Novel, rapid assay for measuring entry of diverse enveloped viruses, including HIV and rabies

Entry is the first and essential step in virus replication and is a target for therapeutic intervention. However, current knowledge on entry mechanism for the majority of viruses is poor, partly due to lack of a simple, sensitive and accurate entry assay that can be applied to diverse viruses. To overcome this obstacle, a novel contents-mixing-based virus entry assay is described that can be broadly applied to many enveloped viruses. By fusing firefly luciferase to the HIV Nef protein, luciferase was directly packaged into HIV particles pseudotyped with envelope proteins of diverse viruses including HIV, rabies and others. Upon cell entry, the luciferase-fusion protein was released into the cell cytoplasm, reacted with its substrates and was detected by light emission. The assay was validated by demonstrating its versatility in measuring virus entry. Entry was detected much more rapidly (in real-time) with higher sensitivity (a multiplicity of infection <0.1 gives a robust signal) and lower background (signal/noise ration >1000) than other comparable assays. In addition to its utility in studying virus entry mechanisms, the assay will aid in screening potential entry/fusion inhibitors and in diagnosis of virus infections.     

HIV: reactive oxygen species, enveloped viruses and hyperbaric oxygen

This paper demonstrates that there are many examples in the literature of contradictory data concerning reactive oxygen intermediates (ROIs), responsible for producing cellular oxidative stress (OS), and their enhancement or diminution of viral replication. Nevertheless, ROIs repeatedly have been shown to be virucidal against enveloped-viruses, like the human immunodeficiency virus (HIV). Hyperbaric oxygen therapy (HBOT) increases the production of ROIs throughout the body, leaving no safe harbor for the virus to hide outside the genome. This technique already has been tried on acquired immune deficiency syndrome (AIDS) patients, with exciting results. Historically, the biggest setback to demonstrating HBO's antiviral effects has been the investigator's folly of studying non-enveloped viruses or failing to initiate ROI production. ROIs specifically attack areas of unsaturation occurring in the polyunsaturated fatty acids of cell membranes and viral envelopes. Moreover, it consistently has been shown that a peroxidized viral envelope breaches, and a breached viral envelope causes viral disintegration.     

Cell culture density modulates the incorporation of HLA antigens by enveloped viruses

Uninfected, as well as feline leukemia virus (FeLV) infected human cells cultured under high cell density conditions undergo changes in the expression of major histocompatibility complex (MHC) antigens, as determined by indirect trace binding radio immunoassay (RIA) using monoclonal anti-HLA antibodies and by decreased sensitivity to complement mediated cytotoxicity by anti-HLA alloantibodies. FeLV particles produced by the viral infected cells are also sensitive to neutralization by anti-HLA antibodies, suggesting that enveloped viral particles incorporated MHC antigens in the viral envelope. The amount of HLA antigens expressed in the viral enveloped, closely reflects the expression of HLA antigens by the virus-producer lymphoid cells. FeLV-infected HsB-2 (T) and SB (B) lymphoid cells cultured under high cell concentration condition show decreased expression of some HLA antigens (A2, B12, B17), and the viral particles produced by those cells also incorporate lower amounts of such antigens. Our results, based on the findings that human lymphoid cells (uninfected, as well as FeLV infected) show decreased expression of some HLA membrane determinants when growth under high cell density conditions, indicate that no viral selective mechanism operates in the incorporation of HLA determinants by enveloped viruses. Instead, our results suggest that viruses pick up MHC antigens from the host cell membrane according to the concentration of those antigens on the surface of the cells at the time of virus budding.     

Human cancers and viruses: a hypothesis for immune destruction of tumours caused by certain enveloped viruses using modified viral antigens.

Certain viruses which have been identified as possible aetiological agents of human malignant tumours have 2 common characteristics: a) they persist in the human body for long periods despite the presence of antibodies to them and b) they all possess viral envelopes. The envelopes, consisting of phospho-lipoproteins are derived from host cells viz nuclear envelope in the case of DNA viruses, and the cell membrane in the case of RNA viruses. These host cell elements on the viral envelope modify the antigenicity of the specific surface antigens which are now perceived by the host immune system as partly self. This in turn blackmails the immune system, if it is to avoid serious auto-immune disease, into producing compromise and ineffective antibodies. The hypothesis proposes the dissolution of the viral envelope in vitro and the re-introduction of the viral core into the host. This should provoke a new uncompromised immune response because it will be directed at the viral core only. This response should eliminate the viral core and with it, the whole enveloped virus, as well as the malignant tumour cells which carry the viral genome derived essentially from the viral core. This approach should introduce a new method for treating and preventing tumours caused by enveloped viruses and the chronic diseases caused by such viruses.     

Current status on the development of pseudoviruses for enveloped viruses

Emerging and reemerging infectious diseases have a strong negative impact on public health. However, because many of these pathogens must be handled in biosafety level, 3 or 4 containment laboratories, research and development of antivirals or vaccines against these diseases are often impeded. Alternative approaches to address this issue have been vigorously pursued, particularly the use of pseudoviruses in place of wild‐type viruses. As pseudoviruses have been deprived of certain gene sequences of the virulent virus, they can be handled in biosafety level 2 laboratories. Importantly, the envelopes of these viral particles may have similar conformational structures to those of the wild‐type viruses, making it feasible to conduct mechanistic investigation on viral entry and to evaluate potential neutralizing antibodies. However, a variety of challenging issues remain, including the production of a sufficient pseudovirus yield and the inability to produce an appropriate pseudotype of certain viruses. This review discusses current progress in the development of pseudoviruses and dissects the factors that contribute to low viral yields.     

Virus-inhibiting surgical glove to reduce the risk of infection by enveloped viruses

Needle puncture and other accidents that occur during surgery and other procedures may lead to viral infections of medical personnel, notably by hepatitis C (HCV) and human immunodeficiency virus (HIV), now that hepatitis B can be prevented by vaccination. A new surgical glove called G-VIR, which contains a disinfecting agent for enveloped viruses, has been developed. Herpes simplex type 1 (HSV) was used as a standard enveloped virus in both in vitro and in vivo tests of the virucidal capacity of the glove. Bovine viral diarrhea virus (BVDV) and feline immunodeficiency virus (FIV) were used as models for HCV and HIV, respectively. For in vitro study, a contaminated needle was passed through a glove and residual virus was titrated; for in vivo studies, animals were stuck with a contaminated needle through a glove. Despite variation in virus enumeration inherent in the puncture technique, statistical evaluation showed that infection was reproducibly and substantially reduced by passage through the virucidal layer. For BVDV, the amount of virus passing through the virucidal glove was reduced in 82% of pairwise comparisons with control gloves that lacked the virucidal agent; when plaque counts were adjusted to a common dilution, the median count for the virucidal glove was on the average reduced >10-fold. In experiments in which the proportion of wells infected with FIV was measured, the ratio of TCID(50) values (control glove to G-VIR) was >15, and probably much higher. For HSV, the amount of virus passing through the virucidal glove was reduced in 81% of comparisons with control gloves; the median of adjusted plaque counts was reduced on the average approximately eightfold or ninefold. In vivo tests with FIV and HSV in cats and mice, respectively, found smaller percentage reductions in infection than the in vitro tests but confirmed the virucidal effect of the gloves.     

Interaction of concanavalin A with enveloped viruses and host cells

HVJ (Sendai virus) and herpes simplex virus, which are both enveloped viruses, have receptors for concanavalin A, but poliovirus, which has no envelope, has no receptors. The HA activity of HVJ was not lost on exposure to concanavalin A; it seems that the sites on HVJ adsorbing concanavalin A differ from the active site of its hemagglutinin.     

Acylation of viral spike glycoproteins: a feature of enveloped RNA viruses

The covalent attachment of fatty acids to the glycoproteins of orthomyxo-, paramyxo, alpha-, and coronavirus was studied. All enveloped viruses analyzed afford covalently bound fatty acid in at least one species of their spike glycoproteins. No internal components of the viruses studied including the hydrophobic M proteins of myxo- and rhabdoviruses contained fatty acid. Analysis of myxovirus particles devoid of the exposed portions of their spikes revealed that fatty acids are linked to the hydrophobic tail fragment of the glycoprotein which is associated with the viral lipid bilayer. With influenza virus hemagglutinin the fatty acid attachment site could be located at the cyanogen bromide peptide of the small subunit (HA₂) which contains the membrane-embedded region of the polypeptide. The binding of fatty acids to viral glycoproteins occurs in a wide range of host cells including mammalian, avian, and insect cells.     

The protease inhibitor p-nitrophenyl-p' -guanidinobenzoate inactivates sindbis and other enveloped viruses.

Sindis virions were inactivated by the protease inhibitor p-nitrophenyl-p′-guanidinobenzoate (NPGB) in a pH-, temperature-, and time-dependent reaction that decreased the titer of infectious virus by 4 to 5 logs. Treated virions remained structurally intact and competent for adsorption and penetration into host cells but failed to induce viral-specific RNA synthesis. Viral RNA from NPGB-treated virions was defective both as a messenger RNA in an in vitro protein synthesizing system and as a source of infectious RNA when tested in BHK cells. Additional studies with NPGB-treated defective-interfering particles and complementation tests with temperature-sensitive mutants of Sindbis virus suggested that NPGB-inactivated RNA could not serve as a template for replication. NPGB inactivated a variety of other enveloped viruses including influenza, Rous sarcoma, and lactic dehydrogenase virus but had no effect on the nonenveloped encephalomyocarditis and reo viruses. Thus, NPGB appears to inactivate virions by penetrating the viral envelope and acylating sensitive sites on viral RNA.     

Competition binding assay in cell culture for identification of epitopes on enveloped and naked viruses

Virus infected monolayers, in wells of 96-well plates, could be used as antigen in competition binding assays to identify epitopes on respectively Semliki Forest virus, encephalomyocarditis virus and mumps virus.     

Two different mechanisms of the inhibition of the multiplication of enveloped viruses by glucosamine.

In Earle's medium containing 10 mM glucose, glucosamine inhibits the multiplication of fowl plague virus (FPV) and Semliki Forest virus (SFV) by interfering with the synthesis of viral glycoproteins, while the synthesis of viral RNA and carbohydrate-free proteins is affected only slightly or not at all. If glucose is replaced by fructose, glucosamine inhibits the synthesis of viral RNA and in this way interferes with the production of all viral proteins. Protein and RNA synthesis of non-infected cells are not significantly impaired under these conditions. The effect on viral RNA synthesis can be readily counteracted by uridine. The inhibition of viral glycoprotein synthesis cannot be reversed at all by uridine and that of viral carbohydrate-free proteins only to a very limited extent. In fructose-containing medium, glucosamine depletes the cells of UTP in such a way that the UTP pool may become rate limiting for viral RNA synthesis but not for cellular RNA synthesis.The multiplication of Newcastle disease virus (NDV) and vesicular stomatitis virus (VSV) is inhibited by glucosamine only in fructose-containing medium but not in glucose-containing medium.     

Cell fusion induced by Germiston and Wesselsbron viruses

Summary Both Germiston and Wesselsbron viruses were found to rapidly induce polykaryocytosis when added to cell cultures; the immediate nature of the reaction suggested that the mechanism was fusion from without. Mouse peritoneal macrophages and Vero, LLC-MK2,Aedes albopictus and L cells were susceptible to fusion. Chloroform treatment did not affect the ability of either virus to induce cell fusion even though the infectivity of Germiston virus was completely destroyed by chloroform. When chloroform-inactivated virus was added to cell cultures at high multiplicities, all cells in the monolayer were fused into a single syncytium.Recipient of a WHO scholarship from Arlangga University, Surabaya, Republic of Indonesia.     

Activation of the alternative complement pathway by enveloped viruses containing limited amounts of sialic acid

We have demonstrated that purified enveloped viruses grown in MDBK cells, such as influenza virus, Simian virus 5 (SV5), and vesicular stomatitis virus (VSV) grown in the presence of SV5 activate the alternative complement pathway, whereas VSV grown in BHK21-F or HKCC cells and Sindbis virus grown in BHK21-F cells do not. A direct correlation between the amount of sialic acid associated with the viral surface and its ability to activate the alternative complement pathway has been demonstrated. Our results indicate that enveloped viruses that lack sialic acid are efficient activators of the alternative complement pathway, whereas those with ≥10 μg of sialic acid/mg of protein do not. Enveloped viruses with 5–10 μg of sialic acid/mg of protein are intermediate in their ability to activate the alternative complement pathway. The results of our experiments employing different enveloped viruses with biologically derived sialic acid content support the hypothesis (D. T. Fearon, 1978, Proc. Nat. Acad. Sci. USA75 1971–1975) that sialic acid is a key membrane constituent for modulating activation of C3 via the alternative complement pathway.     

A proposed treatment for pathogenic enveloped viruses having high rates of mutation or replication

Several enveloped viruses, particularly some RNA viruses, have high rates of mutation or replication, which can make them virulent pathogens in humans and other mammals. A proposed treatment could use synthesized proteins to mask pathogenic viral surface proteins to quickly induce an immune attack on specific enveloped viruses by using existing immune cells. One treatment could inject dual-protein ligand masks into patients' bloodstreams to mask pathogenic surface proteins used to infect mammalian cells. The mammalian immune system already uses an analogous, more complex structure called a pentraxin to neutralize some pathogens by connecting their surface proteins to immune cells. And several types of antiviral peptides have already experimentally demonstrated effectiveness in blocking various viral pathogen infections. These treatments offer advantages, especially for currently untreatable viral pathogens. Furthermore, using dual-protein ligands and the antigenic memory of some sub-populations of NK cells would also allow the creation of defacto vaccines based on a host's NK cells, instead of vaccines utilizing CD4 and CD8 α:β T cells, which are limited by the requirement of MHC presentation of the target antigens to α:β T cells. Targeted NK cell vaccines could attack host cells latently or actively infected by intracellular pathogens, even host cells having pathogen downregulated MHC antigen presentation. Eight postulates concerning the effects of pathogen mutation, or change in phenotype from genetic recombination or rearrangement, and replication rates on pathogen vs host dominance are also listed, which should be applicable to viral and non-viral pathogens.     

Ultrastructural organization of liposomes and isolated glycoproteins of enveloped viruses obtained by using the nonionic detergent MESK

Materials on comparative ultrastructural analysis of isolated glycoproteins (GP) of influenza, parainfluenza, and Venezuelan equine encephalomyelitis viruses as well as liposomes reconstructed on their basis prepared with the use of MECK, a nonionic detergent, are presented. Original virions, subvirion particles, and isolated GP were characterized by typical morphology and remained intact in highly purified and concentrated suspensions owing to the use of the sparing and effective method of treatment with MECK. After removal of the detergent typical micellar aggregates were shown to form from GP monomers for all three viruses under study. The forming rosettes morphologically and biologically are similar to those isolated from ortho-, paramyxo-, and togaviruses using the Triton X-100 detergent. Stages of liposome formation and interaction with cell membrane were studied electron microscopically. A common feature for liposomes from GP of all the three viruses consisted in the formation of two morphological variants of structural complexes: filaments of various lengths and composition in them of GP and vesicles, the arrays of GP in which were similar or identical to those of viral membrane. Liposomes from Sendai virus GP possessed hemolytic activity, however, their interaction with the cell studied by means of the immunoferritine label occurred not by fusion but apparently as a result of destruction of the cell membrane at the sites of protein F contact with plasmalemma and formation in the latter of defects leading to erythrocyte lysis.     

CPF-DD is an inhibitor of infection by human immunodeficiency virus and other enveloped viruses in vitro.

The initial step in the infection cycle of human immunodeficiency virus type 1 (HIV-1) involves binding of its surface glycoprotein gp 120 to the T lymphocyte CD4 antigen. CPF-DD is a low molecular weight inhibitor of HIV infectivity that inhibits gp 120 binding to CD4 in vitro (Finberg et al., Science 249, 287-291, 1990). We find, however, that the actions of CPF-DD are not limited to its ability to interfere with gp 120-CD4 binding; its predominant action is to remove the viral envelope from the underlying core. Subsequently the virions disintegrate. Most enveloped viruses tested were inhibited by CPF-DD, but the infectivity of noneneloped viruses was unaffected or only slightly reduced.     

Interaction of enveloped viruses with planar bilayer membranes: observations on Sendai, influenza, vesicular stomatitis, and Semliki Forest viruses

Exposure of a planar lipid bilayer to Sendai virus at pH 7.0 resulted in conductance increases that continued over several minutes, provided that the virus particles had first been conditioned by freezing and thawing, sonicating, or storing for 2 weeks in the cold. Individual electrical events could not be resolved, even on a millisecond time scale, and thus do not reflect the insertion of structural channels into the lipid bilayer. Prior treatment of the Sendai virions with protease prevented the conductance increases, but exposure of the bilayer to protease after induction of the conductance change did not abolish it. The Sendai-induced conductance change was increased in rate, but qualitatively unchanged in nature, if gangliosides were included in the planar bilayer. Activity for Sendai virus was low at pH 5.0, and increased with increasing pH up to 9.0. Influenza, Semliki Forest virus, and vesicular stomatitis virus all induced similar conductance changes around pH 5.2, but were inactive when tested at pH 7.0. The presence of cholesterol in the bilayer caused marked enhancement (two- to sixfold) of the response to Sendai, influenza and Semliki Forest virus, but caused only slight enhancement of the response to vesicular stomatitis virus. It is concluded that the observed increases in ionic permeability arise from alterations in lipid motions on a submillisecond time scale resulting from the incorporation of damaged viral membranes into the planar bilayer by fusion.