8- and 24-fold), their hypersusceptibility to tenofovir, and strong published in vitro and in vivo data suggest that EFdA is an excellent therapeutic candidate for naive, AZT-, FTC/3TC-, and especially tenofovir-treated patients.

Large Multidomain Protein NMR: HIV-1 Reverse Transcriptase Precursor in Solution.

NMR studies of large proteins, over 100 kDa, in solution are technically challenging and, therefore, of considerable interest in the biophysics field. The challenge arises because the molecular tumbling of a protein in solution considerably slows as molecular mass increases, reducing the ability to detect resonances. In fact, the typical 1H-13C or 1H-15N correlation spectrum of a large protein, using a 13C- or 15N-uniformly labeled protein, shows severe line-broadening and signal overlap. Selective isotope labeling of methyl groups is a useful strategy to reduce these issues, however, the reduction in the number of signals that goes hand-in-hand with such a strategy is, in turn, disadvantageous for characterizing the overall features of the protein. When domain motion exists in large proteins, the domain motion differently affects backbone amide signals and methyl groups. Thus, the use of multiple NMR probes, such as 1H, 19F, 13C, and 15N, is ideal to gain overall structural or dynamical information for large proteins. We discuss the utility of observing different NMR nuclei when characterizing a large protein, namely, the 66 kDa multi-domain HIV-1 reverse transcriptase that forms a homodimer in solution. Importantly, we present a biophysical approach, complemented by biochemical assays, to understand not only the homodimer, p66/p66, but also the conformational changes that contribute to its maturation to a heterodimer, p66/p51, upon HIV-1 protease cleavage.

Interplay between protease and reverse transcriptase dimerization in a model HIV-1 polyprotein.

The Gag-Pol polyprotein in human immunodeficiency virus type I (HIV-1) encodes enzymes that are essential for virus replication: protease (PR), reverse transcriptase (RT), and integrase (IN). The mature forms of PR, RT and IN are homodimer, heterodimer and tetramer, respectively. The precise mechanism underlying the formation of dimer or tetramer is not yet understood. Here, to gain insight into the dimerization of PR and RT in the precursor, we prepared a model precursor, PR-RT, incorporating an inactivating mutation at the PR active site, D25A, and including two residues in the p6* region, fused to a SUMO-tag, at the N-terminus of the PR region. We also prepared two mutants of PR-RT containing a dimer dissociation mutation either in the PR region, PR(T26A)-RT, or in the RT region, PR-RT(W401A). Size exclusion chromatography showed both monomer and dimer fractions in PR-RT and PR(T26A)-RT, but only monomer in PR-RT(W401A). SEC experiments of PR-RT in the presence of protease inhibitor, darunavir, significantly enhanced the dimerization. Additionally, SEC results suggest an estimated PR-RT dimer dissociation constant that is higher than that of the mature RT heterodimer, p66/p51, but slightly lower than the premature RT homodimer, p66/p66. Reverse transcriptase assays and RT maturation assays were performed as tools to assess the effects of the PR dimer-interface on these functions. Our results consistently indicate that the RT dimer-interface plays a crucial role in the dimerization in PR-RT, whereas the PR dimer-interface has a lesser role.

Preclinical assessment of a novel human antibody VH domain targeting mesothelin as an antibody-drug conjugate.

Mesothelin (MSLN) has been a validated tumor-associated antigen target for several solid tumors for over a decade, making it an attractive option for therapeutic interventions. Novel antibodies with high affinity and better therapeutic properties are needed. In the current study, we have isolated and characterized a novel heavy chain variable (VH) domain 3C9 from a large-size human immunoglobulin VH domain library. 3C9 exhibited high affinity (KD [dissociation constant] <3 nM) and binding specificity in a membrane proteome array (MPA). In a mouse xenograft model, 3C9 fused to human IgG1 Fc was detected at tumor sites as early as 8 h post-infusion and remained at the site for over 10 days. Furthermore, 3C9 fused to a human Fc domain drug conjugate effectively inhibited MSLN-positive tumor growth in a mouse xenograft model. The X-ray crystal structure of full-length MSLN in complex with 3C9 reveals interaction of the 3C9 domains with two distinctive residue patches on the MSLN surface. This newly discovered VH antibody domain has a high potential as a therapeutic candidate for MSLN-expressing cancers.

Relative domain orientation of the L289K HIV-1 reverse transcriptase monomer.

HIV-1 reverse transcriptase (RT) is a heterodimer comprised p66 and p51 subunits (p66/p51). Several single amino acid substitutions in RT, including L289K, decrease p66/p51 dimer affinity, and reduce enzymatic functioning. Here, small-angle X-ray scattering (SAXS) with proton paramagnetic relaxation enhancement (PRE), 19 F site-specific NMR, and size exclusion chromatography (SEC) were performed for the p66 monomer with the L289K mutation, p66L289K . NMR and SAXS experiments clearly elucidated that the thumb and RNH domains in the monomer do not rigidly interact with each other but are spatially close to the RNH domain. Based on this structural model of the monomer, p66L289K and p51 were predicted to form a heterodimer while p66 and p51L289K not. We tested this hypothesis by SEC analysis of p66 and p51 containing L289K in different combinations and clearly demonstrated that L289K substitution in the p51 subunit, but not in the p66 subunit, reduces p66/p51 formation. Based on the derived monomer model and the importance of the inter-subunit RNH-thumb domain interaction in p66/p51, validated by SEC, the mechanism of p66 homodimer formation was discussed.

The HIV-1 protein Nef activates the Tec family kinase Btk by stabilizing an intermolecular SH3-SH2 domain interaction.

The Nef protein produced by the viruses HIV-1 and SIV drives efficient viral replication partially by inducing constitutive activation of host cell tyrosine kinases, including members of the Src and Tec families. Here, we uncovered the mechanism by which both HIV-1 and SIV Nef enhanced the activity of the Tec family kinase Btk in vitro and in cells. A Nef mutant that could not bind to the SH3 domain of Src family kinases activated Btk to the same extent as did wild-type Nef, demonstrating that Nef activated Src and Tec family kinases by distinct mechanisms. The Btk SH3-SH2 region formed a homodimer requiring the CD loop in the SH2 domain, which was stabilized by the binding of Nef homodimers. Alanine substitution of Pro327 in the CD loop of the Btk SH2 domain destabilized SH3-SH2 dimers, abolished the interaction with Nef, and prevented activation by Nef in vitro. In cells, Nef stabilized and activated wild-type but not P327A Btk homodimers at the plasma membrane. These data reveal that the interaction with Nef stabilizes Btk dimers through the SH3-SH2 interface to promote kinase activity and show that the HIV-1 Nef protein evolved distinct mechanisms to activate Src and Tec family tyrosine kinases to enhance viral replication.

Inhibitors of HIV-1 Nef-Mediated Activation of the Myeloid Src-Family Kinase Hck Block HIV-1 Replication in Macrophages and Disrupt MHC-I Downregulation.

HIV-1 Nef is an attractive target for antiretroviral drug discovery because of its role in promoting HIV-1 infectivity, replication, and host immune system avoidance. Here, we applied a screening strategy in which recombinant HIV-1 Nef protein was coupled to activation of the Src-family tyrosine kinase Hck, which enhances the HIV-1 life cycle in macrophages. Nef stimulates recombinant Hck activity in vitro, providing a robust assay for chemical library screening. High-throughput screening of more than 730 000 compounds using the Nef·Hck assay identified six unique hit compounds that bound directly to recombinant Nef by surface plasmon resonance (SPR) in vitro and inhibited HIV-1 replication in primary macrophages in the 0.04 to 5 µM range without cytotoxicity. Eighty-four analogs were synthesized around an isothiazolone scaffold from this series, many of which bound to recombinant Nef and inhibited HIV-1 infectivity in the low to submicromolar range. Compounds in this series restored MHC-I to the surface of HIV-infected primary cells and disrupted a recombinant protein complex of Nef with the C-terminal tail of MHC-I and the µ1 subunit of the AP-1 endocytic trafficking protein. Nef inhibitors in this class have the potential to block HIV-1 replication in myeloid cells and trigger recognition of HIV-infected cells by the adaptive immune system in vivo.

Retroviral RNase H: Structure, mechanism, and inhibition.

All retroviruses encode the enzyme, reverse transcriptase (RT), which is involved in the conversion of the single-stranded viral RNA genome into double-stranded DNA. RT is a multifunctional enzyme and exhibits DNA polymerase and ribonuclease H (RNH) activities, both of which are essential to the reverse-transcription process. Despite the successful development of polymerase-targeting antiviral drugs over the last three decades, no bona fide inhibitor against the RNH activity of HIV-1 RT has progressed to clinical evaluation. In this review article, we describe the retroviral RNH function and inhibition, with primary consideration of the structural aspects of inhibition.

A synergy of activity, stability, and inhibitor-interaction of HIV-1 protease mutants evolved under drug-pressure.

A clinically-relevant, drug-resistant mutant of HIV-1 protease (PR), termed Flap+(I54V) and containing L10I, G48V, I54V and V82A mutations, is known to produce significant changes in the entropy and enthalpy balance of drug-PR interactions, compared to wild-type PR. A similar mutant, Flap+(I54A) , which evolves from Flap+(I54V) and contains the single change at residue 54 relative to Flap+(I54V) , does not. Yet, how Flap+(I54A) behaves in solution is not known. To understand the molecular basis of V54A evolution, we compared nuclear magnetic resonance (NMR) spectroscopy, fluorescence spectroscopy, isothermal titration calorimetry, and enzymatic assay data from four PR proteins: PR (pWT), Flap+(I54V) , Flap+(I54A) , and Flap+(I54) , a control mutant that contains only L10I, G48V and V82A mutations. Our data consistently show that selection to the smaller side chain at residue 54, not only decreases inhibitor affinity, but also restores the catalytic activity.

The reduced form of the antibody CH2 domain.

Among the immunoglobulin domains, the CH2 domain has the lowest thermal stability, which also depends on amino acid sequence and buffer conditions. To further identify factors that influence CH2 folding and stability, we characterized the domain in the reduced form using differential scanning fluorimetry and nuclear magnetic resonance. We show that the CH2 domain can fold, similarly to the disulfide-bridged form, without forming a disulfide-bridge, even though the protein contains two Cys residues. Although the reduced form exhibits thermal stability more than 15°C lower than the disulfide-bridged form, it does not undergo immediate full oxidization. To explain this phenomenon, we compared CH2 oxidization at different conditions and demonstrate a need for significant fluctuation of the folded conformation to enhance CH2 disulfide-bridge formation. We conclude that, since CH2 can be purified as a folded, semi-stable, reduced protein that can coexist with the oxidized form, verification of the level of oxidization at each step is critical in CH2 engineering studies.

Effect of Lysyl-tRNA Synthetase on the Maturation of HIV-1 Reverse Transcriptase.

In human immunodeficiency virus-1 (HIV-1), reverse transcriptase (RT) is encoded as a 66 kDa protein, p66, in the Gag-Pol polyprotein. This protein is proteolytically cleaved by HIV-1 protease (PR) to finally generate a mature RT that is a heterodimer, composed of a p66 subunit and a p66-derived 51 kDa subunit, p51. In our prior work, we demonstrated that tRNALys3 binding to p66/p66 facilitates efficient cleavage of p66 to p51 by PR. However, tRNALys3 is known to be recruited to the virus by forming a complex with lysyl-tRNA synthetase (LysRS). Herein, we tested whether LysRS can have an effect on RT maturation in vitro. Importantly, our data show no significant differences in RT maturation in the presence of LysRS. Furthermore, no apparent p66/66 interaction with LysRS was observed. Although PR cleaved LysRS, it did not immediately release tRNALys3 from LysRS. Thus, we conclude that a free fraction of tRNALys3, which is in equilibrium with a LysRS-bound form, interacts with p66/p66 without any additional mechanism involving release of tRNALys3 from LysRS. Given that only transient tRNALys3-p66/p66 interaction is needed for efficient RT maturation, a small amount of free tRNA may be sufficient for this process. These studies reveal molecular level insights into RT maturation and will be useful for the design of cellular/viral experiments to better understand the role of tRNA in HIV-1 replication.

Conformational Changes in HIV-1 Reverse Transcriptase that Facilitate Its Maturation.

HIV-1 reverse transcriptase (RT) is translated as part of the Gag-Pol polyprotein that is proteolytically processed by HIV-1 protease (PR) to finally become a mature heterodimer, composed of a p66 and a p66-derived 51-kDa subunit, p51. Our previous work suggested that tRNALys3 binding to p66/p66 introduces conformational changes in the ribonuclease (RNH) domain of RT that facilitate efficient cleavage of p66 to p51 by PR. In this study, we characterized the conformational changes in the RNH domain of p66/p66 imparted by tRNALys3 using NMR. Moreover, the importance of tRNALys3 in RT maturation was confirmed in cellulo by modulating the levels of Lys-tRNA synthetase, which affects recruitment of tRNALys3 to the virus. We also employed nonnucleoside RT inhibitors, to modulate the p66 dimer-monomer equilibrium and monitor the resulting structural changes. Taken together, our data provide unique insights into the conformational changes in p66/p66 that drive PR cleavage.

Effect of tRNA on the Maturation of HIV-1 Reverse Transcriptase.

The mature HIV-1 reverse transcriptase is a heterodimer that comprises 66 kDa (p66) and 51 kDa (p51) subunits. The latter is formed by HIV-1 protease-catalyzed removal of a C-terminal ribonuclease H domain from a p66 subunit. This proteolytic processing is a critical step in virus maturation and essential for viral infectivity. Here, we report that tRNA significantly enhances in vitro processing even at a substoichiometric tRNA:p66/p66 ratio. Other double-stranded RNAs have considerably less pronounced effect. Our data support a model where interaction of p66/p66 with tRNA introduces conformational asymmetry in the two subunits, permitting specific proteolytic processing of one p66 to provide the mature RT p66/p51 heterodimer.