Infectivity Of Enveloped Viruses Research Articles

Stimulation of Enveloped Virus Infection by β-Amyloid Fibrils

Alzheimer's disease is characterized by deposition of beta-amyloid peptide (Abeta) into plaques in the brain, leading to neuronal toxicity and dementia. Human immunodeficiency virus type 1 (HIV-1) infection of the central nervous system can also cause a dementia, and amyloid deposition in the central nervous system is significantly higher in HIV-1-infected individuals compared with uninfected controls. Here we report that Abeta fibrils stimulated, by 5-20-fold, infection of target cells expressing CD4 and an appropriate coreceptor by multiple HIV-1 isolates but did not permit infection of cells lacking these receptors. Abeta enhanced infection at the stage of virus attachment or entry into the cell. Abeta fibrils also stimulated infection by amphotrophic Moloney leukemia virus, herpes simplex virus, and viruses pseudotyped with the envelope glycoprotein of vesicular stomatitis virus. Other synthetic fibril-forming peptides similarly enhanced viral infection and may be useful in gene delivery applications utilizing retroviral vectors. These data suggest that Abeta deposition may increase the vulnerability of the central nervous system to enveloped viral infection and that amyloidogenic peptides could be useful in enhancing gene transfer by enveloped viral vectors.

Strategies for evaluation of enveloped virus inactivation in red cell concentrates using hypericin.

Photodynamically induced virus inactivation appears promising in preventing transmission of enveloped virus infections in transfusible blood products. The potential for utilizing hypericin as a photosensitizer to inactivate key enveloped viruses in packed red cell concentrates (PRC) was evaluated. In addition to inactivating effectively > or = 10(6) TCID50 of human immunodeficiency virus (HIV), inactivation of bovine viral diarrhea virus (BVDV) in PRC was used as a model for hepatitis C virus to overcome the deficiency in reliable experimental systems for hepatitis C virus (HCV) inactivation. BVDV was two orders of magnitude more sensitive to inactivation by hypericin than HIV. As part of the virucidal efficacy analyses, the effects of photosensitization on hemopoietic cell lines carrying quiescent integrated HIV provirus were studied as models for evaluating virus inactivation in latently infected cells. Phorbol ester-induced virus production by these cells was effectively prevented by photosensitization with hypericin. A refinement of the illumination conditions, incorporating a monochromatic sodium light source with an emission spectrum coinciding with the absorption peak of hypericin, was highly virucidal, however, caused unacceptable levels of hemolysis. Red blood cells could be protected from phototoxic cellular damage by complexing hypericin with human serum albumin (albumin-hypericin), but the decrease in hemolysis was at the expense of virucidal efficacy. Thus, excitation of hypericin with a fluorescent source appears to be useful potentially for virus inactivation in PRC.

Sensitization of rhabdo-, lenti-, and spumaviruses to human serum by galactosyl(alpha1-3)galactosylation.

Vesicular stomatitis virus, human immunodeficiency virus type 2, and human foamy virus, which were produced by cell lines expressing galactosyl(alpha1-3)galactosyl (alphaGal) sugars, were found to be less stable in human serum than those from alphaGal-negative cells, indicating that galactosyl(alpha1-3)galactosylation sensitizes these viruses as well as mammalian type C oncoviruses (Rother et al., J. Exp. Med. 182:1345-1355, 1995; Takeuchi et al., Nature (London) 379:85-88, 1996) to complement killing via natural anti-alphaGal antibodies. Thus, virus killing mediated by anti-alphaGal antibodies may play a role as a barrier to animal-to-human infection of various enveloped viruses. Virus vectors for human in vivo gene therapy based on the viruses mentioned above should be produced from alphaGal-negative cells.

Advances in photochemical approaches for blood sterilization.

Advances in photochemical approaches for blood sterilization.

Safety and Cost-effectiveness of Solvent-Detergent—Treated Plasma

To determine the public health and economic implications of solvent-detergent-treated frozen plasma (SD FP). While this processing technique nearly eliminates the risk of transmitting lipid-enveloped viruses (hepatitis B and C and human immunodeficiency virus), it has associated costs and, because it requires pooling many plasma units, may increase risks of nonenveloped virus transmission. A previously published Markov decision analysis model was modified to assess transfusion-related outcomes in hypothetical cohorts of plasma recipients. In-hospital mortality and other characteristics were determined in 61 patients receiving plasma transfusions at a medium-sized tertiary care center to provide data for the model. Other parameters were obtained from the medical literature. Expected SD FP costs, benefits, and cost-effectiveness, assessed as cost per quality-adjusted life-year saved. Compared with untreated plasma, a unit of SD FP produces a net benefit of 35 minutes in quality-adjusted life expectancy at a cost of about $19. Extrapolated to the 2.2 million plasma units transfused annually in the United States, SD FP would save 147 quality-adjusted life-years at a cost of $42.5 million. The marginal cost-effectiveness, $289,300 per quality-adjusted life-year saved in the baseline analysis, was most sensitive to estimates of SD treatment cost and the clinical setting of plasma use. In sensitivity analysis, the net benefit of SD FP was negated by the existence of even a minute risk of nonenveloped virus infection. From a public health perspective, the relatively high costs and small benefits of reducing enveloped virus infection risks with SD FP (and the additional risks of noneveloped virus transmission) do not appear to justify widespread implementation of this new technology.

Interference with the endosomal acidification by a monoclonal antibody directed toward the 116 (100)-kD subunit of the vacuolar type proton pump.

A monoclonal antibody (OSW2) was prepared by using human osteosarcoma cells. OSW2 was found to be directed toward the 116 (also called 100)- kD protein that uniquely associates to the vacuolar-type proton pump. The antibody specifically localized acidic membrane compartments that could be visualized with acridine orange in many types of human cells. It also reacted with the surface and was internalized along the endosomal pathway. Monitoring the endosome pH by using FITC-dextran and acridine orange suggested that the antibody interfered with low pH. Cell-free experiments indicated that the ATP-dependent acidification was inhibited in endosomes associated with OSW2. In contrast, the antibody gave little effect on the ATPase activity of the solubilized H+ pump. The internalization of OSW2 reduced infectivity of certain enveloped viruses (influenza, SFV, VSV) by 50 to 80%. Inhibition of viral fusion was directly demonstrated by monitoring the fate of octadecylrhodamine-labeled influenza virus fluorescence. These results indicate that the 116 (100)-kD protein is necessary for the control of pH. The antibody represents a novel probe for understanding the role of the endosomal compartments in cellular physiology.

Genetical and biochemical studies on the metabolisms and biological roles of membrane phospholipids in mammalian cells

The genetical and biochemical approach is used as a powerful tool for the elucidation of the metabolic regulation as well as the biological significance of various constituents in living cells. This technique has been successfully applied to the membrane phospholipids in mammalian cells, actually to phosphatidylserine, sphingomyelin, and cardiolipin, all of which are rather minor constituents in membrane phospholipids. Isolation and characterization of the mutant cells defective in biosyntheses and biosynthetic regulations of these phospholipids led to the new findings of the in vivo biosynthetic pathways and also of the biological significance of these phospholipids. By analyzing the five different kinds of mutant cells unusual in metabolisms of phosphatidylserine in CHO-K1 cells, we found a series of the following evidence. Phosphatidylserine is synthesized by the two kinds of base-exchange enzymes, namely serine-exchange enzyme I and II, through the chain reactions in participation of phosphatidylserine decarboxylase; phosphatidylcholine-->phosphatidylserine-->phosphatidylethanolamine--> phosphatidylserine. The substrates of serine-exchange enzyme I are phosphatidylcholine, and either choline, serine, or ethanolamine, while those of serine-exchange enzyme II are phosphatidylethanolamine, and serine or ethanolamine but not choline. The last phosphatidylserine produced in consequence of the chain reaction again becomes a substrate of phosphatidylserine decarboxylase, and so this metabolic flow of the biosynthesis of phosphatidylserine is finally connected to a cyclic reaction between phosphatidylethanolamine and phosphatidylserine catalyzed by serine-exchange enzyme II and phosphatidylserine decarboxylase. Localization of serine-exchange enzyme I is on the cytoplasmic side of endoplasmic reticulum, while that of enzyme II is on its lumen side. There is a lot of evidence including our immunological observation that phosphatidylserine decarboxylase is exclusively located in the inner membrane of mitochondria. Thus, during the process of the above-mentioned reactions, phosphatidylserine have to move from endoplasmic reticulum to mitochondria, and phosphatidylethanolamine does from mitochondria to endoplasmic reticulum. The biological significance of these movements is now under investigation. Enveloped virus infection and its maturation are closely associated with the membrane functions of host mammalian cells. In order to get further information concerning the biological functions of phosphatidylserine and/or phosphatidylethanolamine in mammalian cell membranes, Sindbis virus infection system was examined. Deficiency of phosphatidylserine and/or phosphatidylethanolamine was found to give a profound effect on the maturation of the virus, mainly due to low efficiency of membrane fusion between the viral envelope and endosomal membranes of the host cells. All these results indicate that phosphatidylserine and/or phosphatidylethanolamine are essential to membrane fusion at least in enveloped viral infection.(ABSTRACT TRUNCATED AT 400 WORDS)

Membrane fusion-inhibiting peptides do not inhibit influenza virus fusion or the Ca(2+)-induced fusion of negatively charged vesicles.

Short hydrophobic N-carbobenzoxy oligopeptides are known to inhibit the infectivity of several enveloped viruses. Recently, it was shown that they inhibited the fusion of Sendai virus with N-methyl-dioleoylphosphatidylethanolamine (N-methyl-DOPE) liposomes as well as the low pH-induced fusion of these liposomes with each other (Kelsey, D.R., Flanagan, T. D., Young, J. E., and Yeagle, P. L. (1990) J. Biol. Chem. 265, 12178-12183). Therefore it was concluded that the peptides inhibit membrane fusion, an important step in viral infectivity. Here, it is shown that this peptide and a series of similar peptides did not inhibit influenza virus fusion with N-methyl-DOPE or other liposomes. In fact, some peptides enhanced the overall rate of fusion of influenza virus with N-methyl-DOPE liposomes. In our hands, the peptides did not inhibit influenza infectivity in Madin-Darby canine kidney cells or influenza-induced hemolysis either. They also did not inhibit the Ca(2+)-induced fusion between cardiolipin or phosphatidylserine liposomes. However, the inhibitory effect of one of the peptides on the fusion of Sendai virus with N-methyl-DOPE liposomes and on N-methyl-DOPE liposome-liposome fusion could be reproduced. These data indicate that the peptides do not, as had been suggested (Yeagle, P.L., Young, J.E., Hui, S.W., and Epand, R. M. (1992) Biochemistry 31, 3177-3183), act by preventing the formation of lipid structures with small radii of curvature, such as the inverted phase intermediates that are thought to be involved in N-methyl-DOPE fusion. The results also suggest that the mechanism of inhibition of Sendai virus infection and N-methyl-DOPE fusion by the peptides may be different after all.

Nonoxynol-9 Protects Mice against Vaginal Transmission of Genital Herpes Infections

A vaginal application of a commercially available contraceptive jelly containing nonoxynol-9 (N9) prevented vaginal transmission of herpes simplex virus type 2 (HSV-2) infections in the mouse. When N9 jelly was delivered to the vagina with the virus inoculum, 20 s before inoculum, or 5 min before inoculum, mice were completely protected from visible infection (P < .001). Protection lasted for at least 30 min (P < .03), and significant protection occurred even when the N9 jelly was delivered 15 min after the HSV-2 inoculum (P < .05). These results are consistent with results of studies using N9 products in other animal models and suggest that N9-based contraceptive products can provide significant protection against vaginal transmission of enveloped virus infections in animals.

Photodynamic inactivation of infectivity of human immunodeficiency virus and other enveloped viruses using hypericin and rose bengal: inhibition of fusion and syncytia formation.

The mechanism of the antiviral activity of hypericin was characterized and compared with that of rose bengal. Both compounds inactivate enveloped (but not unenveloped) viruses upon illumination by visible light. Human immunodeficiency and vesicular stomatitis viruses were photodynamically inactivated by both dyes at nanomolar concentrations. Photodynamic inactivation of fusion (hemolysis) by vesicular stomatitis, influenza, and Sendai viruses was induced by both dyes under similar conditions (e.g., I50 = 20-50 nM for vesicular stomatitis virus), suggesting that loss of infectivity resulted from inactivation of fusion. Syncytium formation, between cells activated to express human immunodeficiency virus gp120 on their surfaces and CD4+ cells, was inhibited by illumination in the presence of 1 microM hypericin. Hypericin and rose bengal thus exert similar virucidal effects. Both presumably act by the same mechanism--namely, the inactivation of the viral fusion function by singlet oxygen produced upon illumination. The implications of this photodynamic antiviral action for the potential therapeutic usefulness of both hypericin and rose bengal are discussed.

Effects of protein kinase C inhibitors on viral entry and infectivity

The protein kinase C inhibitor H-7 (2–20 μM) inhibited dose-dependently the infectivity of the vesicular stomatitis virus on cultured human fibroblasts. Electron microscopy showed that H-7 inhibited the viral entry. H-7 also inhibited the infectivity of four other enveloped viruses, herpes simplex I, turkey herpes, vaccinia and Sindbis. Similar results were obtained using staurosporine (2.5 nM), tamoxifen (40 μM), phloretin (140 μM). or W-7 (40 μM). However, the infectivity of non-enveloped viruses (e.g. poliomyelitis I) was not inhibited by H-7. These results show that protein kinase C is critically involved in the infectivity of enveloped viruses, most probably at the level of viral entry (receptor-mediated endocytosis).

Influence of Polyions on the Early Steps of Enterovirus Infection

The influence of electric charged molecules on the early phases of enterovirus infection was studied in order to select antiviral compounds able to prevent viral attachment. The effect of different polyelectrolytes on the multiplication of coxsackie virus B3, echovirus 6 and hepatitis A virus was investigated in susceptible cells by adding the drug before, during or after the viral adsorption period. Among polyanions, the polysaccharides heparin and dextran sulfate inhibited viral infectivity, dextran sulfate being the most effective mainly towards hepatitis A virus infection. DEAE-dextran and protamine sulfate, generally recognized as enhancers of infectivity of naked and enveloped viruses, exhibited an inhibitory effect towards the three picornaviruses tested. Only in the case of hepatitis A did DEAE-dextran slightly improve viral antigen synthesis. The inhibitory effect shown by compounds belonging to positive and negative polyions suggests that the electric charge is not sufficient by itself to explain the antiviral activity of these drugs.

Virus inactivation during production of intravenous immunoglobulin.

The effect of pepsin treatment at pH 4 on the infectivity of several enveloped viruses was assessed under the conditions used during the production of intravenous immunoglobulins. It was shown that the prototypes of four virus families--human immunodeficiency virus (Lentivirinae), herpes simplex virus type 1 and human cytomegalovirus (Herpesviridae), Semliki Forest virus (Togaviridae), and vesicular stomatitis virus (Rhabdoviridae)--were inactivated by this procedure. With vesicular stomatitis virus as a model, the contributions of both low pH and pepsin were demonstrated, and pepsin had a synergistic or additive action.

Inhibition of sendai virus fusion with phospholipid vesicles and human erythrocyte membranes by hydrophobic peptides

Hydrophobic di- and tripeptides which are capable of inhibiting enveloped virus infection of cells are also capable of inhibiting at least three different types of membrane fusion events. Large unilamellar vesicles (LUV) of N-methyl dioleoylphosphatidylethanolamine ( N-methyl DOPE), containing encapsulated 1-aminonaphthalene-3,6,8-trisulfonic acid (ANTS) and/or p-xylene bis(pyridinium bromide) (DPX), were formed by extrusion. Vesicle fusion (contents mixing) and leakage were then monitored with the ANTS/DPX fluorescence assay. Sendai virus fusion with lipid vesicles and Sendai virus fusion with human erythrocyte membranes were measured by following the relief of fluorescence quenching of virus labeled with octadecylrhodamine B chloride (R 18), a lipid mixing assay for fusion. This study found that the effectiveness of the peptides carbobenzoxy- l-Phe- l-Phe (Z- l-Phe- l-Phe), Z- l-Phe, Z-D-Phe, and Z-GIy- l-Phe- l-Phe in inhibiting N-methyl DOPE LUV fusion or fusion of virus with N-methyl DOPE LUV also paralleled their reported ability to block viral infectivity. Furthermore, Z- d-Phe- l-PheGly and Z-GIy- l-Phe inhibited Sendai virus fusion with human erythrocyte membranes with the same relative potency with which they inhibited vesicle-vesicle and virus-vesicle fusion. The evidence suggests a mechanism by which these peptides exert their inhibition of plaque formation by enveloped viruses. This class of inhibitors apparently acts by inhibiting fusion of the viral envelope with the target cell membrane, thereby preventing viral infection. The physical pathway by which these peptides inhibit membrane fusion was investigated. 31 P nuclear magnetic resonance (NMR) of proposed intermediates in the pathway for membrane fusion in LUV revealed that the potent fusion inhibitor Z- d-Phe- l-PheGly selectively altered the structure (or dynamics) of the hypothesized fusion intermediates and that the poor inhibitor Z-GIy- l-Phe did not. One possible interpretation of these 31P NMR results was that the inhibitory peptide stabilized a membrane structure with a large radius of curvature, when the fusion pathway demanded a membrane defect with a small radius of curvature. This hypothesis was tested by determining the influence of an inhibitory and a noninhibitory peptide on the formation of membraneous structures with small radii of curvature, through ultrasonic irradiation of phospholipid dispersions. The inhibitory peptide prevented the formation of membrane structures with small radii of curvature, while the noninhibitory peptide did not prevent the formation of such structures. These data taken collectively suggest that the peptides that inhibit the fusion of enveloped viruses could act, at least in part, by interfering with the formation of intermediates in membrane fusion (possibly punctate membrane defects induced by the virus) that force lipids into structures with small radii of curvature.

Photoinactivation of Viruses in Human Fresh Plasma by Phenothiazine Dyes in Combination with Visible Light

We developed a photodynamic method to inactivate viruses in human fresh plasma. Single plasma bags were illuminated with visible light in the presence of low doses of phenothiazine dyes like methylene blue or toluidine blue. By this treatment the infectivity of different enveloped viruses including the causative agent of AIDS, HIV-1, was completely removable from the plasma. Non enveloped viruses, however, proved to be more stable. The activities of clotting factors and other plasma proteins were only slightly decreased. There was no indication that the procedure led to important structural modifications of plasma proteins. The dyes are photodynamically active at concentrations much lower than those at which they are therapeutically used as antidots in the treatment of methemoglobinemia.

Peptide inhibitors of enveloped virus infection inhibit phospholipid vesicle fusion and Sendai virus fusion with phospholipid vesicles.

Small hydrophobic peptides that are capable of inhibiting Sendai virus infection of cells (Richardson, C. D., Scheid, A., and Choppin, P. W. (1980) Virology 105, 205-222) are also capable of inhibiting membrane fusion in a pure lipid vesicle system. Large unilamellar vesicles of N-methyl dioleoylphosphatidylethanolamine containing encapsulated 1-aminonaphthalene-3,6,8-trisulfonic acid and/or p-xylene bis (pyridinium bromide) were formed by extrusion. Vesicle fusion (contents mixing) and leakage were then monitored with the 1-aminonaphthalene-3,6,8-trisulfonic acid/p-xylene bis(pyridinium bromide) fluorescence assay. Sendai virus fusion with lipid vesicles was measured by following the relief of fluorescence quenching of virus labeled with octadecylrhodamine B chloride, a lipid mixing assay for fusion. The efficiency with which the peptides carbobenzoxy-D-Phe-L-PheGly, carbobenzoxy-L-Phe-L-Tyr, and carbobenz-oxy-Gly-L-Phe inhibit fusion of N-methyl dioleoyl-phosphatidylethanolamine large unilamellar vesicles directly paralleled their previously known effectiveness in blocking virus infectivity of cultured cells. In addition, above a certain concentration threshold, the inhibitory peptides decreased the initial rate of leakage from lipid vesicles. The inhibition by these peptides of virus-vesicle fusion followed the same order of potency as for vesicle-vesicle fusion. The observation of the same relative potency of these peptides toward inhibition of virus-cell infection, and virus-vesicle and vesicle-vesicle membrane fusion suggested that these peptides inhibited virus-cell infection by inhibiting the ability of the virus to fuse with the cell. Furthermore, these results suggest that the mechanism of inhibition of all three fusion events may have steps in common.

An approach to AIDS therapy using hyperthermia and membrane modification

Altering the biophysical characteristics of cell membranes by diet and membrane perturbing agents markedly influences thermosensitivity of cells. Likewise, manipulation of viral envelopes either by altering their lipid composition by diet or by the use of agents that perturb the lipid envelope influence infectivity of enveloped viruses and the progression of viral disease. The use of hyperthermia and envelope modification as a combined approach to treat AIDS has until now neither been suggested nor attempted. On the basis of my previous work and a review of the literature, I theorize that the combination of hyperthermia with procedures designed to alter the viral envelope will likely result in an increased viral sensitivity and be useful clinically for treatment of patients with enveloped viral diseases such as AIDS.

Basic monosaccharide derivatives: tools for exploring the active site of glycohydrolases and for studies in glycoprotein biosynthesis

The majority of glycoside hydrolases is strongly inhibited by monosaccaride derivatives bearing a basic NH—group adjacent to C—i, i. e. by glycosylanines, 5—amino—5—deoxyhexopyranoses, and 1,5—iminoalditols. This inhibition with Kr—values up to 104—tines smaller than those of neutral analogs is explained by a catalytic site which features an acid and a carboxylated in iuxtaposition close to the anoneric carbon of the bound inhibitor or substrate. The inhibitor protonated by the acid forms an ion pair with the carboxylate. The stability of this ion pair and the lack of influence of added salt on the inhibition calls for an active site strongly shielded from the aqueous environment. The acid/carboxylate arrangement of functional groups is supported by inhibition studies with substrate related epoxides that are activated by proton transfer from the acid and form an ester with carboxylate. N—Alkylation of the basic inhibitors leads, in many cases, to an enhanced inhibition. Structural variation of the alkyl substituents permits a detailed characterisation of the aglycon site as exemplified by studies on lysosomal —glucosidase Inhibition by 1,5—dideoxy—i,5—imino—D—glucitol and —mannitol of D(—glucosi— dases and —mannosidases participating in the biosynthesis of carbohydrate chains linked to asparagine residues of glycoproteins results in altered glycan structures of newly synthesized proteins. The effects of these alteration on surrace expression and secretion of the proteins, on intracellular compartmentation and on the formation and infectivity of enveloped viruses is discussed.

Effect of sucrose phosphate and sorbitol on infectivity of enveloped viruses during storage.

Cytomegalovirus and varicella-zoster virus recovery from sucrose phosphate (0.2 M SP) and 70% sorbitol (sorbitol) was compared after storage at -70, 4, and 20 degrees C over time. Recovery from 0.2 M SP was uniformly better. More tissue culture infective doses and infectious foci were recovered in cell monolayers inoculated with 0.2 M SP virus stocks as compared with viruses stored in 70% sorbitol. Although both viruses were isolated from diluted fresh stocks (10(-1) through 10(-4], freezing diluted virus suspensions generally resulted in diminished recovery. Similar stabilizing effects on respiratory syncytial virus and herpes simplex virus type 1 infectivities were observed when stocks were preserved in 0.2 M SP, as compared with 70% sorbitol. Overall, 0.2 M SP was better than 70% sorbitol for stabilizing cytomegalovirus, varicella-zoster virus, respiratory syncytial virus, and herpes simplex virus type 1 infectivities under the conditions tested.