Ionic Contra-Viral Therapy (ICVT); a new approach to the treatment of DNA virus infections.

The sequestration of cellular K(+) has been shown elsewhere to elicit a broad spectrum of antiviral activity. The obligatory, coupled cotransports of Na(+), K(+) and Cl(-) (NKCC1) and of Na(+) and K(+) (NKATPase) effect net cellular K(+) influx. We examined the effects of specific inhibitors of these transports; a cardiac glycoside (Digoxin) and a loop diuretic (Furosemide) on virus replication in vitro. The replication of the DNA viruses, herpes simplex virus, varicella zoster virus, human cytomegalovirus and adenovirus was inhibited. There was normal replication of the RNA virus encephalomyocarditis virus. Antiviral activities of both drugs were influenced by extracellular K(+). Antiviral effects were most potent when Digoxin and Furosemide were used in combination. Targeting the host cell in this way is fundamentally different to other antiviral drug developments to date and we propose the descriptive term Ionic Contra Viral Therapy (ICVT) for the purpose of definition. We believe that specific inhibitors of coupled K(+) transports merit controlled clinical trial for a broad spectrum of DNA virus infections by local application.

Functional and genetic integrity of the CD8 T-cell repertoire in advanced HIV infection.

BACKGROUND: HIV-specific cytotoxic T lymphocytes (CTL) can restrict HIV replication in acute and chronic infection, but disease progression occurs in parallel with declining CTL activity. An understanding of why CTL fail to control HIV replication might reveal important mechanisms of disease progression and enhance prospects for developing effective CTL-based immunotherapies. OBJECTIVES: To investigate the functional integrity, T-cell repertoire diversity, and HIV reactivity of CD8 T lymphocytes in individuals with advanced HIV infection. METHODS: Individuals were considered to have progressed to advanced HIV infection if their total T-cell count was < 500 x 10(6) cells/(l) on at least two successive clinic visits. CD8 T cells from these individuals were analyzed for CTL function, HIV reactivity and T-cell receptor (TCR) diversity by chromium release assays and reverse transcriptase polymerase chain reaction. RESULTS: CD8 T cells from all individuals with advanced HIV infection proliferated and differentiated into functional CTL in vitro. Despite extremely low T-cell counts and previous AIDS-defining illnesses, six individuals had inducible anti-HIV CTL responses. In two additional cases, HIV-specific CTL activity became detectable following significant treatment-associated remission of T-cell lymphopenia. Assessment of TCRbetaV gene family representation and betaV gene intrafamily diversity indicated CD8 T-cell repertoire diversity is maintained through advanced HIV infection. CONCLUSIONS: These data suggest that HIV-specific CTL activity can be selectively compromised while the functional and genetic integrity of the CD8 population as a whole remains intact. A substantial fraction of individuals retain inducible anti-HIV CTL activity through advanced HIV infection and, in at least some cases, effective treatment can restore HIV-specific CTL responses even at this late stage of disease.

Occult lifelong persistence of infectious hepadnavirus and residual liver inflammation in woodchucks convalescent from acute viral hepatitis.

Traces of hepatitis B virus (HBV) genome can persist for years following recovery from hepatitis B. To determine overall duration, molecular characteristics, and pathological implications of this serologically undetectable form of hepadnaviral carriage, we have analyzed the expression of transcriptionally active virus genomes, their infectivity, and examined liver alterations during the natural lifespan of woodchucks convalescent from acute infection with HBV- related woodchuck hepatitis virus (WHV). In this study, we document lifelong persistence of scanty amounts of replicating virus both in the liver and lymphatic system after spontaneous resolution of an episode of experimental hepadnaviral hepatitis. Antibodies to virus nucleocapsid (core) were found to be the most reliable immunovirological marker coexisting with occult infection. In the majority of convalescent woodchucks, serial liver biopsies showed protracted minimal to mild necroinflammation with periods of normal morphology; however, hepatocellular carcinoma (HCC) ultimately developed in 2 of 9 animals studied. Inocula derived from lymphoid cells of convalescent animals induced classical acute hepatitis in virus-naive woodchucks that progressed to chronic hepatitis and HCC in 1 of the animals, demonstrating infectivity and pathogenic competence of the carried virus. Our results reveal that low levels of infectious WHV and residual hepatic inflammation usually continue for life after resolution of hepatitis and that this recovery does not avert HCC development. They also demonstrate that, in addition to the liver, the lymphatic system is the site of the occult lifelong maintenance of replicating hepadnavirus.

Detection of hepatitis B and woodchuck hepatitis viral DNA in plasma and mononuclear cells from heparinized blood by the polymerase chain reaction.

Amplification by the polymerase chain reaction (PCR) of hepatitis B virus (HBV) DNA extracted from parallel samples of serum and heparinized plasma gave contradictory results, indicating that heparin inhibits virus detection. Similarly, analysis of PCR products of woodchuck hepatitis virus (WHV) DNA showed that heparinization of blood abolished WHV DNA amplification, while anticoagulation with sodium EDTA or acid citrate dextrose did not. Amplification of recombinant WHV and HBV DNA in the presence of increasing concentrations of sodium heparin progressively inhibited and finally abolished virus genome detection. The inhibitory effect of heparin was reversed by treatment of either plasma or isolated DNA with heparinase (5 U/reaction, 1 h at 28 degrees C) prior to PCR. In contrast, heparin did not influence the detection of hepadnavirus in peripheral blood mononuclear cells (PBMC), even after prolonged incubation of the cells with heparin in culture. These findings confirm that heparin exerts a dramatic inhibitory effect on hepadnaviral DNA detection by PCR and they demonstrate that this effect can be reversed by heparinase. The findings also show that extensively washed PBMC derived from heparinized blood can be a reliable source of nucleic acids for amplification of hepadnavirus genome. These results imply that previous data should be reassessed if samples of heparinized plasma were found hepadnavirus DNA nonreactive by PCR or when these samples were used as a starting material for PCR quantitation of viral genome.

Identification and characterization of the cell surface 70-kilodalton sialoglycoprotein(s) as a candidate receptor for encephalomyocarditis virus on human nucleated cells.

The attachment of encephalomyocarditis (EMC) virus to human nucleated cells susceptible to virus infection was examined with HeLa and K562 cell lines. Both cell types showed specific virus binding competitively blocked by unlabeled virions. The number of binding sites for EMC virus on HeLa and K562 cells were approximately 1.6 x 10(5) and 3.5 x 10(5) per cell, respectively, and dissociation binding constants were 1.1 and 2.7 nM, respectively. Treatment of cells with cycloheximide after pretreatment with trypsin eliminated EMC virus attachment, suggesting that the virus-binding moiety is proteinaceous in nature. Digestion of cells, cell membranes, and sodium deoxycholate-solubilized cell membranes with proteases or neuraminidases or treatment of cells with lectins demonstrated that the EMC virus-cell interaction is mediated by a sialoglycoprotein. Proteins with a molecular mass of 70 kDa were isolated from detergent-solubilized cell membranes of both HeLa and K562 cells by EMC virus affinity chromatography. The purified proteins, as well as their 70-kDa-molecular-mass equivalents detected in intact surface membranes of HeLa and K562 cells, specifically bound EMC virus in a virus overlay protein blot assay, whereas membranes from nonpermissive K562 D clone cells did not. Western immunoblot analysis with glycophorin A-specific antibody confirmed that the identified 70-kDa binding site on K562 cells is not glycophorin A, which is the EMC virus receptor molecule on virus-nonpermissive human erythrocytes (HeLa cells do not express glycophorin A). These results indicate that EMC virus attachment to permissive human cells is mediated by a cell surface sialoglycoprotein(s) with a molecular mass of 70 kDa.

Persistent infection of K562 cells by encephalomyocarditis virus.

Infection of human erythroleukemic K562 cells by encephalomyocarditis virus readily resulted in establishment of persistently infected cultures. In contrast to the usual typical lytic infection by encephalomyocarditis virus, in which trypan blue staining of cells reaches close to 100% by about 15 h postinfection, K562 cell cultures required 3 to 4 days postinfection to reach a maximum of about 80 to 90% cell staining. The proportion of K562 cells taking up stain gradually decreased to about 10% of those present by about 13 days postinfection; during this time, virus yield per day measured by either plaque or hemagglutination titration fell about 10-fold. The decrease in percent staining was followed by waves of increased staining accompanied by increased virus production. Virus-producing cultures were maintained for over 3 months. Evolution of both virus and cells accompanied establishment of persistence in that plaque size changed from about 7 mm in diameter for the original virus to less than 1.5 mm by day 20 postinfection and most of the cells cloned from persistently infected cultures were resistant to superinfection with the original virus. Resistance was due, at least in part, to reduced virus attachment in that binding of 3H-labeled virus to cloned resistant cells was about 2% of that to uninfected cells.

Genetic stability of Ross River virus during epidemic spread in nonimmune humans.

We have examined the rate of evolution of Ross River virus, a mosquito-borne RNA virus, during epidemic spread through tens of thousands of nonimmune humans over a period of 10 months. Two regions of the Ross River virus genome were sequenced: the E2 gene (1.2 kb in length), which encodes the major neutralization determinant of the virus, and 0.4 kb of the 3'-untranslated region. In the E2 gene, a single nucleotide change was selected which led to a predicted amino acid change at residue 219. No changes were selected in the 3'-untranslated region. By comparison with rates of evolution reported for non-arthropod-borne RNA viruses, the rate for Ross River virus is surprisingly low. We identify three features of the Ross River virus replication and transmission cycle which may limit the rate of evolution of arthropod-borne viruses in the field.

Aggregation of encephalomyocarditis virus induced by radio-iodination.

Radio-iodination causes encephalomyocarditis virus to behave aberrantly when examined by affinity chromatography and to sediment rapidly during analysis on sucrose density gradients suggesting that aggregation had taken place. The change in physical properties of the virus occurred whether iodination was carried out with 125I or 131I, with radio-iodine from two different sources, or using two different iodination procedures. The changes were not observed in virus subjected to an iodination procedure in the absence of radio-iodine suggesting that modification of tyrosine residues was involved rather than a side reaction such as amino acid oxidation. It is recommended that caution be exercised when following the fate of radio-iodinated virus in any particular study because its behaviour may not reflect that of normal, non-iodinated virus present.

A sialoglycopeptide from human erythrocytes with receptor-like properties for encephalomyocarditis and influenza viruses.

Encephalomyocarditis and influenza viruses attach to human erythrocytes causing haemagglutination. The receptor for both viruses on these cells is the major membrane sialoglycoprotein, glycophorin, solubilized preparations of which inhibit haemagglutination by either virus. We show here that glycophorin preparations inhibited haemagglutination of both viruses, even after the preparations were digested with chymotrypsin. To determine which component(s) in the digest exhibited activity, peptides separated by gel filtration were assayed for haemagglutination inhibition; one peptide only, CH-0, was active. A tentative structure was deduced for CH-0 from amino acid and sialic acid analyses. It was already known that neuraminidase treatment of erythrocytes or glycophorin prevents interaction with either virus, suggesting that sialic acid may form part of the active binding site in the receptor. However, receptor activity requires more than the presence of a particular arrangement of sialic acid since the arrangement in CH-0 was identical to that in two other inactive chymotryptic peptides. Examination by gel filtration, sucrose density gradient centrifugation and SDS-polyacrylamide gel electrophoresis demonstrated that Ch-0 readily aggregated, unlike the inactive peptides. It was proposed that the CH-0 chymotryptic peptide showed receptor-like activity (inhibited haemagglutination) because its tendency to aggregate allowed strong multivalent binding with virus particles.

Chromatofocusing of sialoglycoproteins.

Sialoglycoproteins of different sialic acid contents have been separated from each other by chromatofocusing on the ion exchanger PBE 94 using gradients of pH 4.00 down to pH 1.00. The technique is much faster than isoelectric focusing, apparently does not result in desialylation of the sialoglycoproteins and can handle with ease 10 ng to 50 mg quantities of protein on a 22 X 0.9 cm column. The technique revealed that commercial preparations of fetuin and human acid glycoprotein contained several components. Glycophorin, desialylated by controlled neuraminidase treatment, was fractionated by chromatofocusing into several components which differed in sialic acid content and in ability to inhibit haemagglutination by wheat germ agglutinin and encephalomyocarditis and influenza viruses.

The interaction of encephalomyocarditis virus with its erythrocyte receptor on affinity chromatography columns.

Glycophorin, the major sialoglycoprotein in the human erythrocyte surface membrane, can serve as a red cell receptor for both wheat-germ agglutinin (WGA) and encephalomyocarditis (EMC) virus since glycophorin bound to WGA--Sepharose can at the same time bind EMC virus. In contrast, glycophorin bound to WGA--Sepharose cannot bind EMC virus in the presence of SDS. The evidence suggests that virus binding to glycophorin-WGA--Sepharose occurred in the absence of SDS because glycophorin was present in aggregated complexes which were large enough either to accommodate both EMC virus and WGA at the same time, or alternatively to provide sufficient attachment sites for multivalent binding of virions.

Effect of enzymes on the attachment of influenza and encephalomyocarditis viruses to erythrocytes.

Encephalomyocarditis (EMC) and influenza viruses attach to human erythrocytes causing haemagglutination of the cells. Sialoglycoproteins, containing predominantly glycophorin A, from these cells behave as soluble virus receptors and inhibit haemagglutination by both viruses. Removal of 43% of the sialic acid from erythrocytes with neuraminidase prevented their haemagglutination by EMC virus loss of 40% of glycophorin sialic acid destroyed its inhibitory properties against this virus. However, about 80% of the sialic acid had to be removed from erythrocytes or from glycophorin to achieve the same results for influenza virus. Trypsin treatment of erythrocytes or glycophorin had little effect on haemagglutination or inhibition involving either virus, although the glycopeptides released contain up to 70% of the total sialic acid, and despite the fact that glycophorin was drastically reduced in size as shown by SDS--polyacrylamide gel electrophoresis. It is concluded that not all of the sialic acid present in erythrocyte sialoglycoprotein receptors is involved in attachment of EMC or influenza viruses and that the attachment sites on erythrocytes for these viruses are not identical.

Preparation of erythrocyte receptors for viruses by affinity chromatography.

A comparatively simple method for the purification of human erythrocyte receptors for encephalomyocarditis and influenza viruses is described. The procedure utilises the fact that these viruses share in common the erythrocyte receptor for wheat germ agglutinin (WGA), which enables commercially available WGA-Sepharose to be used in the purification of receptors for these viruses by affinity chromatography. Conditions are also described for introducing either 125I into the receptor in situ, or 3H-acetyl residues into the solubilised receptor.

The size and location of the poly(A) tract in EMC virus RNA.

Encephalomyocarditis (EMC) virus RNA, selected by its affinity for oligo(dT)-cellulose, contains poly(A) of size : (i) about 14 nucleotide residues long, based on the percentage of radioactivity in the RNA resistant to digestion by a mixture of pancreatic and T1 RNases; (ii) about 15 residues long, as measured by the ratio of the amount of terminal adenosine to internal adenylic acid in isolated poly(A); and (III) in the range 12 to 45 residues, the majority of tracts being about 16 to 18 residues long, based upon electrophoretic mobility on polyacrylamide gels using poly(A) molecules of known size as mol. wt. markers. The poly(A) appears to be located at the 3'-terminus of the virus genome since the tract, liberated by digestion with a mixture of pancreatic and T1 RNases, was shown by compositional analysis to contain a non-phosphorylated 3'-terminus and only adenine residues. The size heterogeneity in the poly(A) tracts revealed by gel electrophoresis is also consistent with a terminal location. Comparison of our data for EMC virus with published data for other picornaviruses suggests that the sizes of poly(A) tracts in polio- and Mengovirus RNA have been overestimated; poly(A) tracts in cardioviruses appear to be smaller than those in poliovirus; the minimum size of poly(A) required for full infectivity of picornavirus RNA has also been overestimated; a tract of at least 13 adenine residues long is required for full infectivity of EMC virus RNA.

Requirement of an adenylic acid-rich segment for the infectivity of encephalomyocarditis virus RNA.

About 80% of the RNA molecules extracted from encephalomyocarditis (EMC) virus were bound by oligo(dT)-cellulose under conditions which bind poly(A) but not poly(C) nor ribosomal RNA. This shows that most EMC virus RNA molecules contain a poly(A) tract. Both bound and unbound fractions contained RNA molecules of apparently the same size when examined by sucrose gradient sedimentation, but the bound fraction contained an adenylic acid-rich segment of about 20 nucleotides long, whereas the unbound RNA did not. The bound RNA had 200 times the specific infectivity of the unbound RNA which suggests that the poly(A) tract present in EMC virus RNA is required for infectivity.

Chromatographic studies on picornavirus capsid polypeptides.

The polypeptides of encephalomyocarditis, Mouse-Elberfeld and type 5 rhinoviruses behave similarly when chromatographed on calcium phosphate (brushite), each being eluted by a linear phosphate buffer gradient containing sodium dodecyl sulphate in three major peaks, CI, C2 and C3. Analysis of the peaks by polyacrylamide gel electrophoresis suggests that the major capsid polypeptides of these three picornaviruses elute in the order: delta (peak CI), gamma with (peak C2) and alpha (peak C3).