Block HIV-1 Replication Research Articles

Blocking HIV-1 replication: are Fc-Fcγ receptor interactions required?

Interactions between IgG Fc and its receptors (FcγRs) have been shown to augment broadly neutralizing Ab-mediated (BnAb-mediated) protection from simian-human immunodeficiency virus (SHIV) challenge. In the current issue of the JCI, Parsons and collaborators compared the BnAb PGT121 with a version engineered to have impaired FcγR binding for their ability to protect macaques from an intravenous challenge with SHIV-infected cells as well as to treat already infected animals. Unexpectedly, and in contrast to previous studies, both versions of the Ab were equally able to prevent infection and decrease viral loads in infected animals. Thus, FcγR engagement does not always improve the in vivo antiviral activity of BnAbs.

Antibodies and Antibody Derivatives: New Partners in HIV Eradication Strategies.

Promptly after primoinfection, HIV generates a pool of infected cells carrying transcriptionally silent integrated proviral DNA, the HIV-1 reservoir. These cells are not cleared by combined antiretroviral therapy (cART), and persist lifelong in treated HIV-infected individuals. Defining clinical strategies to eradicate the HIV reservoir and cure HIV-infected individuals is a major research field that requires a deep understanding of the mechanisms of seeding, maintenance and destruction of latently infected cells. Although CTL responses have been classically associated with the control of HIV replication, and hence with the size of HIV reservoir, broadly neutralizing antibodies (bNAbs) have emerged as new players in HIV cure strategies. Several reasons support this potential role: (i) over the last years a number of bNAbs with high potency and ability to cope with the extreme variability of HIV have been identified; (ii) antibodies not only block HIV replication but mediate effector functions that may contribute to the removal of infected cells and to boost immune responses against HIV; (iii) a series of new technologies have allowed for the in vitro design of improved antibodies with increased antiviral and effector functions. Recent studies in non-human primate models and in HIV-infected individuals have shown that treatment with recombinant bNAbs isolated from HIV-infected individuals is safe and may have a beneficial effect both on the seeding of the HIV reservoir and on the inhibition of HIV replication. These promising data and the development of antibody technology have paved the way for treating HIV infection with engineered monoclonal antibodies with high potency of neutralization, wide coverage of HIV diversity, extended plasma half-life in vivo and improved effector functions. The exciting effects of these newly designed antibodies in vivo, either alone or in combination with other cure strategies (latency reversing agents or therapeutic vaccines), open a new hope in HIV eradication.

Discovery of a Branched Peptide That Recognizes the Rev Response Element (RRE) RNA and Blocks HIV-1 Replication.

We synthesized and screened a unique 46 656-member library composed of unnatural amino acids that revealed several hits against RRE IIB RNA. Among the hit peptides identified, peptide 4A5 was found to be selective against competitor RNAs and inhibited HIV-1 Rev-RRE RNA interaction in cell culture in a p24 ELISA assay. Biophysical characterization in a ribonuclease protection assay suggested that 4A5 bound to the stem-loop region in RRE IIB while SHAPE MaP probing with 234 nt RRE RNA indicated additional interaction with secondary Rev binding sites. Taken together, our investigation suggests that HIV replication is inhibited by 4A5 blocking binding of Rev and subsequent multimerization.

Developing and Evaluating Inhibitors against the RNase H Active Site of HIV-1 Reverse Transcriptase.

We tested three compounds for their ability to inhibit the RNase H (RH) and polymerase activities of HIV-1 reverse transcriptase (RT). A high-resolution crystal structure (2.2 Å) of one of the compounds showed that it chelates the two magnesium ions at the RH active site; this prevents the RH active site from interacting with, and cleaving, the RNA strand of an RNA-DNA heteroduplex. The compounds were tested using a variety of substrates: all three compounds inhibited the polymerase-independent RH activity of HIV-1 RT. Time-of-addition experiments showed that the compounds were more potent if they were bound to RT before the nucleic acid substrate was added. The compounds significantly inhibited the site-specific cleavage required to generate the polypurine tract (PPT) RNA primer that initiates the second strand of viral DNA synthesis. The compounds also reduced the polymerase activity of RT; this ability was a result of the compounds binding to the RH active site. These compounds appear to be relatively specific; they do not inhibit either Escherichia coli RNase HI or human RNase H2. The compounds inhibit the replication of an HIV-1-based vector in a one-round assay, and their potencies were only modestly decreased by mutations that confer resistance to integrase strand transfer inhibitors (INSTIs), nucleoside analogs, or nonnucleoside RT inhibitors (NNRTIs), suggesting that their ability to block HIV replication is related to their ability to block RH cleavage. These compounds appear to be useful leads that can be used to develop more potent and specific compounds.IMPORTANCE Despite advances in HIV-1 treatment, drug resistance is still a problem. Of the four enzymatic activities found in HIV-1 proteins (protease, RT polymerase, RT RNase H, and integrase), only RNase H has no approved therapeutics directed against it. This new target could be used to design and develop new classes of inhibitors that would suppress the replication of the drug-resistant variants that have been selected by the current therapeutics.

Development and Evaluation of Peptide-Functionalized Gold Nanoparticles for HIV Integrase Inhibition

The HIV integrase enzyme represents an important target for HIV inhibition because it is essential for viral replication and there is no cellular counterpart. Blocking HIV replication at this early stage of the viral life cycle can also prevent the establishment of viral reservoirs in the form of latently infected cells. Complications such as drug resistance and other adverse side effects associated with HIV treatment necessitate novel approaches for HIV inhibition. Nanoparticle-based systems are fast revolutionizing the biomedical field with applications in infectious disease diagnosis and treatment, and are important tools to investigate for HIV targeting. This study explores the development of gold nanoparticles functionalized with a hexapeptide, previously demonstrated to inhibit integrase at low micromolar concentrations. We also sought to investigate the effect of Tat peptide incorporation to facilitate both cellular and nuclear entry, so that both cytoplasmic and nuclear events mediated by integrase, could be targeted. Therefore, the aim of this study is to synthesize, characterize and evaluate the antiviral activity of nanoparticle-peptide complexes. Our results showed no obvious HIV inhibitory activity, but did provide original and fundamental in vitro data for this potent hexapeptide when challenged with HIV infection. Further insight into the optimization of gold nanoparticle functionalization with peptides is also provided.

P21 Restricts HIV-1 in Monocyte-Derived Dendritic Cells through the Reduction of Deoxynucleoside Triphosphate Biosynthesis and Regulation of SAMHD1 Antiviral Activity.

HIV-1 infection of noncycling cells, such as dendritic cells (DCs), is impaired due to limited availability of deoxynucleoside triphosphates (dNTPs), which are needed for HIV-1 reverse transcription. The levels of dNTPs are tightly regulated during the cell cycle and depend on the balance between dNTP biosynthesis and degradation. SAMHD1 potently blocks HIV-1 replication in DCs, although the underlying mechanism is still unclear. SAMHD1 has been reported to be able to degrade dNTPs and viral nucleic acids, which may both hamper HIV-1 reverse transcription. The relative contribution of these activities may differ in cycling and noncycling cells. Here, we show that inhibition of HIV-1 replication in monocyte-derived DCs (MDDCs) is associated with an increased expression of p21cip1/waf, a cell cycle regulator that is involved in the differentiation and maturation of DCs. Induction of p21 in MDDCs decreases the pool of dNTPs and increases the antiviral active isoform of SAMHD1. Although both processes are complementary in inhibiting HIV-1 replication, the antiviral activity of SAMHD1 in our primary cell model appears to be, at least partially, independent of its dNTPase activity. The reduction in the pool of dNTPs in MDDCs appears rather mostly due to a p21-mediated suppression of several enzymes involved in dNTP synthesis (i.e., RNR2, TYMS, and TK-1). These results are important to better understand the interplay between HIV-1 and DCs and may inform the design of new therapeutic approaches to decrease viral dissemination and improve immune responses against HIV-1.IMPORTANCE DCs play a key role in the induction of immune responses against HIV. However, HIV has evolved ways to exploit these cells, facilitating immune evasion and virus dissemination. We have found that the expression of p21, a cyclin-dependent kinase inhibitor involved in cell cycle regulation and monocyte differentiation and maturation, potentially can contribute to the inhibition of HIV-1 replication in monocyte-derived DCs through multiple mechanisms. p21 decreased the size of the intracellular dNTP pool. In parallel, p21 prevented SAMHD1 phosphorylation and promoted SAMHD1 dNTPase-independent antiviral activity. Thus, induction of p21 resulted in conditions that allowed the effective inhibition of HIV-1 replication through complementary mechanisms. Overall, p21 appears to be a key regulator of HIV infection in myeloid cells.

The Dynamic Interplay between HIV-1, SAMHD1, and the Innate Antiviral Response.

The innate immune response constitutes the first cellular line of defense against initial HIV-1 infection. Immune cells sense invading virus and trigger signaling cascades that induce antiviral defenses to control or eliminate infection. Professional antigen-presenting cells located in mucosal tissues, including dendritic cells and macrophages, are critical for recognizing HIV-1 at the site of initial exposure. These cells are less permissive to HIV-1 infection compared to activated CD4+ T-cells, which is mainly due to host restriction factors that serve an immediate role in controlling the establishment or spread of viral infection. However, HIV-1 can exploit innate immune cells and their cellular factors to avoid detection and clearance by the host immune system. Sterile alpha motif and HD-domain containing protein 1 (SAMHD1) is the mammalian deoxynucleoside triphosphate triphosphohydrolase responsible for regulating intracellular dNTP pools and restricting the replication of HIV-1 in non-dividing myeloid cells and quiescent CD4+ T-cells. Here, we review and analyze the latest literature on the antiviral function of SAMHD1, including the mechanism of HIV-1 restriction and the ability of SAMHD1 to regulate the innate immune response to viral infection. We also provide an overview of the dynamic interplay between HIV-1, SAMHD1, and the cell-intrinsic antiviral response to elucidate how SAMHD1 modulates HIV-1 infection in non-dividing immune cells. A more complete understanding of SAMHD1’s role in the innate immune response to HIV-1 infection may help develop stratagems to enhance its antiviral effects and to more efficiently block HIV-1 replication and avoid the pathogenic result of viral infection.

Cellular fatty acid synthase is required for late stages of HIV-1 replication

BackgroundLike all viruses, HIV-1 relies on host systems to replicate. The human purinome consists of approximately two thousand proteins that bind and use purines such as ATP, NADH, and NADPH. By virtue of their purine binding pockets, purinome proteins are highly druggable, and many existing drugs target purine-using enzymes. Leveraging a protein affinity media that uses the purine-binding pocket to capture the entire purinome, we sought to define purine-binding proteins regulated by HIV-1 infection.ResultsUsing purinome capture media, we observed that HIV-1 infection increases intracellular levels of fatty acid synthase (FASN), a NADPH-using enzyme critical to the synthesis of de novo fatty acids. siRNA mediated knockdown of FASN reduced HIV-1 particle production by 80%, and treatment of tissue culture cells or primary PBMCs with Fasnall, a newly described selective FASN inhibitor, reduced HIV-1 virion production by 90% (EC50 = 213 nM). Despite the requirement of FASN for nascent virion production, FASN activity was not required for intracellular Gag protein production, indicating that FASN dependent de novo fatty acid biosynthesis contributes to a late step of HIV-1 replication.ConclusionsHere we show that HIV-1 replication both increases FASN levels and requires host FASN activity. We also report that Fasnall, a novel FASN inhibitor that demonstrates anti-tumor activity in vivo, is a potent and efficacious antiviral, blocking HIV-1 replication in both tissue culture and primary cell models of HIV-1 replication. In adults, most fatty acids are obtained exogenously from the diet, thus making FASN a plausible candidate for pharmacological intervention. In conclusion, we hypothesize that FASN is a novel host dependency factor and that inhibition of FASN activity has the potential to be exploited as an antiretroviral strategy.

Inhibition of Non Canonical HIV-1 Tat Secretion Through the Cellular Na+,K+-ATPase Blocks HIV-1 Infection

Besides its essential role in the activation of HIV-1 gene expression, the viral Tat protein has the unusual property of trafficking in and out of cells. In contrast to Tat internalization, the mechanism involved in extracellular Tat release has so far remained elusive. Here we show that Tat secretion occurs through a Golgi-independent pathway requiring binding of Tat with three short, non-consecutive intracytoplasmic loops at the C-terminus of the cellular Na+,K+-ATPase pump alpha subunit. Ouabain, a pump inhibitor, blocked this interaction and prevented Tat secretion; virions produced in the presence of this drug were less infectious, consistent the capacity of virion-associated Tat to increase HIV-1 infectivity. Treatment of CD4+ T-cells with short peptides corresponding to the Tat-binding regions of the pump alpha subunit impaired extracellular Tat release and blocked HIV-1 replication. Thus, non canonical, extracellular Tat secretion is essential for viral infectivity.

Interferon epsilon protects primary macrophages against HIV infection

Abstract Interferon epsilon (IFNe) is a unique type I IFN that is not induced by pattern-recognition response elements. IFNɛ is constitutively expressed in mucosal tissues including the female genital mucosa. Although the direct anti-viral activity of IFNe was thought to be weak compared to IFNa, IFNe controls Chlamydia muridarum and herpes simplex virus 2 in mice, possibly through modulation of immune response. We show here that IFNe induces an antiviral state in human macrophages that blocks HIV-1 replication. IFNe had little or no protective effect in activated CD4+ T cells or transformed cell lines unless activated CD4+ T cells were infected with replication competent HIV-1 at a low MOI. The block to HIV infection of macrophages was maximal after 24 h of treatment and was reversible. IFNe acted on early stages of the HIV life cycle including viral entry, reverse transcription and nuclear import. The protection did not appear to operate through known type I IFN-induced HIV host restriction factors such as APOBEC3A and SAMHD1. IFNe-stimulated immune mediators and pathways had the signature of type I IFNs but were distinct from IFNα2. IFNe induced significant phagocytosis and reactive oxygen species, which contributed to the block to HIV replication in macrophages. These findings indicate that IFNe induces an antiviral state in macrophages that is mediated by different factors than those induced by IFNa2. Understanding the mechanism of IFNe-mediated HIV inhibition through immune modulation has implications for prevention.

Synthesis, biological evaluation and molecular docking study of N-(2-methoxyphenyl)-6-((4-nitrophenyl)sulfonyl)benzamide derivatives as potent HIV-1 Vif antagonists

Viral infectivity factor (Vif) is protective against APOBEC3G (A3G)-mediated viral cDNA hypermutations, and development of molecules that inhibit Vif mediated A3G degradation is a novel strategy for blocking HIV-1 replication. Through optimizations of the central ring of N-(2-methoxyphenyl)-2-((4-nitrophenyl)thio)benzamide (RN-18), we found a potent compound 12c with EC50 value of 1.54 μM, enhancing the antiviral activity more than 150-fold compared with RN-18 in nonpermissive H9 cells. 12c protected A3G from degradation by inhibiting Vif function. Besides, 12c suppressed different HIV-1 clinical strains (HIV-1KM018, HIV-1TC-1 and HIV-1WAN) and drug-resistant strains (NRTI, NNRTI, PI, and FI) with relatively high activities. Amidation of 12c with glycine gave a prodrug 13a, improving the water solubility about 2600-fold compared with 12c. Moreover, 13a inhibited the virus replication efficiently with an EC50 value of 0.228 μM. These results suggested that the prodrug 13a is a promising candidate agent for the treatment of AIDS.

Intracellular Reassociation of RNA-DNA Hybrids that Activates RNAi in HIV-Infected Cells.

Human immunodeficiency virus Type 1 (HIV-1) is the major cause of acquired immune deficiency syndrome (AIDS). In 2014, it was estimated that 1.2 million people died from AIDS-related illnesses. RNA interference-based therapy to block HIV replication is a field that, as of now, is without any FDA-approved drugs available for clinical use. In this chapter we describe a protocol for testing and utilizing a new approach that relies on reassociation of RNA-DNA hybrids activating RNAi and blocking HIV replication in human cells.

The Genome Segments of Bluetongue Virus Differ in Copy Number in a Host-Specific Manner

HIV-1 infection of noncycling cells, such as dendritic cells (DCs), is impaired due to limited availability of deoxynucleoside triphosphates (dNTPs), which are needed for HIV-1 reverse transcription. The levels of dNTPs are tightly regulated during the cell cycle and depend on the balance between dNTP biosynthesis and degradation. SAMHD1 potently blocks HIV-1 replication in DCs, although the underlying mechanism is still unclear. SAMHD1 has been reported to be able to degrade dNTPs and viral nucleic acids, which may both hamper HIV-1 reverse transcription. The relative contribution of these activities may differ in cycling and noncycling cells. Here, we show that inhibition of HIV-1 replication in monocyte-derived DCs (MDDCs) is associated with an increased expression of p21cip1/waf, a cell cycle regulator that is involved in the differentiation and maturation of DCs. Induction of p21 in MDDCs decreases the pool of dNTPs and increases the antiviral active isoform of SAMHD1. Although both processes are complementary in inhibiting HIV-1 replica-tion, the antiviral activity of SAMHD1 in our primary cell model appears to be, at least partially, independent of its dNTPase activity. The reduction in the pool of dNTPs in MDDCs appears rather mostly due to a p21-mediated suppression of several enzymes involved in dNTP synthesis (i.e., RNR2, TYMS, and TK-1). These results are important to better understand the interplay between HIV-1 and DCs and may inform the design of new therapeutic approaches to decrease viral dissemination and improve immune responses against HIV-1. IMPORTANCE DCs play a key role in the induction of immune responses against HIV. However, HIV has evolved ways to exploit these cells, facilitating immune evasion and virus dissemination. We have found that the expression of p21, a cyclin-dependent kinase inhibitor involved in cell cycle regulation and monocyte differentiation and maturation, potentially can contribute to the inhibition of HIV-1 replication in monocyte-derived DCs through multiple mechanisms. p21 decreased the size of the intracellular dNTP pool. In parallel, p21 prevented SAMHD1 phos-phorylation and promoted SAMHD1 dNTPase-independent antiviral activity. Thus, induction of p21 resulted in conditions that allowed the effective inhibition of HIV-1 replication through complementary mechanisms. Overall, p21 appears to be a key regulator of HIV infection in myeloid cells.

IFN-ε protects primary macrophages against HIV infection.

IFN-ε is a unique type I IFN that is not induced by pattern recognition response elements. IFN-ε is constitutively expressed in mucosal tissues, including the female genital mucosa. Although the direct antiviral activity of IFN-ε was thought to be weak compared with IFN-α, IFN-ε controls Chlamydia muridarum and herpes simplex virus 2 in mice, possibly through modulation of immune response. We show here that IFN-ε induces an antiviral state in human macrophages that blocks HIV-1 replication. IFN-ε had little or no protective effect in activated CD4+ T cells or transformed cell lines unless activated CD4+ T cells were infected with replication-competent HIV-1 at a low MOI. The block to HIV infection of macrophages was maximal after 24 hours of treatment and was reversible. IFN-ε acted on early stages of the HIV life cycle, including viral entry, reverse transcription, and nuclear import. The protection did not appear to operate through known type I IFN-induced HIV host restriction factors, such as APOBEC3A and SAMHD1. IFN-ε-stimulated immune mediators and pathways had the signature of type I IFNs but were distinct from IFN-α in macrophages. IFN-ε induced significant phagocytosis and ROS, which contributed to the block to HIV replication. These findings indicate that IFN-ε induces an antiviral state in macrophages that is mediated by different factors than those induced by IFN-α. Understanding the mechanism of IFN-ε-mediated HIV inhibition through immune modulation has implications for prevention.

Characterization of two distinct early post-entry blocks to HIV-1 in common marmoset lymphocytes.

In nature, primate lentiviruses infect humans and several Old World monkeys and apes. However, to date, lentiviruses infecting New World monkeys have not been described. We studied the susceptibility of common marmoset cells to HIV-1 infection and observed the presence of post-entry blocks to the early phase of HIV-1 infection in peripheral blood lymphocytes (PBLs) and a B lymphocytic cell line (B-LCL). The blocks present in these cells are dominant and phenotypically different from each other. In PBLs, the block occurs at the level of reverse transcription, reducing the accumulation of early and late transcripts, similar to the block imposed by TRIM5α. However, we have found that marmoset TRIM5α does not block HIV-1. In contrast, the restriction factor present in B-LCLs blocks HIV-1 replication at a later step, after nuclear entry, and inhibits integration. Additionally, we have identified an HIV-1 capsid mutant, N74D, that is able to escape the restriction in the marmoset B-LCLs. Our results suggest that the factors responsible for the blocks present in marmoset PBLs and B-LCLs are different. We propose the existence of at least two new restriction factors able to block HIV-1 infection in marmoset lymphocytes.

687. Development of a Safe and Effective Combinatorial Foamy Virus Vector for HIV Gene Therapy

The Berlin patient and successes in retroviral hematopoietic stem cell (HSC) gene therapy suggest that gene therapy may provide a functional cure for HIV. However, retroviral-HSC gene therapy has resulted in serious adverse side effects due to vector mediated genotoxicity, including leukemia. Foamy virus (FV) vectors have a promising integration profile and may be safer than retroviral vectors. FV vectors also can efficiently deliver anti-HIV transgenes that can reduce the titer of HIV-1 based lentiviral vectors. We report a novel combinatorial anti-HIV FV vector that uses a housekeeping elongation factor 1 alpha (EF1α) promoter but still potently blocks HIV infection. We first evaluated the relative potency of various previously described anti-HIV transgenes, including the C46 fusion inhibitor, the F12-Vif derivative Chim3, lens epithelium derived growth factor-integrase binding domain (LIBD) and TRIM5α-CyclophilinA fusion (TCypA) in a FV vector background. We found that C46 was the most potent anti-HIV transgene, followed by TCypA and LEDGF-IBD. Next, we hypothesized that using a less-genotoxic internal promoter to drive the transgenes would reduce vector-mediated genotoxicity. Therefore, a direct comparison was made between the efficacy of housekeeping gene promoters (EF1α and Ubiquitin C; UbC) and a highly genotoxic SFFV promoter in expressing the C46 transgene and subsequently blocking HIV replication. We observed that C46 EF1α had ~2 to 4 fold higher anti-HIV effect than C46 driven by either SFFV or UbC promoters, respectively. Based on these results, we designed a novel combinatorial FV vector expressing three anti-HIV transgenes: C46, TcypA and LIBD. The gene cassette was driven by an EF1α promoter and also has mCherry as a reporter gene (FV-E C46TLmC-W). This vector can be produced at high titer, 1.4 × 107 transducing units/ml which is critical for clinical translation. This novel combinatorial anti-HIV FV vector showed a higher potency in blocking HIV replication than C46 alone at a late time point, 21 days post HIV infection (Figure 1Figure 1). An in vitro competitive survival advantage assay indicated that cells transduced with our novel combinatorial anti-HIV FV vectors are highly resistant to HIV infection compared to cells transduced with FV expressing a control EGFP reporter gene alone (FV-EG-W) (Figure 2Figure 2). Further studies will be focused on the efficacy of our novel combinatorial anti-HIV FV vector in human CD34+ cells transplanted in a mouse xenotransplant in vivo model. Our goal is to develop a safe and potent combinatorial FV vector for clinical studies.View Large Image | Download PowerPoint SlideView Large Image | Download PowerPoint Slide

333. Intracellular Ankyrin Specific to p24 and zfp Specific to 2-LTR Are Alternatives to CCR5 Knockdown for Stem Cell Gene Therapy

HIV-1 genome integration and capsid assembly are some of the most important steps to viral life cycle. Designed scaffold proteins, zinc finger and ankyrin repeat protein, were characterized as intracellular antiviral agents that can interfere in these steps. Zinc finger protein, 2LTRZFP, plays a vital role in blocking HIV integration. This protein targets the 2-LTR-circle junction of the HIV-1 DNA but it would have no effect on the HIV-infected cells. Another protein, AnkGAG1D4, will then specifically bind to the viral capsid resulting in inhibition of HIV-1 assembly. Since only one particular scaffold protein cannot confer full protection against the virus. Combination of these two proteins became the key to enhance inhibitory function to block HIV-1 replication at early and late infection stage and also help eliminate latent viral reservoirs. The human T cell line modified by using a lentiviral vector bearing 2LTRZFP alone or combination with N-myristoylatedMyr(+)AnkGAG1D4 were protected from viral infection in HIV-1 challenge. Interestingly, the Myr(+)AnkGAG1D4 showed significantly antiviral effect in HIV-1-preinfected SupT1 cells and also possessed a broad antiviral activity against SIV, and SHIV. These results demonstrate that 2LTRZFP and Myr(+)AnkGAG1D4 proteins can be the novel anti-HIV-1 therapeutic agents for human applications.

A Parallel Synthesis Approach to the Identification of Novel Diheteroarylamide-Based Compounds Blocking HIV Replication: Potential Inhibitors of HIV-1 Pre-mRNA Alternative Splicing.

A 256-compound library was evaluated in an anti-HIV screen to identify structural "mimics" of the fused tetracyclic indole compound 1 (IDC16) that conserve its anti-HIV activity without associated cytotoxicity. Four diheteroarylamide-type compounds, containing a common 5-nitroisobenzothiazole motif, were identified as active. In subsequent screens, the most potent compound 9 (1C8) was active against wild-type HIV-1IIIB (subtype B, X4-tropic) and HIV-1 97USSN54 (subtype A, R5-tropic) with EC50's of 0.6 and 0.9 μM, respectively. Compound 9 also inhibited HIV strains resistant to drugs targeting HIV reverse transcriptase, protease, integrase, and coreceptor CCR5 with EC50's ranging from 0.9 to 1.5 μM. The CC50 value obtained in a cytotoxicity assay for compound 9 was >100 μM, corresponding to a therapeutic index (CC50/EC50) of approximately 100. Further comparison studies revealed that, whereas the anti-HIV activity for compound 9 and the parent molecule 1 are similar, the cytotoxic effect for compound 9 was, as planned, markedly suppressed.

Identification of the HIV-1 Vif and Human APOBEC3G Protein Interface.

Human cells express natural antiviral proteins, such as APOBEC3G (A3G), that potently restrict HIV replication. As a counter-defense, HIV encodes the accessory protein Vif, which binds A3G and mediates its proteasomal degradation. Our structural knowledge on how Vif and A3G interact is limited, because a co-structure is not available. We identified specific points of contact between Vif and A3G by using functional assays with full-length A3G, patient-derived Vif variants, and HIV forced evolution. These anchor points were used to model and validate the Vif-A3G interface. The resultant co-structure model shows that the negatively charged β4-α4 A3G loop, which contains primate-specific variation, is the core Vif binding site and forms extensive interactions with a positively charged pocket in HIV Vif. Our data present a functional map of this viral-host interface and open avenues for targeted approaches to block HIV replication by obstructing the Vif-A3G interaction.

Subtle Dynamic Changes Accompany Hck Activation by HIV-1 Nef and are Reversed by an Antiretroviral Kinase Inhibitor.

The HIV-1 virulence factor Nef interacts with the macrophage Src-family kinase Hck, resulting in constitutive kinase activation that contributes to viral replication and immune escape. Previous chemical library screens identified the diphenylfuranopyrimdine kinase inhibitor DFP-4AB, which selectively inhibits Nef-dependent Hck activity in biochemical assays and potently blocks HIV replication in vitro. In the present study, hydrogen exchange mass spectrometry (HX MS) was used to study conformational changes in downregulated Hck that result from Nef binding, as well as the impact of DFP-4AB on these changes. Remarkably, interaction with Nef induced only subtle changes in deuterium uptake by Hck, with the most significant changes in the N-lobe of the kinase domain adjacent to the docking site for Nef on the SH3 domain. No changes in hydrogen exchange were observed in the Hck SH2 domain or C-terminal tail, indicating that this regulatory interaction is unaffected by Nef binding. When HX MS was performed in the presence of DFP-4AB, the effect of Nef on Hck N-lobe dynamics was completely reversed. These results show that constitutive activation of Hck by HIV-1 Nef requires only modest changes to the conformational dynamics of the overall kinase structure. DFP-4AB reverses these effects, consistent with its activity against this Nef-induced signaling event in HIV-infected cells.