Tetraspanin CD37 Regulates ß2 Integrin-Mediated Adhesion and Migration in Neutrophils.

Deciphering the molecular basis of leukocyte recruitment is critical to the understanding of inflammation. In this study, we investigated the contribution of the tetraspanin CD37 to this key process. CD37-deficient mice showed impaired neutrophil recruitment in a peritonitis model. Intravital microscopic analysis indicated that the absence of CD37 impaired the capacity of leukocytes to follow a CXCL1 chemotactic gradient accurately in the interstitium. Moreover, analysis of CXCL1-induced leukocyte-endothelial cell interactions in postcapillary venules revealed that CXCL1-induced neutrophil adhesion and transmigration were reduced in the absence of CD37, consistent with a reduced capacity to undergo ß2 integrin-dependent adhesion. This result was supported by in vitro flow chamber experiments that demonstrated an impairment in adhesion of CD37-deficient neutrophils to the ß2 integrin ligand, ICAM-1, despite the normal display of high-affinity ß2 integrins. Superresolution microscopic assessment of localization of CD37 and CD18 in ICAM-1-adherent neutrophils demonstrated that these molecules do not significantly cocluster in the cell membrane, arguing against the possibility that CD37 regulates ß2 integrin function via a direct molecular interaction. Moreover, CD37 ablation did not affect ß2 integrin clustering. In contrast, the absence of CD37 in neutrophils impaired actin polymerization, cell spreading and polarization, dysregulated Rac-1 activation, and accelerated ß2 integrin internalization. Together, these data indicate that CD37 promotes neutrophil adhesion and recruitment via the promotion of cytoskeletal function downstream of integrin-mediated adhesion.

Monocytes from HIV-infected individuals show impaired cholesterol efflux and increased foam cell formation after transendothelial migration.

DESIGN: HIV-infected (HIV+) individuals have an increased risk of atherosclerosis and cardiovascular disease which is independent of antiretroviral therapy and traditional risk factors. Monocytes play a central role in the development of atherosclerosis, and HIV-related chronic inflammation and monocyte activation may contribute to increased atherosclerosis, but the mechanisms are unknown. METHODS: Using an in-vitro model of atherosclerotic plaque formation, we measured the transendothelial migration of purified monocytes from age-matched HIV+ and uninfected donors and examined their differentiation into foam cells. Cholesterol efflux and the expression of cholesterol metabolism genes were also assessed. RESULTS: Monocytes from HIV+ individuals showed increased foam cell formation compared with controls (18.9 vs. 0%, respectively, P = 0.004) and serum from virologically suppressed HIV+ individuals potentiated foam cell formation by monocytes from both uninfected and HIV+ donors. Plasma tumour necrosis factor (TNF) levels were increased in HIV+ vs. control donors (5.9 vs. 3.5 pg/ml, P = 0.02) and foam cell formation was inhibited by blocking antibodies to TNF receptors, suggesting a direct effect on monocyte differentiation to foam cells. Monocytes from virologically suppressed HIV+ donors showed impaired cholesterol efflux and decreased expression of key genes regulating cholesterol metabolism, including the cholesterol transporter ABCA1 (P = 0.02). CONCLUSION: Monocytes from HIV+ individuals show impaired cholesterol efflux and are primed for foam cell formation following transendothelial migration. Factors present in HIV+ serum, including elevated TNF levels, further enhance foam cell formation. The proatherogenic phenotype of monocytes persists in virologically suppressed HIV+ individuals and may contribute mechanistically to increased atherosclerosis in this population.

Properties of HIV-1 associated cholesterol in addition to raft formation are important for virus infection.

The overall HIV-1 membrane lipid contents resemble lipid rafts, and we have previously demonstrated that raft-promoting properties of virus-associated cholesterol (with modifications in either the 3ß-OH group or AB rings) are important for HIV-1 infectivity. As cholesterol is present in both rafts and non-rafts domains of HIV-1 membrane, we question whether the interpretation of rafts property of virus-associated cholesterol being an absolute requirement for HIV-1 function is too simplistic. The carbon side chain of cholesterol is the third component of cholesterol that can affect the fluidity of membrane depending on its context within the lipid membrane bilayers. In this work, we have used synthetic cholesterol analogues that have different lengths of carbon side chain for our investigation. In contrast to our previous report, we have found that cholesterol side chain analogues that lack in vitro defined raft promoting-property is able to support HIV-1 replication. More specifically, cholesterol analogues with side chains of intermediate length have greater capacity to support HIV-1 infection, suggesting HIV-1 is able to maintain function using cholesterol variants that promote a range of non-rafts- to rafts-properties. Our data demonstrate cholesterol properties other than raft-promoting function also contribute to the infectivity of HIV-1.

Visualising single molecules of HIV-1 and miRNA nucleic acids.

BACKGROUND: The scarcity of certain nucleic acid species and the small size of target sequences such as miRNA, impose a significant barrier to subcellular visualization and present a major challenge to cell biologists. Here, we offer a generic and highly sensitive visualization approach (oligo fluorescent in situ hybridization, O-FISH) that can be used to detect such nucleic acids using a single-oligonucleotide probe of 19-26 nucleotides in length. RESULTS: We used O-FISH to visualize miR146a in human and avian cells. Furthermore, we reveal the sensitivity of O-FISH detection by using a HIV-1 model system to show that as little as 1-2 copies of nucleic acids can be detected in a single cell. We were able to discern newly synthesized viral cDNA and, moreover, observed that certain HIV RNA sequences are only transiently available for O-FISH detection. CONCLUSIONS: Taken together, these results suggest that the O-FISH method can potentially be used for in situ probing of, as few as, 1-2 copies of nucleic acid and, additionally, to visualize small RNA such as miRNA. We further propose that the O-FISH method could be extended to understand viral function by probing newly transcribed viral intermediates; and discern the localisation of nucleic acids of interest. Additionally, interrogating the conformation and structure of a particular nucleic acid in situ might also be possible, based on the accessibility of a target sequence.

HIV taken by STORM: super-resolution fluorescence microscopy of a viral infection.

BACKGROUND: The visualization of viral proteins has been hindered by the resolution limit of conventional fluorescent microscopes, as the dimension of any single fluorescent signal is often greater than most virion particles. Super-resolution microscopy has the potential to unveil the distribution of proteins at the resolution approaching electron microscopy without relying on morphological features of existing characteristics of the biological specimen that are needed in EM. RESULTS: Using direct stochastic optical reconstruction microscopy (dSTORM) to achieve a lateral resolution of 15-20 nm, we quantified the 2-D molecular distribution of the major structural proteins of the infectious human immunodeficiency virus type 1 (HIV-1) before and after infection of lymphoid cells. We determined that the HIV-1 matrix and capsid proteins undergo restructuring soon after HIV-1 infection. CONCLUSIONS: This study provides the proof-of-concept for the use of dSTORM to visualize the changes in the molecular distribution of viral proteins during an infection.

Early events of HIV-1 infection: can signaling be the next therapeutic target?

Intracellular signaling events are signposts of biological processes, which govern the direction and action of biological activities. Through millions of years of evolution, pathogens, such as viruses, have evolved to hijack host cell machinery to infect their targets and are therefore dependent on host cell signaling for replication. This review will detail our current understanding of the signaling events that are important for the early steps of HIV-1 replication. More specifically, the therapeutic potential of signaling events associated with chemokine coreceptors, virus entry, viral synapses, and post-entry processes will be discussed. We argue that these pathways may represent novel targets for antiviral therapy.

Labeling of multiple HIV-1 proteins with the biarsenical-tetracysteine system.

Due to its small size and versatility, the biarsenical-tetracysteine system is an attractive way to label viral proteins for live cell imaging. This study describes the genetic labeling of the human immunodeficiency virus type 1 (HIV-1) structural proteins (matrix, capsid and nucleocapsid), enzymes (protease, reverse transcriptase, RNAse H and integrase) and envelope glycoprotein 120 with a tetracysteine tag in the context of a full-length virus. We measure the impact of these modifications on the natural virus infection and, most importantly, present the first infectious HIV-1 construct containing a fluorescently-labeled nucleocapsid protein. Furthermore, due to the high background levels normally associated with the labeling of tetracysteine-tagged proteins we have also optimized a metabolic labeling system that produces infectious virus containing the natural envelope glycoproteins and specifically labeled tetracysteine-tagged proteins that can easily be detected after virus infection of T-lymphocytes. This approach can be adapted to other viral systems for the visualization of the interplay between virus and host cell during infection.

Coinfection of hepatic cell lines with human immunodeficiency virus and hepatitis B virus leads to an increase in intracellular hepatitis B surface antigen.

Liver-related mortality is increased in the setting of HIV-hepatitis B virus (HBV) coinfection. However, interactions between HIV and HBV to explain this observation have not been described. We hypothesized that HIV infection of hepatocytes directly affects the life cycle of HBV. We infected human hepatic cell lines expressing HBV (Hep3B and AD38 cells) or not expressing HBV (Huh7, HepG2, and AD43 cells) with laboratory strains of HIV (NL4-3 and AD8), as well as a vesicular stomatitis virus (VSV)-pseudotyped HIV expressing enhanced green fluorescent protein (EGFP). Following HIV infection with NL4-3 or AD8 in hepatic cell lines, we observed a significant increase in HIV reverse transcriptase activity which was infectious. Despite no detection of surface CD4, CCR5, and CXCR4 by flow cytometry, AD8 infection of AD38 cells was inhibited by maraviroc and NL4-3 was inhibited by AMD3100, demonstrating that HIV enters AD38 hepatic cell lines via CCR5 or CXCR4. High-level infection of AD38 cells (50%) was achieved using VSV-pseudotyped HIV. Coinfection of the AD38 cell line with HIV did not alter the HBV DNA amount or species as determined by Southern blotting or nucleic acid signal amplification. However, coinfection with HIV was associated with a significant increase in intracellular HBsAg when measured by Western blotting, quantitative HBsAg, and fluorescence microscopy. We conclude that HIV infection of HBV-infected hepatic cell lines significantly increased intracellular HBsAg but not HBV DNA synthesis and that increased intrahepatic HBsAg secondary to direct infection by HIV may contribute to accelerated liver disease in HIV-HBV-coinfected individuals.

In vivo targeting of DC-SIGN-positive antigen-presenting cells in a nonhuman primate model.

In vivo targeting of antigen-presenting cells (APCs) with antigens coupled to antibodies directed against APC-specific endocytic receptors is a simple and a promising approach to induce or modulate immune responses against those antigens. In a recent in vitro study, we have shown that targeting of APCs with an antigen coupled to an antibody directed against the endocytic receptor DC-SIGN effectively induces a specific immune response against that antigen. The aim of the present study was to determine the ability of the murine antihuman DC-SIGN antibody AZN-D1 to target APCs in a cynomolgus macaque model after its administration in vivo. Immunohistochemical analysis demonstrated that macaques injected intravenously with AZN-D1 have AZN-D1-targeted APCs in all lymph nodes (LNs) tested and in the liver. DC-SIGN-positive cells were mainly located in the medullary sinuses of the LNs and in the hepatic sinusoids in the liver. No unlabeled DC-SIGN molecules were found in the LN of AZN-D1-injected macaques. Morphologic criteria and staining of sequential LN sections with a panel of antibodies indicated that the DC-SIGN-targeted cells belong to the myeloid lineage of APCs. In conclusion, this is the first study that shows specific targeting of APCs in vivo by using antibodies directed against DC-SIGN.

Spectrum of antiviral activity of o-(acetoxyphenyl)hept-2-ynyl sulphide (APHS).

Since some antiviral drugs have a broad spectrum of action, the aim of this study was to assess whether o-(acetoxyphenyl)hept-2-ynyl sulphide (APHS), a recently described inhibitor of human immunodeficiency virus type 1 (HIV-1) replication, has an effect on the replication of other retroviruses, (-) and (+) RNA viruses and DNA viruses. APHS did not affect the replication of feline immunodeficiency virus, HIV-2 and a HIV-1 strain resistant to non-nucleoside reverse transcriptase inhibitors (NNRTI). APHS could also not inhibit the replication of the RNA viruses, respiratory syncytium virus or mouse hepatitis virus. In contrast, APHS did inhibit the replication of wild-type herpes simplex virus type 1 (HSV-1) as well as acyclovir-resistant HSV-1 and HSV-2 mutant. These results suggest that APHS is a NNRTI of HIV-1 replication, but not HIV-2 replication, and that APHS is an inhibitor of both HSV-1 and HSV-2 replication.

Anti-HIV drug development--an overview.

Highly active antiretroviral therapy (HAART) has markedly decreased mortality and morbidity in the developed world. HAART consists of a combination of three or more of the following classes of antiretroviral (ARV) drug: reverse transcriptase inhibitors, protease inhibitors and a recently approved fusion inhibitor. However, HAART cannot completely eradicate HIV from the body, results in long-term toxicity and eventually leads to the emergence of drug-resistant HIV strains. These problems prompt the search for potent new drugs that are active against drug-resistant viral strains and that can safely be combined with other ARV drugs. The aim of this review was to give an overview of new compounds in preclinical or early clinical development that interact with various steps in the HIV life cycle: virus-cell attachment; gp120-CD4 binding; gp120-coreceptor binding; viral fusion; viral assembly and disassembly; reverse transcription; nuclear import of the pre-integration complex; proviral integration; viral transcription; processing of viral transcripts and nuclear export; assembly of new virions; cellular factors involved in HIV replication.

Aspirin-like molecules that inhibit human immunodeficiency virus 1 replication.

Some anti-inflammatory molecules are also known to possess anti-human immunodeficiency virus (HIV) activity. We found that o-(acetoxyphenyl)hept-2-ynyl sulfide (APHS), a recently synthesized non-steroidal anti-inflammatory molecule can inhibit HIV-1 replication. The aim of this study was to clarify the mechanism of action of APHS. When administered during the first steps of the infection, APHS was capable of inhibiting the replication of several HIV-1 strains (macrophage-tropic and/or lymphocytotropic) in a dose-dependent manner in both peripheral blood mononuclear cells (PBMC), monocyte-derived macrophages and peripheral blood lymphocytes with 50% inhibitory concentration values of approximately 10 microM. The 50% toxic concentration of APHS varied between 100 and 200 microM in the different primary cells tested. APHS did not affect HIV-1 replication once the provirus was already inserted into the cellular genome. APHS also did not inhibit HIV-1 entry into the host cells as determined by quantification of gag RNA inside PBMC 2h after infection. However, APHS did inhibit gag DNA synthesis during reverse transcription in primary cells, which indicates that APHS may target the reverse transcription process.

APHS can act synergically with clinically available HIV-1 reverse transcriptase and protease inhibitors and is active against several drug-resistant HIV-1 strains in vitro.

OBJECTIVES: The use of multiple drug combinations in current anti-human immunodeficiency virus (HIV) therapy allows lower dosages of individual drugs and results in enhancement of the therapeutic effect due to synergic interactions between different drugs. We have shown that o-(acetoxyphenyl)hept-2-ynyl sulphide (APHS), a recently developed non-steroidal anti-inflammatory drug, shows anti-HIV activity in a dose-dependent manner. The first aim of this study was to investigate whether APHS can act synergically with the clinically available reverse transcriptase and protease inhibitors (RTIs and PIs, respectively) in vitro. Because of the increasing prevalence of RTI- and PI-resistant HIV-1 strains, the second aim of this study was to assess the antiviral activity of APHS against drug-resistant HIV-1 strains in vitro. MATERIALS AND METHODS: HIV-infected peripheral blood mononuclear cells (PBMC) were treated for 7 days with different combinations of APHS and RTIs or PIs. The MT-2 cell line was infected with different HIV-1 strains and treated with APHS for 5 days. RESULTS: APHS showed synergic interactions with the RTIs zidovudine, lamivudine and efavirenz and with the PIs indinavir and ritonavir. The 50% inhibitory concentration (IC50) of APHS in this assay dropped from 13 microM when used alone, to 5 micro M after combination with an RTI or PI. In combination with APHS the IC50 of the RTI and PI drugs tested also dropped. APHS inhibits the replication of HIV-1 strains resistant to zidovudine, lamivudine, stavudine, didanosine, zalcitabine and ritonavir. CONCLUSIONS: These results indicate that APHS can be combined with RTIs and PIs and can inhibit several NRTI and PI-resistant HIV-1 strains.