Characterization of Bombyx mori nucleopolyhedrovirus with a knockout of Bm17

Open reading frame 17 (Bm17) gene of Bombyx mori nucleopolyhedrovirus is a highly conserved gene in lepidopteran nucleopolyhedroviruses, but its function remains unknown. In this report, transient-expression and superinfection assays indicated that BM17 localized in the nucleus and cytoplasm of infected BmN cells. To determine the role of Bm17 in baculovirus life cycle, we constructed a Bm17 knockout virus and characterized its properties in cells. Analysis of the production and infection of budded virions, the level of viral DNA replication revealed showed that there was no significant difference among the mutant, the control, and the Bm17 repaired virus strains. These results suggest that BM17 is not essential for virus replication in cultured cells.     

Suppression of HIV-1 protease inhibitor resistance by phosphonate-mediated solvent anchoring

The introduction of human immunodeficiency virus type 1 (HIV-1) protease inhibitors (PIs) markedly improved the clinical outcome and control of HIV-1 infection. However, cross-resistance among PIs due to a wide spectrum of mutations in viral protease is a major factor limiting their broader clinical use. Here we report on the suppression of PI resistance using a covalent attachment of a phosphonic acid motif to a peptidomimetic inhibitor scaffold. The resulting phosphonate analogs maintain high binding affinity to HIV-1 protease, potent antiretroviral activity, and unlike the parent molecules, display no loss of potency against a panel of clinically important PI-resistant HIV-1 strains. As shown by crystallographic analysis, the phosphonate moiety is highly exposed to solvent with no discernable interactions with any of the enzyme active site or surface residues. We term this effect "solvent anchoring" and demonstrate that it is driven by a favorable change in the inhibitor binding entropy upon the interaction with mutant enzymes. This type of thermodynamic behavior, which was not found with the parent scaffold fully buried in the enzyme active site, is a result of the increased degeneracy of inhibitor binding states, allowing effective molecular adaptation to the expanded cavity volume of mutant proteases. This strategy, which is applicable to various PI scaffolds, should facilitate the design of novel PIs and potentially other antiviral therapeutics.     

The role of <Emphasis Type="Italic">N</Emphasis>-glycans of HIV-1 gp41 in virus infectivity and susceptibility to the suppressive effects of carbohydrate-binding agents

Background

Carbohydrate-binding agents (CBAs) are potent antiretroviral compounds that target the N-glycans on the HIV-1 envelope glycoproteins. The development of phenotypic resistance to CBAs by the virus is accompanied by the deletion of multiple N-linked glycans of the surface envelope glycoprotein gp120. Recently, also an N-glycan on the transmembrane envelope glycoprotein gp41 was shown to be deleted during CBA resistance development.

Results

We generated HIV-1 mutants lacking gp41 N-glycans and determined the influence of these glycan deletions on the viral phenotype (infectivity, CD4 binding, envelope glycoprotein incorporation in the viral particle and on the transfected cell, virus capture by DC-SIGN⁺ cells and transmission of DC-SIGN-captured virions to CD4⁺ T-lymphocytes) and on the phenotypic susceptibility of HIV-1 to a selection of CBAs. It was shown that some gp41 N-glycans are crucial for the infectivity of the virus. In particular, lack of an intact N616 glycosylation site was shown to result in the loss of viral infectivity of several (i.e. the X4-tropic III_B and NL4.3 strains, and the X4/R5-tropic HE strain), but not all (i.e. the R5-tropic ADA strain) studied HIV-1 strains. In accordance, we found that the gp120 levels in the envelope of N616Q mutant gp41 strains NL4.3, III_B and HE were severely decreased. In contrast, N616Q gp41 mutant HIV-1_ADA contained gp120 levels similar to the gp120 levels in WT HIV-1_ADA virus. Concomitantly deleting multiple gp41 N-glycans was often highly detrimental for viral infectivity. Using surface plasmon resonance technology we showed that CBAs have a pronounced affinity for both gp120 and gp41. However, the antiviral activity of CBAs is not dependent on the concomitant presence of all gp41 glycans. Single gp41 glycan deletions had no marked effects on CBA susceptibility, whereas some combinations of two to three gp41 glycan-deletions had a minor effect on CBA activity.

Conclusions

We revealed the importance of some gp41 N-linked glycans, in particular the N616 glycan which was shown to be absolutely indispensable for the infectivity potential of several virus strains. In addition, we demonstrated that the deletion of up to three gp41 N-linked glycans only slightly affected CBA susceptibility.

     

Accumulation and activation of natural killer cells in local intraperitoneal HIV-1/MuLV infection results in early control of virus infected cells

Natural killer (NK) cells are important effectors in resistance to viral infections. The role of NK cells in the acute response to human immunodeficiency virus 1 (HIV-1) infected cells was investigated in a mouse model based on a HIV-1/murine leukemia virus (MuLV) pseudovirus. Splenocytes infected with HIV-1/MuLV were injected intraperitoneally and local immunologic responses and persistence of infected cells were investigated. In vivo depletion with an anti-NK1.1 antibody showed that NK cells are important in resistance to virus infected cells. Moreover, NK cell frequency in the peritoneal cavity increased in response to infected cells and these NK cells had a more mature phenotype, as determined by CD27 and Mac-1 expression. Interestingly, after injection of HIV-1/MuLV infected cells, but not MuLV infected cells, peritoneal NK cells had an increased cytotoxic activity. In conclusion, NK cells play a role in the early control of HIV-1/MuLV infected cells in vivo.     

RNA interference as a tool for exploring HIV-1 robustness

Short interfering RNAs (siRNAs) that target viral genes can efficiently inhibit human immunodeficiency virus type 1 (HIV-1) replication. Nevertheless, there is the potential for viral escape, particularly with a highly mutable target such as HIV-1. We present a novel strategy for anticipating and preventing viral escape using second-generation siRNAs. The evolutionary capacity of HIV-1 was tested by exerting strong selective pressure on a highly conserved sequence in the HIV-1 genome. We assayed the antiviral efficacy of five overlapping siRNAs directed against an essential region of the HIV-1 protease. Serial viral transfers in U87-CD4-CXCR4 cells were performed using four of the siRNAs. This procedure was repeated until virus breakthrough was detected. After several serial culture passages, resistant virus with a single point mutation in the targeted region was detected in the culture supernatants. The emergence of resistant virus was confirmed by molecular cloning and DNA sequencing of viral RNA. The most common escape route was the D30N mutation. Importantly, the addition of a second-generation siRNA that matched the D30N mutation restored viral inhibition and delayed development of escape variants. Passages performed with both siRNAs prevented the emergence of the D30N escape mutant and forced the virus to develop new escape routes. Thus, second-generation siRNAs can be used to block escape from RNA interference (RNAi) and to search for new RNAi escape routes. The protocol described here may be useful for exploring the sequence space available for HIV-1 evolution and for producing attenuated or deleterious viruses.     

Requirement of human immunodeficiency virus type 1 nef for in vivo replication and pathogenicity.

The role of human immunodeficiency virus type 1 (HIV-1) accessory genes in pathogenesis has remained unclear because of the lack of a suitable in vivo model. The most controversial of these genes is nef. We investigated the requirement for Nef for in vivo replication and pathogenicity of two isolates of HIV-1 (HIV-1JR-CSF and HIV-1NL4-3) in human fetal thymus and liver implants in severe combined immunodeficient mice. HIV-1JR-CSF and HIV-1NL4-3 differ in their in vitro phenotypes in that HIV-1JR-CSF does not induce syncytia and is relatively noncytopathic, while HIV-1NL4-3 is highly cytopathic and readily induces syncytia. The nef mutants of both isolates grew with kinetics similar to those of parental virus strains in stimulated peripheral blood lymphocytes but demonstrated attenuated growth properties in vivo. HIV-1NL4-3 induced severe depletion of human thymocytes within 6 weeks of infection, whereas its nef mutant did not. Thus, HIV-1 Nef is required for efficient in vivo viral replication and pathogenicity.     

Hepatitis C Virus Resistance to Carbohydrate-Binding Agents

Carbohydrate binding agents (CBAs), including natural lectins, are more and more considered as broad-spectrum antivirals. These molecules are able to directly inhibit many viruses such as Human Immunodeficiency Virus (HIV), Hepatitis C Virus (HCV), Dengue Virus, Ebola Virus or Severe Acute Respiratory Syndrome Coronavirus through binding to envelope protein N-glycans. In the case of HIV, it has been shown that CBAs select for mutant viruses with N-glycosylation site deletions which are more sensitive to neutralizing antibodies. In this study we aimed at evaluating the HCV resistance to CBAs in vitro. HCV was cultivated in the presence of increasing Galanthus nivalis agglutinin (GNA), Cyanovirin-N, Concanavalin-A or Griffithsin concentrations, during more than eight weeks. At the end of lectin exposure, the genome of the isolated strains was sequenced and several potential resistance mutations in the E1E2 envelope glycoproteins were identified. The effect of these mutations on viral fitness as well as on sensitivity to inhibition by lectins, soluble CD81 or the 3/11 neutralizing antibody was assessed. Surprisingly, none of these mutations, alone or in combination, conferred resistance to CBAs. In contrast, we observed that some mutants were more sensitive to 3/11 or CD81-LEL inhibition. Additionally, several mutations were identified in the Core and the non-structural proteins. Thus, our results suggest that in contrast to HIV, HCV resistance to CBAs is not directly conferred by mutations in the envelope protein genes but could occur through an indirect mechanism involving mutations in other viral proteins. Further investigations are needed to completely elucidate the underlying mechanisms.     

High mannose-specific lectin (KAA-2) from the red alga Kappaphycus alvarezii potently inhibits influenza virus infection in a strain-independent manner

The carbohydrate binding profile of the red algal lectin KAA-2 from Kappaphycus alvarezii was evaluated by a centrifugal ultrafiltration–HPLC method using pyridylaminated oligosaccharides. KAA-2 bound exclusively to high mannose type N-glycans, but not to other glycans such as complex type, hybrid type, or the pentasaccharide core of N-glycans. This lectin exhibited a preference for an exposed α1–3 Man on a D2 arm in a similar manner to Eucheuma serra agglutinin (ESA-2), which shows various biological activities, such as anti-HIV and anti-carcinogenic activity. We tested the anti-influenza virus activity of KAA-2 against various strains including the recent pandemic H1N1-2009 influenza virus. KAA-2 inhibited infection of various influenza strains with EC₅₀s of low nanomolar levels. Immunofluorescence microscopy using an anti-influenza antibody demonstrated that the antiviral activity of KAA-2 was exerted by interference with virus entry into host cells. This mechanism was further confirmed by the evidence of direct binding of KAA-2 to a viral envelope protein, hemagglutinin (HA), using an ELISA assay. These results indicate that this lectin would be useful as a novel antiviral reagent for the prevention of infection.     

HIV-1 Nef Dimerization Is Required for Nef-Mediated Receptor Downregulation and Viral Replication

Nef, a human immunodeficiency virus type 1 (HIV-1) accessory factor capable of interaction with a diverse array of host cell signaling molecules, is essential for high-titer HIV replication and AIDS progression. Previous biochemical and structural studies have suggested that Nef may form homodimers and higher-order oligomers in HIV-infected cells, which may be required for both immune and viral receptor downregulation as well as viral replication. Using bimolecular fluorescence complementation, we provide the first direct evidence for Nef dimers within HIV host cells and identify the structural requirements for dimerization in vivo. Bimolecular fluorescence complementation analysis shows that the multiple hydrophobic and electrostatic interactions found within the dimerization interface of the Nef X-ray crystal structure are essential for dimerization in cells. Nef dimers localized to the plasma membrane as well as the trans-Golgi network, two subcellular localizations essential for Nef function. Mutations in the Nef dimerization interface dramatically reduced both Nef-induced CD4 downregulation and HIV replication. Viruses expressing dimerization-defective Nef mutants were disabled to the same extent as HIV that fails to express Nef in terms of replication. These results identify the Nef dimerization region as a potential molecular target for antiretroviral drug discovery.     

Abrogation of in vitro suppression of human immunodeficiency virus type 1 (HIV-1) replication mediated by CD8+ T lymphocytes of asymptomatic HIV-1 carriers by staphylococcal enterotoxin B and phorbol esters through induction of tumor necrosis factor alpha.

CD8+ T lymphocytes of asymptomatic human immunodeficiency virus type 1 (HIV-1) carriers (AC) suppress HIV-1 replication in vitro. Failure of host defense mechanisms and increased virus proliferation are associated with disease progression. The exact mechanisms inducing these changes at the advanced stage of the disease are still obscure. In this study, we searched for experimental conditions favoring the abrogation of the suppression of viral replication in peripheral blood mononuclear cells (PBMC) of AC by using various pharmacological and biological probes modifying cell activation. Among such agents, staphylococcal enterotoxin B (SEB) and phorbol 12-myristate 13-acetate (PMA) markedly increased otherwise low levels of HIV-1 replication in cultures of phytohemagglutinin-stimulated AC PBMC following in vitro HIV-1 LAI infection. A similar but less pronounced virus induction was also observed in macrophage-tropic HIV-1. Individual pretreatment of CD4+ and CD8+ PBMC fractions with these agents caused a reduction in CD8+ cell proliferation and enhanced HIV-1 replication in CD4+ cells. SEB- and PMA-mediated augmentation of HIV-1 replication in AC PBMC was significantly blocked by neutralizing antibody to tumor necrosis factor-alpha (TNF-alpha), although recombinant TNF-alpha alone failed to reproduce the effects of SEB or PMA. Our results suggest that the induction of TNF-alpha may be one of the mechanisms that overcomes the CD8+-induced suppression of HIV-1 replication in AC and that it may induce HIV-1 replication.     

Early therapy of vertical human immunodeficiency virus type 1 (HIV-1) infection: control of viral replication and absence of persistent HIV-1-specific immune responses

Studies of potent antiretroviral combination regimens were undertaken in young infants to evaluate the potential for long-term suppression of viral replication and to evaluate the immune consequences of such therapies. Early combination antiretroviral therapy led to a loss of plasma viremia, cultivable virus, and labile extrachromosomal replication intermediates. Despite preservation of immune function, persistent human immunodeficiency type 1 (HIV-1)-specific immune responses were not detected in most infants. The absence of detectable, persisting immune responses in most HIV-1-infected infants treated early contrasts with what is typically seen in adults who are treated early. These results are consistent with the notion that early combination antiretroviral therapy of HIV-1-infected infants allows the long-term suppression of viral replication.