A broad antiviral neutral glycolipid, fattiviracin FV-8, is a membrane fluidity modulator

To screen for an effective antiviral compound which acts as a membrane fluidity modulator, dichotomous effects on human immunodeficiency virus type 1 (HIV-1) infection due to different treatments of several glycolipids and lipids were examined. Continuous treatment of infected cells with 40 microg ml(-1) fattiviracin FV-8, a neutral glycolipid isolated from Streptomycetes, inhibited HIV-1 infection by 96%, whereas pretreatment with 400 microg ml(-1) enhanced infectivity 4.7-fold. The glycolipid showed similar effects as glycyrrhizin; it inhibited infection by broad enveloped viruses, blocked cell-cell fusion, reduced the infectivity of treated virions and enhanced susceptibility to viral infection and cell-cell fusion of cells pretreated with high doses of the compound. Suppression and enhancement was correlated with decreased and increased fluidity of plasma membrane of the fattiviracin FV-8-treated cells. Restricted movement of membrane molecules might impede the formation of a wide fusion pore, and therefore be critical to the entry of viruses. Thus, this can be applied as a new strategy to inhibit viral infections.     

Biosynthesis of fattiviracin FV-8, an antiviral agent

Streptomyces microflavus strain No. 2445 produces many derivatives of fattiviracin antibiotics. The major product of these derivatives is fattiviracin FV-8, which consists of four glucose and two trihydroxy fatty acid residues. We found that this strain has the ability to convert several sugars in the culture medium to glucose, and the glucose added to the medium is directly incorporated into the FV-8 molecule. Two trihydroxy fatty acid residues in the FV-8 molecule are derived from acetic acid, and production of FV-8 is inhibited by the addition of cerulenin, which is an inhibitor of fatty acid biosynthesis.     

The enantiomers of carbocyclic 5'-norguanosine: activity towards Epstein-Barr virus

(-)-5'-noraristeromycin (1) has shown antiviral activity towards, particularly cytomegalovirus, vaccinia virus and measles while its (+)-enantiomer (2) is effective towards hepatitis B virus. To determine if the antiviral characteristics of 1 and 2 extended to the guanine analogues (3 and 4), these enantiomers were prepared and evaluated against herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2), cytomegalovirus (CMV), varicella zoster virus (VZV), Epstein-Barr virus (EBV), human herpes virus type 6 (HHV-6), human herpes virus type 8 (HHV-8), vaccinia virus (VV), cowpox virus (CV), vesicular stomatitis virus (VSV), respiratory syncytial virus (RSV), hepatitis B virus (HBV), and human immunodeficiency virus type 1 (HIV-1) and type 2 (HIV-2). The only activity found for 3 was for Epstein-Barr virus in VCA Elisa (EC50 0.78 microg/mL), immunofluorescence assay for VCA or gp 350/250 (1.8-4.0 microg/mL) and DNA hybridization (EC50 0.82 microg/mL) assays with no accompanying toxicity seen in the host Daudi cells. No activity was noted for 4.     

Broad-Spectrum Inhibition of Retroviral and Filoviral Particle Release by Tetherin

The expression of many putative antiviral genes is upregulated when cells encounter type I interferon (IFN), but the actual mechanisms by which many IFN-induced gene products inhibit virus replication are poorly understood. A recently identified IFN-induced antiretroviral protein, termed tetherin (previously known as BST-2 or CD317), blocks the release of nascent human immunodeficiency virus type 1 (HIV-1) particles from infected cells, and an HIV-1 accessory protein, Vpu, acts as a viral antagonist of tetherin. Here, we show that tetherin is capable of blocking not only the release of HIV-1 particles but also the release of particles assembled using the major structural proteins of a variety of prototype retroviruses, including members of the alpharetrovirus, betaretrovirus, deltaretrovirus, lentivirus, and spumaretrovirus families. Moreover, we show that the release of particles assembled using filovirus matrix proteins from Marburg virus and Ebola virus is also sensitive to inhibition by tetherin. These findings indicate that tetherin is a broadly specific inhibitor of enveloped particle release, and therefore, inhibition is unlikely to require specific interactions with viral proteins. Nonetheless, tetherin colocalized with nascent virus-like particles generated by several retroviral and filoviral structural proteins, indicating that it is present at, or recruited to, sites of particle assembly. Overall, tetherin is potentially active against many enveloped viruses and likely to be an important component of the antiviral innate immune defense.     

Initial HIV-1 antigen-specific CD8+ T cells in acute HIV-1 infection inhibit transmitted/founder virus replication

CD8-mediated virus inhibition can be detected in HIV-1-positive subjects who naturally control virus replication. Characterizing the inhibitory function of CD8(+) T cells during acute HIV-1 infection (AHI) can elucidate the nature of the CD8(+) responses that can be rapidly elicited and that contribute to virus control. We examined the timing and HIV-1 antigen specificity of antiviral CD8(+) T cells during AHI. Autologous and heterologous CD8(+) T cell antiviral functions were assessed longitudinally during AHI in five donors from the CHAVI 001 cohort using a CD8(+) T cell-mediated virus inhibition assay (CD8 VIA) and transmitted/founder (T/F) viruses. Potent CD8(+) antiviral responses against heterologous T/F viruses appeared during AHI at the first time point sampled in each of the 5 donors (Fiebig stages 1/2 to 5). Inhibition of an autologous T/F virus was durable to 48 weeks; however, inhibition of heterologous responses declined concurrent with the resolution of viremia. HIV-1 viruses from 6 months postinfection were more resistant to CD8(+)-mediated virus inhibition than cognate T/F viruses, demonstrating that the virus escapes early from CD8(+) T cell-mediated inhibition of virus replication. CD8(+) T cell antigen-specific subsets mediated inhibition of T/F virus replication via soluble components, and these soluble responses were stimulated by peptide pools that include epitopes that were shown to drive HIV-1 escape during AHI. These data provide insights into the mechanisms of CD8-mediated virus inhibition and suggest that functional analyses will be important for determining whether similar antigen-specific virus inhibition can be induced by T cell-directed vaccine strategies.     

Human immunodeficiency virus type 1 escapes from RNA interference-mediated inhibition

Short-term assays have suggested that RNA interference (RNAi) may be a powerful new method for intracellular immunization against human immunodeficiency virus type 1 (HIV-1) infection. However, RNAi has not yet been shown to protect cells against HIV-1 in long-term virus replication assays. We stably introduced vectors expressing small interfering RNAs (siRNAs) directed against the HIV-1 genome into human T cells by retroviral transduction. We report here that an siRNA directed against the viral Nef gene (siRNA-Nef) confers resistance to HIV-1 replication. This block in replication is not absolute, and HIV-1 escape variants that were no longer inhibited by siRNA-Nef appeared after several weeks of culture. These RNAi-resistant viruses contained nucleotide substitutions or deletions in the Nef gene that modified or deleted the siRNA-Nef target sequence. These results demonstrate that efficient inhibition of HIV-1 replication through RNAi is possible in stably transduced cells. Therefore, RNAi could become a realistic gene therapy approach with which to overcome the devastating effect of HIV-1 on the immune system. However, as is known for antiviral drug therapy against HIV-1, antiviral approaches involving RNAi should be used in a combined fashion to prevent the emergence of resistant viruses.     

Characterization of in vitro inhibition of human immunodeficiency virus by purified recombinant CD4.

The first step in infection of human T cells with human immunodeficiency virus (HIV) is binding of viral envelope glycoprotein gp120 to its cellular receptor, CD4. The specificity of this interaction has led to the development of soluble recombinant CD4 (rCD4) as a potential antiviral and therapeutic agent. We have previously shown that crude preparations of rCD4 can indeed block infection of T cells by HIV type 1 (HIV-1). Here we present a more detailed analysis of this antiviral activity, using HIV-1 infection of the T lymphoblastoid cell line H9 as a model. Purified preparations of rCD4 blocked infection in this system at nanomolar concentrations; combined with the known affinity of the CD4-gp120 interaction, this finding suggests that the inhibition is simply due to competition for gp120 binding. As predicted, rCD4 had comparable activity against all strains of HIV-1 tested and significant activity against HIV-2. Higher concentrations of rCD4 blocked infection even after the virus had been adsorbed to the cells. These findings imply that the processes of viral adsorption and penetration require different numbers of gp120-CD4 interactions. Recombinant CD4 was able to prevent the spread of HIV infection in mixtures of uninfected and previously infected cells. Our studies support the notion that rCD4 is a potent antiviral agent, effective against a broad range of HIV-1 isolates, and demonstrate the value of purified rCD4 as an experimental tool for studying the mechanism of virus entry into cells.     

Synthesis and antiviral activity of hydrazides and substituted benzalhydrazides of betulinic acid and its derivatives

New nitrogen-containing derivatives of betulinic and betulonic acids, hydrazides and N'-benzalhydrazides, were synthesized. Their antiviral activities toward of influenza A virus, herpes simplex type I virus, enterovirus ECHO6, and HIV-1 were studied in vitro. Betulinic acid 3-oxime was found to have the highest activity against the influenza virus. Betulonic acid, betulinic acid 4-chlorobenzalhydrazide, betulonic acid 3-oxime benzalhydrazide, and betulinic acid hydrazide inhibited the replication of herpes simplex type I virus. Betulinic acid hydrazide also showed antiviral activity toward HIV-1. All the derivatives of betulinic acid under study displayed a low antiviral activity toward enterovirus ECHO6.     

Proline-rich tandem repeats of antibody complementarity-determining regions bind and neutralize human immunodeficiency virus type 1 particles.

The proline-rich tandem repeat domain of human mucin MUC1 forms an extended structure containing large repeating loops that are crested by a turn. We show that the repeating-loop structure of MUC1 can be replaced by an antibody complementarity-determining region loop of a human immunodeficiency virus type 1 (HIV-1)-specific neutralizing antibody to create a chimeric, multivalent, mucin-like, anti-HIV-1 compound. We used 8 residues of an antibody molecule to replace 8 of 20 residues of the MUC1 tandem-repeat sequence. The antiviral peptide discussed here contains three copies of a 20-residue tandem repeat, (IYYDYEEDPAPGSTAPPAHG)3, for a total of 60 residues. We demonstrate that the mucin-antibody chimera retains the binding specificity of the parent antibody (monoclonal antibody F58), GPGR of the HIV-1 gp120 V3 neutralizing epitope, and the ability to neutralize virus particles. In inhibition enzyme-linked immunosorbent assay, the mucin-antibody chimeric peptide could inhibit 71 to 84% of binding to a V3 loop peptide by monoclonal antibodies known to be specific for GPGR in the V3 loop. The mucin-antibody chimeric peptide could also inhibit monoclonal antibody binding to native gp120 captured from virus particles. In addition, the chimeric peptide neutralized the homologous HIV-IIIB virus in a standard neutralization assay. The methods of antiviral peptide design and construction presented here are general and theoretically limited only by the size of the antibody repertoire. This approach could be used to synthesize peptides for a variety of therapeutic applications.     

Reversal of HIV-1 latency with anti-microRNA inhibitors

Human immunodeficiency virus type 1 (HIV-1) latency is achieved when host cells contain integrated proviral DNA but do not produce viral particles. The virus remains in resting CD4 T-lymphocytes, evading host immune surveillance and antiviral drugs. When resting cells are activated, infectious viral particles are produced. Latency is critical for the survival of all HIV-1 strains in vivo. Recently, it has been reported that a cluster of cellular microRNAs (miRNAs) enriched specifically in resting CD4+ T-cells suppresses translation of most HIV-1-encoded proteins in the cytoplasm, sustaining HIV-1 escape from the host immune response. Complementary antisense miRNA inhibitors block the inhibitory effect of miRNAs and drive viral production from the resting T-lymphocytes without activating the cells. Therefore, inhibition of these HIV-1-specific cellular miRNAs is of great therapeutic significance for eliminating the HIV-1 reservoir in HIV-1-infected individuals receiving suppressive highly active antiretroviral therapy (HAART).     

Expression and immunogenicity of human immunodeficiency virus type 1 Gag expressed by a replication-competent rhabdovirus-based vaccine vector

A replication-competent rhabdovirus-based vector expressing human immunodeficiency virus type 1 (HIV-1) Gag protein was characterized on human cell lines and analyzed for the induction of a cellular immune response in mice. We previously described a rabies virus (RV) vaccine strain-based vector expressing HIV-1 gp160. The recombinant RV was able to induce strong humoral and cellular immune responses against the HIV-1 envelope protein in mice (M. J. Schnell et al., Proc. Natl. Acad. Sci. USA 97:3544-3549, 2000; J. P. McGettigan et al., J. Virol. 75:4430-4434, 2001). Recent research suggests that the HIV-1 Gag protein is another important target for cell-mediated host immune defense. Here we show that HIV-1 Gag can efficiently be expressed by RV on both human and nonhuman cell lines. Infection of HeLa cells with recombinant RV expressing HIV-1 Gag resulted in efficient expression of HIV-1 precursor protein p55 as indicated by both immunostaining and Western blotting. Moreover, HIV-1 p24 antigen capture enzyme-linked immunosorbent assay and electron microscopy showed efficient release of HIV-1 virus-like particles in addition to bullet-shaped RV particles in the supernatants of the infected cells. To initially screen the immunogenicity of this new vaccine vector, BALB/c mice received a single vaccination with the recombinant RV expressing HIV-1 Gag. Immunized mice developed a vigorous CD8(+) cytotoxic T-lymphocyte response against HIV-1 Gag. In addition, 26.8% of CD8(+) T cells from mice immunized with RV expressing HIV-1 Gag produced gamma interferon after challenge with a recombinant vaccinia virus expressing HIV-1 Gag. These results further confirm and extend the potency of RV-based vectors as a potential HIV-1 vaccine.     

Effect of macromolecular crowding agents on human immunodeficiency virus type 1 capsid protein assembly in vitro

Previous studies on the self-assembly of capsid protein CA of human immunodeficiency virus type 1 (HIV-1) in vitro have provided important insights on the structure and assembly of the mature HIV-1 capsid. However, CA polymerization in vitro was previously observed to occur only at very high ionic strength. Here, we have analyzed the effects on CA assembly in vitro of adding unrelated, inert macromolecules (crowding agents), aimed at mimicking the crowded (very high macromolecular effective concentration) environment within the HIV-1 virion. Crowding agents induced fast and efficient polymerization of CA even at low (close to physiological) ionic strength. The hollow cylinders thus assembled were indistinguishable in shape and dimensions from those formed in dilute protein solutions at high ionic strength. However, two important differences were noted: (i) disassembly by dilution of the capsid-like particles was undetectable at very high ionic strength, but occurred rapidly at low ionic strength in the presence of a crowding agent, and (ii) a variant CA from a presumed infectious HIV-1 with mutations at the CA dimerization interface was unable to assemble at any ionic strength in the absence of a crowding agent; in contrast, this mutation allowed efficient assembly, even at low ionic strength, when a crowding agent was used. The use of a low ionic strength and inert macromolecules to mimic the crowded environment inside the HIV-1 virion may lead to a better in vitro evaluation of the effects of conditions, mutations or/and other molecules, including potential antiviral compounds, on HIV-1 capsid assembly, stability and disassembly.     

Cyclophilin A and TRIM5alpha independently regulate human immunodeficiency virus type 1 infectivity in human cells

Cyclophilin A (CypA), a cytoplasmic, human immunodeficiency virus type 1 (HIV-1) CA-binding protein, acts after virion membrane fusion with human cells to increase HIV-1 infectivity. HIV-1 CA is similarly greeted by CypA soon after entry into rhesus macaque or African green monkey cells, where, paradoxically, the interaction decreases HIV-1 infectivity by facilitating TRIM5alpha-mediated restriction. These observations conjure a model in which CA recognition by the human TRIM5alpha orthologue is precluded by CypA. Consistent with the model, selection of a human cell line for decreased restriction of the TRIM5alpha-sensitive, N-tropic murine leukemia virus (N-MLV) rendered HIV-1 transduction of these cells independent of CypA. Additionally, HIV-1 virus-like particles (VLPs) saturate N-MLV restriction activity, particularly when the CA-CypA interaction is disrupted. Here the effects of CypA and TRIM5alpha on HIV-1 restriction were examined directly. RNA interference was used to show that endogenous human TRIM5alpha does indeed restrict HIV-1, but the magnitude of this antiviral activity was not altered by disruption of the CA-CypA interaction or by elimination of CypA protein. Conversely, the stimulatory effect of CypA on HIV-1 infectivity was completely independent of human TRIM5alpha. Together with previous reports, these data suggest that CypA protects HIV-1 from an unknown antiviral activity in human cells. Additionally, target cell permissivity increased after loading with heterologous VLPs, consistent with a common saturable target that is epistatic to both TRIM5alpha and the putative CypA-regulated restriction factor.