Disparate outcomes in Hispanic patients with metabolic dysfunction-associated steatotic liver disease/steatohepatitis and type 2 diabetes: Large cohort study.

BACKGROUND: Metabolic dysfunction-associated steatotic liver disease (MASLD) and metabolic dysfunction-associated steatohepatitis (MASH) are a growing health burden across a significant portion of the global patient population. However, these conditions seem to have disparate rates and outcomes between different ethnic populations. The combination of MASLD/MASH and type 2 diabetes increases the risk of hepatocellular carcinoma (HCC), and Hispanic patients experience the greatest burden, particularly those in South Texas. AIM: To compare outcomes between Hispanic and non-Hispanic patients in the United States, while further focusing on the Hispanic population within Southeast Texas to determine whether the documented disparity in outcomes is a function of geographical circumstance or if there is a more widespread reason that all clinicians must account for in prognostic consideration. METHODS: This cohort analysis was conducted with data obtained from TriNetX, LLC ("TriNetX"), a global federated health research network that provides access to deidentified medical records from healthcare organizations worldwide. Two cohort networks were used: University of Texas Medical Branch (UTMB) hospital and the United States national database collective to determine whether disparities were related to geographic regions, like Southeast Texas. RESULTS: This study findings revealed Hispanics/Latinos have a statistically significant higher occurrence of HCC, type 2 diabetes mellitus, and liver fibrosis/cirrhosis in both the United States and the UTMB Hispanic/Latino groups. All-cause mortality in Hispanics/Latinos was lower within the United States group and not statistically elevated in the UTMB cohort. CONCLUSION: This would appear to support that Hispanic patients in Southeast Texas are not uniquely affected compared to the national Hispanic population.

Nuclear trafficking of the HIV-1 pre-integration complex depends on the ADAM10 intracellular domain.

Previously, we showed that ADAM10 is necessary for HIV-1 replication in primary human macrophages and immortalized cell lines. Silencing ADAM10 expression interrupted the HIV-1 life cycle prior to nuclear translocation of viral cDNA. Furthermore, our data indicated that HIV-1 replication depends on the expression of ADAM15 and γ-secretase, which proteolytically processes ADAM10. Silencing ADAM15 or γ-secretase expression inhibits HIV-1 replication between reverse transcription and nuclear entry. Here, we show that ADAM10 expression also supports replication in CD4(+) T lymphocytes. The intracellular domain (ICD) of ADAM10 associates with the HIV-1 pre-integration complex (PIC) in the cytoplasm and immunoprecipitates and co-localizes with HIV-1 integrase, a key component of PIC. Taken together, our data support a model whereby ADAM15/γ-secretase processing of ADAM10 releases the ICD, which then incorporates into HIV-1 PIC to facilitate nuclear trafficking. Thus, these studies suggest ADAM10 as a novel therapeutic target for inhibiting HIV-1 prior to nuclear entry.

Gp120 in the pathogenesis of human immunodeficiency virus-associated pain.

OBJECTIVE: Chronic pain is a common neurological comorbidity of human immunodeficiency virus (HIV)-1 infection, but the etiological cause remains elusive. The objective of this study was to identify the HIV-1 causal factor that critically contributes to the pathogenesis of HIV-associated pain. METHODS: We first compared the levels of HIV-1 proteins in postmortem tissues of the spinal cord dorsal horn (SDH) from HIV-1/acquired immunodeficiency syndrome patients who developed chronic pain (pain-positive HIV-1 patients) and HIV-1 patients who did not develop chronic pain (pain-negative HIV-1 patients). Then we used the HIV-1 protein that was specifically increased in the pain-positive patients to generate mouse models. Finally, we performed comparative analyses on the pathological changes in the models and the HIV-1 patients. RESULTS: We found that HIV-1 gp120 was significantly higher in pain-positive HIV-1 patients (vs pain-negative HIV-1 patients). This finding suggested that gp120 was a potential causal factor of the HIV-associated pain. To test this hypothesis, we used a mouse model generated by intrathecal injection of gp120 and compared the pathologies of the model and the pain-positive human HIV-1 patients. The results showed that the mouse model and pain-positive human HIV-1 patients developed extensive similarities in their pathological phenotypes, including pain behaviors, peripheral neuropathy, glial reactivation, synapse degeneration, and aberrant activation of pain-related signaling pathways in the SDH. INTERPRETATION: Our findings suggest that gp120 may critically contribute to the pathogenesis of HIV-associated pain.

Cultivated vaginal microbiomes alter HIV-1 infection and antiretroviral efficacy in colonized epithelial multilayer cultures.

There is a pressing need for modeling of the symbiotic and at times dysbiotic relationship established between bacterial microbiomes and human mucosal surfaces. In particular clinical studies have indicated that the complex vaginal microbiome (VMB) contributes to the protection against sexually-transmitted pathogens including the life-threatening human immunodeficiency virus (HIV-1). The human microbiome project has substantially increased our understanding of the complex bacterial communities in the vagina however, as is the case for most microbiomes, very few of the community member species have been successfully cultivated in the laboratory limiting the types of studies that can be completed. A genetically controlled ex vivo model system is critically needed to study the complex interactions and associated molecular dialog. We present the first vaginal mucosal culture model that supports colonization by both healthy and dysbiotic VMB from vaginal swabs collected from routine gynecological patients. The immortalized vaginal epithelial cells used in the model and VMB cryopreservation methods provide the opportunity to reproducibly create replicates for lab-based evaluations of this important mucosal/bacterial community interface. The culture system also contains HIV-1 susceptible cells allowing us to study the impact of representative microbiomes on replication. Our results show that our culture system supports stable and reproducible colonization by VMB representing distinct community state types and that the selected representatives have significantly different effects on the replication of HIV-1. Further, we show the utility of the system to predict unwanted alterations in efficacy or bacterial community profiles following topical application of a front line antiretroviral.

The dual role of tetraspanin CD63 in HIV-1 replication.

BACKGROUND: Previously, we showed that the tetraspanin membrane protein CD63 mediates both early and post-integration stages of the HIV-1 replication cycle. The temporal roles of CD63 were discerned using monoclonal antibodies and small interfering RNAs (siRNAs) to block CD63 function, and determining which of the sequential steps in HIV-1 replication were disrupted. Inhibition was shown to occur during early infection, suggestive of involvement in virus entry or reverse transcription. In addition, we have shown that treatment with CD63 siRNA post-infection, significantly inhibited virus production in supernatant, suggesting an important role for CD63 in macrophages during HIV-1 replication events occurring after proviral integration, and possibly during egress. RESULTS: In this study we used CD63 siRNA to investigate the infectivity of pseudotyped viruses (carrying an NL4-3 Env-negative luciferase backbone) in primary human macrophages. We demonstrated that lab adapted R5- and R5X4-tropic HIV-1 strains are significantly inhibited by CD63 silencing. However, the infectivity of MLV or VSV-pseudotyped strains, which enter though receptor-mediated endocytosis, is unaffected by silencing CD63. These results indicate that CD63 may support Env-mediated entry or fusion events facilitated though CD4 and CCR5. Also, antibody and siRNA-based CD63 inhibition studies indicate a potential role for CD63 following proviral integration. Further, we show that CD63 expression is key for efficient replication in primary CD4⁺ T cells, complementing our prior studies with primary human macrophages and immortalized cell lines. CONCLUSIONS: Collectively, these findings indicate that CD63 may support Env-mediated fusion as well as a late (post-integration) step in the HIV-1 replication cycle.

In vitro model of mycobacteria and HIV-1 co-infection for drug discovery.

Tuberculosis (TB) has become a global health threat in the wake of the Human Immunodeficiency Virus (HIV) pandemic and is the leading cause of death in people with HIV/AIDS. Treatment of patients with Mycobacterium tuberculosis (Mtb)/HIV co-infection is complicated by drug interactions and toxicity that present huge challenges for clinical intervention. Discovery efforts to identify novel compounds with increased effectiveness and decreased drug-drug interactions against Mtb, HIV-1, or both, would be greatly aided by the use of a co-infection model for screening drug libraries. Currently, inhibitors of Mtb are screened independently in mycobacterial cell cultures or target based biochemical screens and less often in macrophages or peripheral blood leukocytes. Similarly, HIV-1 drugs are screened in vitro independently from anti-mycobacterial compounds. Here, we describe an in vitro model where primary human peripheral blood mononuclear cells or monocyte-derived macrophages are infected with Mycobacterium bovis BCG and HIV-1, and used to evaluate drug toxicity and activity in a co-infection setting. Our results with standard compounds (e.g. Azidothymidine, Rifampicin) demonstrate the utility of this in vitro model to evaluate drug effectiveness relevant to cellular toxicity, HIV-1 replication, and intracellular mycobacterial growth, through the use of ELISA, bacterial enumeration, and multi-variate flow cytometry. This model and associated assays have great value in accelerating the discovery of compounds for use in Mtb/HIV-1 co-infected patients.

Identification of cellular proteins required for replication of human immunodeficiency virus type 1.

Cellular proteins are essential for human immunodeficiency virus type 1 (HIV-1) replication and may serve as viable new targets for treating infection. Using gene trap insertional mutagenesis, a high-throughput approach based on random inactivation of cellular genes, candidate genes were found that limit virus replication when mutated. Disrupted genes (N=87) conferring resistance to lytic infection with several viruses were queried for an affect on HIV-1 replication by utilizing small interfering RNA (siRNA) screens in TZM-bl cells. Several genes regulating diverse pathways were found to be required for HIV-1 replication, including DHX8, DNAJA1, GTF2E1, GTF2E2, HAP1, KALRN, UBA3, UBE2E3, and VMP1. Candidate genes were independently tested in primary human macrophages, toxicity assays, and/or Tat-dependent ß-galactosidase reporter assays. Bioinformatics analyses indicated that several host factors present in this study participate in canonical pathways and functional processes implicated in prior genome-wide studies. However, the genes presented in this study did not share identity with those found previously. Novel antiviral targets identified in this study should open new avenues for mechanistic investigation.

Host factors mediating HIV-1 replication.

Human immunodeficiency virus type 1(HIV-1) infection is the leading cause of death worldwide in adults attributable to infectious diseases. Although the majority of infections are in sub-Saharan Africa and Southeast Asia, HIV-1 is also a major health concern in most countries throughout the globe. While current antiretroviral treatments are generally effective, particularly in combination therapy, limitations exist due to drug resistance occurring among the drug classes. Traditionally, HIV-1 drugs have targeted viral proteins, which are mutable targets. As cellular genes mutate relatively infrequently, host proteins may prove to be more durable targets than viral proteins. HIV-1 replication is dependent upon cellular proteins that perform essential roles during the viral life cycle. Maraviroc is the first FDA-approved antiretroviral drug to target a cellular factor, HIV-1 coreceptor CCR5, and serves to intercept viral-host protein-protein interactions mediating entry. Recent large-scale siRNA and shRNA screens have revealed over 1000 candidate host factors that potentially support HIV-1 replication, and have implicated new pathways in the viral life cycle. These host proteins and cellular pathways may represent important targets for future therapeutic discoveries. This review discusses critical cellular factors that facilitate the successive steps in HIV-1 replication.

A functional role for ADAM10 in human immunodeficiency virus type-1 replication.

BACKGROUND: Gene trap insertional mutagenesis was used as a high-throughput approach to discover cellular genes participating in viral infection by screening libraries of cells selected for survival from lytic infection with a variety of viruses. Cells harboring a disrupted ADAM10 (A Disintegrin and Metalloprotease 10) allele survived reovirus infection, and subsequently ADAM10 was shown by RNA interference to be important for replication of HIV-1. RESULTS: Silencing ADAM10 expression with small interfering RNA (siRNA) 48 hours before infection significantly inhibited HIV-1 replication in primary human monocyte-derived macrophages and in CD4⁺ cell lines. In agreement, ADAM10 over-expression significantly increased HIV-1 replication. ADAM10 down-regulation did not inhibit viral reverse transcription, indicating that viral entry and uncoating are also independent of ADAM10 expression. Integration of HIV-1 cDNA was reduced in ADAM10 down-regulated cells; however, concomitant 2-LTR circle formation was not detected, suggesting that HIV-1 does not enter the nucleus. Further, ADAM10 silencing inhibited downstream reporter gene expression and viral protein translation. Interestingly, we found that while the metalloprotease domain of ADAM10 is not required for HIV-1 replication, ADAM15 and γ-secretase (which proteolytically release the extracellular and intracellular domains of ADAM10 from the plasma membrane, respectively) do support productive infection. CONCLUSIONS: We propose that ADAM10 facilitates replication at the level of nuclear trafficking. Collectively, our data support a model whereby ADAM10 is cleaved by ADAM15 and γ-secretase and that the ADAM10 intracellular domain directly facilitates HIV-1 nuclear trafficking. Thus, ADAM10 represents a novel cellular target class for development of antiretroviral drugs.

A post-entry role for CD63 in early HIV-1 replication.

Macrophages and CD4(+) lymphocytes are the major reservoirs for HIV-1 infection. CD63 is a tetraspanin transmembrane protein, which has been shown to play an essential role during HIV-1 replication in macrophages. In this study, we further confirm the requirement of CD63 in early HIV-1 replication events in both macrophages and a CD4(+) cell line. Further analysis revealed that viral attachment and cell-cell fusion were unaffected by CD63 silencing. However, CD63-depleted macrophages showed a significant decrease in the initiation and completion of HIV-1 reverse transcription, affecting subsequent events of the HIV-1 life cycle. Integration of HIV-1 cDNA as well as the formation of 2-LTR circles was notably reduced. Reporter assays showed that CD63 down regulation reduced production of the early HIV protein Tat. In agreement, CD63 silencing also inhibited production of the late protein p24. These findings suggest that CD63 plays an early post-entry role prior to or at the reverse transcription step.

Quantitative PCR used to assess HIV-1 integration and 2-LTR circle formation in human macrophages, peripheral blood lymphocytes and a CD4+ cell line.

BACKGROUND: Integration is an intermediate step in the HIV life cycle and is defined as the insertion of HIV-1 proviral DNA into the host chromosome. If integration does not occur when HIV-1 cDNA enters the nucleus, it circularizes upon itself and forms a 2-LTR circle. Monitoring the level of integrated HIV-1 cDNA in different primary cell subsets is very important, particularly regarding the effect of HAART in HIV-1 infected individuals. Because of limitations of prior HIV-1 integration assays, there is limited data on the level of integration and 2-LTR circle formation in primary cell subsets, particularly in human monocyte-derived macrophages and peripheral blood lymphocytes (PBL). RESULTS: In this study, we utilized a well-defined, sensitive two-step quantitative real-time PCR method to detect HIV-1 integration as well as conventional real-time PCR to detect 2-LTR circle formation in human macrophages and PBL isolated from six different healthy donors, as well as U373 CD4+ cells by infecting with HIV-1SX (R5) or dual-tropic isolate HIV-189.6 (R5/X4) virus strains. We used the FDA-approved integrase inhibitor, raltegravir, to determine quantitative differences of integrated HIV viral cDNA in HIV-1 infected cells with and without raltegravir treatment. Our results show that integration and 2-LTR circle formation can be assessed in primary macrophages, PBL, and a CD4+ cell line by this method. Specifically, our results demonstrate that this two-step real-time PCR method can distinguish between HIV-1 integrated viral cDNA and non-integrated nuclear HIV-1 2-LTR circles caused by impaired integration with raltegravir-treatment. This further confirms that only integrated HIV-1 cDNA can be specifically amplified and quantified by two-step PCR without non-specifically detecting non-integrated viral cDNA. CONCLUSION: These results consistently demonstrate that the well-established real-time PCR assays used are robust, sensitive and quantitative for the detection of HIV-1 integration and 2-LTR circle formation in physiologically relevant human macrophages and PBL using lab-adapted virus strains, instead of pseudovirus. With two-step real-time PCR, we show that unintegrated, nuclear HIV-1 cDNA is not detected in raltegravir-treated cells, while specific for only integrated HIV-1 cDNA in non-treated cells. These methods could be applied as a useful tool in further monitoring specific therapy in HIV-1 infected individuals.

Non-structural and nucleocapsid proteins of Punta Toro virus induce apoptosis of hepatocytes through both intrinsic and extrinsic pathways.

Punta Toro virus (PTV; family Bunyaviridae, genus Phlebovirus) causes severe hepatic damage through brisk apoptosis of hepatocytes. In the present study, two viral proteins encoded by the S segment of the viral genome, non-structural (NSs) and nucleocapsid protein (N), were examined for their roles in apoptosis. Expression of NSs in HepG2 cells led to apoptosis in 45% of transfected cells, and with N, 28%, on average. These levels represent a four- to an eightfold increase over cells transfected with the mutated protein vectors. Caspase-3, -8 and -9 activities were increased by N protein when compared with the control NC (P < 0.05), and by NSsA and NSsB, as compared to control NSsC (P < 0.01). Treatment of the transfected cells with caspase-8 or -9 inhibitors markedly decreased apoptosis. Neutralization of TNF-alpha or Fas ligand had no effect on apoptosis. These results indicate that both NSs and N are responsible for causing hepatocyte apoptosis by triggering the extrinsic caspase-8 and intrinsic caspase-9 pathways.

Dried-plasma transport using a novel matrix and collection system for human immunodeficiency virus and hepatitis C virus virologic testing.

A novel method for the collection and transportation of dried-blood-plasma samples, SampleTanker (ST), was developed and compared to standard shipping protocols for frozen-plasma specimens containing human immunodeficiency virus type 1 (HIV-1) and/or hepatitis C virus (HCV). Matched frozen and dried 1-ml EDTA-containing plasma samples were collected and analyzed by several molecular-based virologic assays. After addition of 1.175 ml of reconstitution buffer, 1.035 ml of dried plasma was recovered. Mean intra-assay variances were 0.05, 0.05, and 0.06 log(10) copies/ml for the Versant, Amplicor, and NucliSens QT HIV-1 load assays, respectively (P, not significant). However, mean HIV-1 viral load was consistently reduced in dried samples by 0.32 to 0.51 log(10) copies/ml, depending on assay type (P < 0.05). Infectious HIV-1 was not recovered from dried ST plasma. There was no significant difference in HIV-1 viral load results obtained using ST after 8 weeks of storage at ambient temperature. Compared to frozen plasma, HIV-1 genotypic results were >99% concordant at the nucleotide and amino acid levels, as well as for resistance-associated mutations. We further demonstrated successful detection of multiple analytes, including HIV-1 viral load, HIV-1 antiretroviral resistance genotype, and HCV genotype, from a single ST unit. Dried plasma collected with ST yielded comparable results to frozen samples for multiple-analyte clinical testing. As such, ST could be a useful alternative for virologic tests and clinical trials worldwide by significantly diminishing transportation cost and the sample volume restrictions associated with dried-blood-spot technology.

Localization of West Nile Virus in monkey brain: double staining antigens immunohistochemically of neurons, neuroglia cells and West Nile Virus.

West Nile virus (WNV) can cause encephalitis or meningitis that affects brain tissue, which can also lead to permanent neurological damage that can be fatal. To our knowledge, no consistent double immunohistochemical staining of neurons, neuroglia cells, and WNV has yet been reported. To establish a method for performing double-label immunohistochemical detection of neurons, neuroglia cells and WNV, examining the pathological characteristics of WNV-infected neurons, neuroglia cells, and investigating distribution of WNV in monkey brain, paraffin-embedded monkey brain tissue were retrospectively studied by immunohistochemical staining of neurons, neuroglia cells and WNV. Antibodies against neuron-specific enolase (NSE), glial fibrillary acidic protein (GFAP) and WNV were used to develop the method of double-label immunohistochemical staining, which allowed independent assessment of neuron status and WNV distribution. A range of immunohistochemical WNV infection in monkey brain was observed in both neurons and neuroglia cells in terms of the thickness of lesion staining, and the WNV staining was slightly higher in neuroglia cells than in neurons. All these findings suggest that WNV invasion in the brain plays a crucial role in neurological damage by inducing central nervous system (CNS) cell dysfunction or cell death directly.

Impact of human immunodeficiency virus type 1 reverse transcriptase inhibitor drug resistance mutation interactions on phenotypic susceptibility.

The role specific reverse transcriptase (RT) drug resistance mutations play in influencing phenotypic susceptibility to RT inhibitors in virus strains with complex resistance interaction patterns was assessed using recombinant viruses that consisted of RT-PCR-amplified pol fragments derived from plasma HIV-1 RNA from two treatment-experienced patients. Specific modifications of key RT amino acids were performed by site-directed mutagenesis. A panel of viruses with defined genotypic resistance mutations was assessed for phenotypic drug resistance. Introduction of M184V into several different clones expressing various RT resistance mutations uniformly decreased susceptibility to abacavir, lamivudine, and didanosine, and increased susceptibility to zidovudine, stavudine, and tenofovir; replication capacity was decreased. The L74V mutation had similar but slightly different effects, contributing to decreased susceptibility to abacavir, lamivudine, and didanosine and increased susceptibility to zidovudine and tenofovir, but in contrast to M184V, L74V contributed to decreased susceptibility to stavudine. In virus strains with the nonnucleoside reverse transcriptase inhibitor (NNRTI) mutations K101E and G190S, the L74V mutation increased replication capacity, consistent with published observations, but replication capacity was decreased in strains without NNRTI resistance mutations. K101E and G190S together tend to decrease susceptibility to all nucleoside RT inhibitors, but the K103N mutation had little effect on nucleoside RT inhibitor susceptibility. Mutational interactions can have a substantial impact on drug resistance phenotype and replication capacity, and this has been exploited in clinical practice with the development of fixed-dose combination pills. However, we are the first to report these mutational interactions using molecularly cloned recombinant strains derived from viruses that occur naturally in HIV-infected individuals.

A critical role for CD63 in HIV replication and infection of macrophages and cell lines.

HIV infection typically involves interaction of Env with CD4 and a chemokine coreceptor, either CCR5 or CXCR4. Other cellular factors supporting HIV replication have also been characterized. We previously demonstrated a role for CD63 in early HIV infection events in macrophages via inhibition by anti-CD63 antibody pretreatment. To confirm the requirement for CD63 in HIV replication, we decreased CD63 expression using CD63-specific short interfering RNAs (siRNA), and showed inhibition of HIV replication in macrophages. Surprisingly, pretreatment with CD63 siRNA not only silenced CD63 expression by 90%, but also inhibited HIV-1 replication in a cultured cell line (U373-MAGI) which had been previously shown to be insensitive to CD63 monoclonal antibody inhibition. Although the anti-CD63 antibody was previously shown to inhibit early HIV infection events only in macrophages, we now show a potential role for CD63 in later HIV replication events in macrophages and cell lines. Further delineation of the role of CD63 in HIV replication may lead to development of novel therapeutic compounds.

Evolution of HIV resistance mutations in patients maintained on a stable treatment regimen after virologic failure.

OBJECTIVE: We compared the rate of emergence of thymidine analogue mutations (TAMs) and major protease inhibitor mutations in adherent patients who remained on stable treatment with a thymidine analogue and/or protease inhibitor after the onset of virologic failure. DESIGN: Follow-up genotypic resistance testing was done using archived plasma obtained from patients having 0 or 1 TAM and/or 0 or 1 major protease inhibitor resistance mutation at the onset of virologic failure. RESULTS: The median duration of observed failure was 691 days. There were 41 thymidine analogue regimens and 34 protease inhibitor regimens; concomitant ritonavir was used 4 times. New major protease inhibitor mutations emerged more rapidly than did new TAMs (P = 0.0019); new TAMs emerged more rapidly in thymidine analogue regimens that did not include lamivudine (P = 0.0073). The emergence of TAMs and major protease inhibitor mutations did not differ if lamivudine was not part of the thymidine analogue regimen. The evolution of CD4 cell counts and plasma viral loads (pVLs) during virologic failure was similar regardless of whether or not a new TAM or major protease inhibitor mutations emerged or, for thymidine analogue-containing regimens, whether lamivudine was or was not used. CONCLUSIONS: Major protease inhibitor mutations arose more frequently and rapidly than did TAMs in patients with sustained virologic failure who received lamivudine.

Delivery of double-stranded DNA thioaptamers into HIV-1 infected cells for antiviral activity.

Oligonucleotide agents (ODN) are emerging as attractive alternatives to chemical drugs. However, the clinical use of ODNs as therapeutics has been hindered by their susceptibility to degradation by cellular enzymes and their limited ability to penetrate intact cells. We have used various liposome-mediated transfection agents, for the in vitro delivery of DNA thioaptamers into U373-MAGI-CCR5 cells. Our lead thioaptamer, R12-2, targets the RNase H domain of the HIV-1 reverse transcriptase (RT) and inhibits viral infection in U373-MAGI-CCR5 cells. R12-2, a 62-base-pair, double-stranded DNA molecule with a monothio-phosphate modified backbone, was selected through a novel combinatorial selection method. We studied the use of oligofectamine (OF), TFX-20, Transmessenger (TM), and Gene Jammer (GJ) for transfection of the thio-modified DNA aptamers. OF-transfected U373-MAGI-CCR5 cells resulted in 68% inhibition of HIV infection in the treated cells compared to the untreated control. Inhibition was observed in a dose-dependent manner with maximal inhibition of 83%. In this report, we demonstrate that monothioate-modified DNA duplex oligonucleotides can be efficiently delivered into cells by liposome-based transfection agents to inhibit HIV replication.

Combinatorial selection, inhibition, and antiviral activity of DNA thioaptamers targeting the RNase H domain of HIV-1 reverse transcriptase.

Despite the key role played by the RNase H of human immunodeficiency virus-1 reverse transcriptase (HIV-1 RT) in viral proliferation, only a few inhibitors of RNase H have been reported. Using in vitro combinatorial selection methods and the RNase H domain of the HIV RT, we have selected double-stranded DNA thioaptamers (aptamers with selected thiophosphate backbone substitutions) that inhibit RNase H activity and viral replication. The selected thioaptamer sequences had a very high proportion of G residues. The consensus sequence for the selected thioaptamers showed G clusters separated by single residues at the 5'-end of the sequence. Gel electrophoresis mobility shift assays and nuclear magnetic resonance spectroscopy showed that the selected thioaptamer binds to the isolated RNase H domain, but did not bind to a structurally similar RNase H from Escherichia coli. The lead thioaptamer, R12-2, showed specific binding to HIV-1 RT with a binding constant (K(d)) of 70 nM. The thioaptamer inhibited the RNase H activity of intact HIV-1 RT. In cell culture, transfection of thioaptamer R12-2 (0.5 microg/mL) markedly inhibited viral production and exhibited a dose response of inhibition with R12-2 concentrations ranging from 0.03 to 2.0 microg/mL (IC(50) < 100 nM). Inhibition was also seen across a wide range of virus inoculum, ranging from a multiplicity of infection (moi) of 0.0005 to 0.05, with a reduction of the level of virus production by more than 50% at high moi. Suppression of virus was comparable to that seen with AZT when moi

Directed discovery of bivalent peptide ligands to an SH3 domain.

The Caenorhabditis elegans SEM-5 SH3 domains recognize proline-rich peptide segments with modest affinity. We developed a bivalent peptide ligand that contains a naturally occurring proline-rich binding sequence, tethered by a glycine linker to a disulfide-closed loop segment containing six variable residues. The glycine linker allows the loop segment to explore regions of greatest diversity in sequence and structure of the SH3 domain: the RT and n-Src loops. The bivalent ligand was optimized using phage display, leading to a peptide (PP-G(4)-L) with 1000-fold increased affinity for the SEM-5 C-terminal SH3 domain over that of a natural ligand. NMR analysis of the complex confirms that the peptide loop segment is targeted to the RT and n-Src loops and parts of the beta-sheet scaffold of this SH3 domain. This binding region is comparable to that targeted by a natural non-PXXP peptide to the p67(phox) SH3 domain, a region not known to be targeted in the Grb2 SH3 domain family. PP-G(4)-L may aid in the discovery of additional binding partners of Grb2 family SH3 domains.