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Virus adsorption and uptake of human rhinovirus 14 (HRV14) were studied with HeLa cells and baby hamster kidney (BHK) cells which were transfected with the HRV14 receptor intercellular adhesion molecule-1 (ICAM-1). Transmission electron microscopy of HeLa cells revealed that HRV14 was internalized via clathrin-coated pits and -coated vesicles. A minority of virus particles also used uncoated vesicles for entry. The internalization showed the characteristics of receptor-mediated endocytosis. Presence of the carboxylic ionophore monensin inhibited viral uncoating, indicating a pH-dependent entry mechanism. The expression of ICAM-1 on the surface of the ICAM-1 transfected baby hamster kidney cells (BHK-ICAM cells) allowed extensive virus adsorption and internalization through membrane channels. Virus particles were lined up in these channels like pearls on a string, but did not induce a productive infection. Although ICAM-1 was expressed to the same degree on BHK-ICAM and HeLa cells, HRV14 induced neither viral protein and RNA syntheses nor infectious virus progeny in BHK-ICAM cells. ICAM-1 on the transfected BHK cells was a functional active receptor as it rendered these cells permissive to coxsackievirus A21. These results suggest that HRV14 uptake into BHK-ICAM cells is blocked directly in or shortly after its final step of internalization, the uncoating. Our findings underline that the receptor ICAM-1 determines virus uptake into cells, however, is not sufficient to confer susceptibility of BHK cells to HRV14 infection.

We have previously shown that the pseudorabies virus (PrV) glycoproteins gB and gC (former PrV-gII and PrV-gIII) exhibit heparin-binding properties. While PrV-gC functions as the major adsorption protein, the biological role of the heparin-binding properties of PrV-gB are not understood. We used a gC-deleted PrV-mutant, PrV (dlg92/dltk), to analyse the heparin-binding properties of PrV-gB and the biological role of the PrV-gB-protein in adsorption. PrV-gB was the only glycoprotein of this vaccine strain binding to immobilised heparin in in vitro assays. Presence of the gC-protein was not necessary for the interaction of gB with heparin. Soluble heparin also interfered with adsorption of this mutant virus to a similar extent as it blocked adsorption of wild-type PrV (Ka), but it had only a minor inhibitory effect on infectivity of the mutant strain. These results show that PrV-gB interacts specifically with immobilized heparin and heparin-like structures on the cell surface, but this interaction is not required for a productive infection.

Pseudorabies virus (PrV), an alphaherpesvirus of swine, uses cellular heparan sulfate residues as a receptor for attachment. Interaction of the virus with its receptor is mediated by the envelope glycoprotein C (PrV-gC), a protein with heparin-binding properties. We have previously shown that a region of this protein shows structural similarities to the high-affinity heparin-binding site of the serum protease-inhibitor antithrombin III (ATII). In this publication, we describe the effect of ATIII on interaction of PrV with its cellular receptor. ATIII bound specifically to heparan sulfate residues on the surface of herpesvirus-permissive RK13 cells. Binding of ATIII to RK13 cells interfered with adsorption of radioactively labelled PrV to these cells. Enzymatic treatment using heparinase I (E.C. 4.2.2.7) removed the receptor for PrV as well as the receptor for ATIII. Since amino acids 130-137 of the high affinity heparin-binding site of ATIII show structural similarities to amino acids 134-141 of PrV-gC, both sequences were synthesized as synthetic peptides. Although interaction of the peptide derived from ATIII with heparin was significantly stronger, both peptides interacted specifically with heparin in assays in vitro. These results suggest that PrV and ATIII interact with the same structure on the cellular surface.

Previous studies from Spain, Italy, and France have demonstrated a high prevalence (71% to 91%) of antibodies against hepatitis C virus in patients with porphyria cutanea tarda (PCT). To determine the role of hepatitis C virus (HCV) in PCT in Germany, we have assessed the prevalence of antibodies against HCV and hepatitis B virus (HBV) in 106 patients (mean age, 60 +/- 14 years) with the disease. Eight of 106 patients (8%) were positive for HCV antibodies and HCV RNA using second-generation enzyme-linked immunosorbent assay (ELISA), recombinant immunoblot assay, and polymerase chain reaction. Antibodies against HBV core antigen were found in 14 patients (13%). Of the patients with antibodies against HCV alanine transaminase (ALT) (aspartate transaminase [AST]) levels above normal occurred in 71% (86%). Because elevated ALT (AST) levels were also found in 51% (64%) of 88 patients without markers of HCV or HBV, we suggest that liver damage in PCT may exist in absence of these viruses. This is supported by the finding that in patients without HCV or HBV markers, higher serum ALT and AST activities were found in patients with overt disease or relapse (ALT, 59 +/- 44 U/L; AST, 37 +/- 21 U/L), whereas patients in remission displayed significantly lower serum enzyme activities (ALT, 16 +/- 8 U/L; AST, 16 +/- 7 U/L), (P < .001). These results indicate that HCV infection does not play a major role in the pathogenesis of PCT in Germany.

The pseudorabies virus glycoprotein III (PrV-gIII) has been identified previously as the major viral component binding to a heparin-like receptor on the surface of target cells. The amino acid sequence of gIII contains three regions corresponding to consensus sequences for heparin binding. A synthetic peptide corresponding to amino acids 134 to 141 of PrV-gIII bound heparin in a dot blot assay. In contrast, a synthetic peptide derived from amino acids 290-299 of PrV-gIII did not bind heparin. We therefore conclude that the region containing amino acid 134-141 is involved in binding to the heparin-like cellular receptor.

Objective: To evaluate changes in serum HIV p24-antigen levels in a subset of patients who participated in a European/Australian double-blind, placebo-controlled trial evaluating the efficacy of zidovudine (250 mg every 6 h) alone or in combination with acyclovir (800 mg every 6 h) in patients with AIDS, AIDS-related complex (ARC) or Kaposi's sarcoma (KS)., Design: Double-blind, placebo-controlled randomized clinical trial of less than or equal to 6 months' therapy., Setting: Samples were obtained from patients attending teaching hospital outpatient clinics in seven European countries and Australia., Subjects: One hundred and ninety-seven HIV-infected patients (60 with AIDS and 137 with ARC or KS)., Main Outcome Measures: Serum HIV p24-antigen levels measured using the Abbott HIV solid-phase enzyme immunoassay., Results: Of 76 ARC/KS patients who were initially HIV p24-antigen-positive, one out of 25 randomized to placebo, eight out of 23 to zidovudine and 11 out of 28 to the zidovudine/acyclovir combination became antigen-negative. The proportion of patients who became antigen-negative was significantly higher in both the zidovudine group (P = 0.016) and the zidovudine/acyclovir group (P = 0.004), compared with the placebo group. There were no statistical differences between the zidovudine and the zidovudine/acyclovir groups. During the trial p24-antigen levels in the zidovudine-treated patients reached their minimum after 4-8 weeks of therapy, and tended to increase gradually thereafter. Disease progression occurred irrespective of whether p24-antigen levels declined during therapy. No association between p24-antigen responses to therapy and baseline disease stage, Karnofsky score or baseline CD4 cell count was detectable., Conclusion: Acyclovir does not potentiate the effect of zidovudine on p24-antigen levels. Change in antigen level in response to antiviral therapy needs further investigation before it is used as a surrogate marker for clinical efficacy of antiviral therapy.

Glycoproteins in the range 50 and 23/25 kDa were identified as poliovirus specific binding sites on HeLa cells with the monoclonal antibody mAb 122. mAb 122 is characterized by its partial inhibiting effect on poliovirus reproduction and adsorption when prebound to HeLa cells. The binding sites are endocytosed in native cells and specific for poliovirus as mAb 122 did not interfere with the adsorption of human rhinovirus type 14 (HRV 14). The poliovirus binding sites are present also on nonprimate so called nonsusceptible cells, e.g., mouse L-cells, as could be shown with sensitive ELISA based binding assays and performance of binding studies with fixed cells at 37 degrees.

A combined assay consisting of a pre-cpe-protection assay and a double-antibody sandwich ELISA for detecting poliovirus was developed on a microtiter scale in order to quantify inhibition of virus replication caused by cell protective antibodies. The system was of high sensitivity and allowed the measurement of the protecting effect caused by a broad range of antibody concentrations before appearance of cytopathic effects. It was applied as a screening test for a large number of hybridomas secreting antibodies specific to the surface of HeLa cells and allowed the identification of four monoclonal antibodies (mAbs) with partial protection activity against poliovirus infection. One of the antibodies, mAb 122, detected SDS-PAGE-separated HeLa cell membrane proteins of 23-25 kDa and 50 kDa by immunoblot, indicating that these proteins are involved in poliovirus adsorption.

The glycoprotein complex gII of pseudorabies virus was isolated by immunoprecipitation with the monoclonal antibody M5, which was covalently linked to protein A-Sepharose. After sodium dodecyl sulfate-polyarylamide gel electrophoresis under reducing conditions and blotting onto poly(vinylidene difluoride) membrane, its subunits, gIIa, gIIb, and gIIc, were subjected to N-terminal sequencing. gIIa and gIIb start at position 59 and gIIc starts at position 503 according to the amino acid sequence deduced from the gene, indicating that there is one major protein (gIIa) which is cleaved into the two protein fragments gIIb and gIIc. Protein labeling with 14C-amino acids gave no indication that the three proteins (gIIa, gIIb, and gIIc) of the complex are present in equimolar ratios. It seems that gIIa is only a minor component of the complex, whereas gIIb and gIIc are contained in equimolar amounts.

To determine whether heparan sulphate residues on the cellular surface could serve as an attachment receptor for pseudorabies virus (PRV), the effect of heparin on PRV in plaque reduction and adsorption tests was investigated. PRV was significantly less sensitive to heparin than was herpes simplex virus type 1 (HSV-1). At concentrations of 500 micrograms/ml heparin the number of plaques formed by PRV was reduced to 7% of the untreated control whereas the number of plaques formed by HSV-1 was reduced to below 0.1%. Adsorption of PRV to host cells was also less sensitive to heparin treatment than was adsorption of HSV-1. Experiments concerning the binding sites of PRV showed that heparin binds to the disulphide-linked glycoprotein complex gII (PRV gB), gIII (PRV gC) and probably gV.

Infection with herpes simplex virus type 1 (HSV-1) induces different morphological changes in different cell lines. This is demonstrated by comparative scanning (SEM and transmission (TEM) electron microscopic investigations of cell cultures prepared under identical conditions. SEM of HSV-1 infected HEp-2 cells reveals a slightly altered cell surface: only the number of the microvilli is reduced. Large amounts of released virions are detectable adhering to the outer plasma membrane. Ultra-thin sections show typical virus maturation steps in the nuclei (formation of nucleocapsids and virus budding from the inner lamella of the nuclear membrane) and in the cytoplasm (egress of enveloped nucleocapsids through membranous structures). HSV-infected primary chick embryo fibroblast (CEF) cells are characterized by crumpled and rough surfaces without virus particles adhering to the membrane. Ultra-thin sections exhibit atypical virus maturation with many unenveloped nucleocapsids within the cytoplasm. The distribution of HSV-induced antigen(s) on the surface of the infected cells is identical in the two cell systems as determined by the peroxidase labelling technique. The c.p.e. (as seen by phase contrast light microscopy) is similar in both HEp-2 and CEF cells: both fusion and rounding up is induced in the infected cells.

The replication of Mouse Elberfeld (ME) virus was accelerated when HEp-2 cells were mixedly infected with poliovirus in the presence of guanidine. The latent period of the replication of ME virus was shortened by 3 h when cells were preinfected for at least 2 h with poliovirus and inhibited by guanidine. Simultaneous infection with poliovirus and ME virus resulted in a shortening by 1 h of the latent period of ME virus replication. The accelerated replication of ME virus was shown to be due to modification and exploitation of a membrane complex induced by poliovirus in the presence of guanidine; on superinfection ME virus successively modified this poliovirus-induced complex of 470S ("light' complex) into a "heavy' complex of 700S specific for ME virus.

Poliovirus type 1 appeared from electron microscope studies to enter HEp-2 cells by receptor-mediated endocytosis. On adsorption the virus was evenly distributed over the cell surface, with some preference for the microvilli and their bases. Invagination of the cell surface membrane with the attached virus commenced at coated pits and led to the formation of virus-containing coated vesicles in the cytoplasm. These coated vesicles fused with intracellular vesicles to form endosomes. When cells infected with poliovirus or Mouse Elberfeld virus were treated with the weak bases chloroquine, NH4Cl or the ionophore monensin to raise the intraendosomal and intralysosomal pH above 6, virus-directed macromolecular synthesis and production of progeny were prevented. These results suggest that the virus genomes are released to the cytoplasm via endosomes and/or lysosomes by a pH-dependent process.

The preexistence of a cytoplasmic membrane complex in HEp-2 cells, induced by poliovirus when inhibited in its reproduction by guanidine, was a prerequisite for accelerated reproduction of superinfecting Mouse Elberfeld (ME) virus. Guanidine-inhibited poliovirus induced a membrane complex of 470S that was successively modified into a faster sedimenting membrane complex (up to 700S) by superinfecting ME virus and exploited for ME virus reproduction. The modified membrane complex was the site for ME virus-specific RNA polymerization characterized by the existence of in vivo and in vitro activity of ME virus RNA polymerase associated with the modified membrane complex. Proof of membrane-bound RNA polymerase and newly synthesized ME virus RNA including replicative intermediate led to the conclusion that superinfecting ME virus exploits the 'poliovirus/guanidine'-induced complex as the site of action of its replication complex.

The surface alterations of HEp-2 cells induced by mixed infection with two different picornaviruses (poliovirus and ME virus) were compared by scanning electron microscopic and transmission electron microscopic studies and by 51Cr-release assay. The contribution of each of the viruses to the resulting surface changes was discernible, as investigations on the chronology of the cytopathic alterations demonstrated that the changes were distinct for either virus. The surface of ME virus-infected cells was characterized by large membranous structures ('sheets' and blebs) representing huge vacuoles. These sheets were not seen in poliovirus-infected cells. Poliovirus induced more prominent cell pycnosis, elongation of filopodia and condensation of collapsed microvilli on the cell surface than ME virus. Mixed infection with these two viruses led to surface alterations typical for ME virus. These ME virus-specific changes occurred irrespective of poliovirus reproduction or its inhibition by guanidine. ME virus-specific alterations also predominated in cytolytic membrane damage as expressed by 51Cr-release from infected cells. 51Cr-release was more pronounced from ME virus than from poliovirus-infected cells, even when ME virus reproduction was suppressed by interfering poliovirus. However, alteration of the internal structures of the infected cells was only dominated by ME virus when the reproduction of poliovirus was suppressed.

Infection of different strains of tissue culture cells with herpes simplex virus type 1 (HSV-1) resulted in a reduced 51Cr-permeability. A stability of the cellular membrane to Triton X-100, toxic sera and HSV-specific complement-mediated immune-cytolysis could be observed simultaneously. The results differed with respect to the cell strain used in the experiments.

Previously we have reported that among the proteins of purified pseudorabies virions there are four major glycoproteins (T. Ben-Porat and A. S. Kaplan, Virology 41:265-273, 1970). Several minor glycoproteins can also be identified by two-dimensional gel electrophoresis. Removal of the viral envelope with Triton X-100 selectively removes from the virions all of the glycoproteins as well as several non-glycosylated proteins. Sedimentation analysis or chromatography of these proteins reveals that several are complexed with one another, some being covalently linked via disulfide bridges. Analysis of the proteins by immunoprecipitation with monoclonal antibodies reactive with the membrane proteins showed also that three of the four major virus glycoproteins (125K, 74K, and 58K; gIIa, gIIb, and gIIc, respectively) are linked covalently by disulfide bridges. Furthermore, all three share extensive sequence homology as indicated by the identity of their antigenic determinants and by partial peptide mapping; they probably originate from a single protein precursor. The fourth major glycoprotein (98K; gIII) is not complexed to any other protein. Three minor glycoproteins (130K [gI], 98K [gIV], and 62K [gV]), which form a noncovalently linked complex with a 115K nonglycosylated protein, have also been identified. Of the monoclonal antibodies used in this study, only those reactive with the major 98K glycoprotein (gIII) inhibit virus adsorption and neutralize virus infectivity in the absence of complement. However, all react with surface components of the virion, indicating that the proteins with which they react are exposed on the surface of the virions. A nomenclature for the pseudorabies virus glycoproteins is proposed.

Poliovirus capsid proteins comprise 15.1 lysines in VP1, 5.6 lysines in VP2, 11.7 lysines in VP3 and 5.5 lysines in VP4. Treatment with monofunctional reagent N-succinimidyl 2,3-3H-proprionate leads to the modification of 3.4 lysines in VP1, 0.6 lysines in VP2, 2.0 lysines in VP3 and 0.03 lysines in VP4. Chemical modification with the monofunctional reagent N-succinimidyl 3-(4-hydroxy,5-125I-iodophenyl)propionate results in a predominant labelling of VP1 and VP3, whereas VP2 is less accessible and VP4 is not modified. Cross-linking of poliovirus with bifunctional imidoesters, dimethyl suberimidate (DMS, 1.1 nm) and dimethyl adipimidate (DMA, 0.8 nm) leads to a new protein complex of mol. wt. which corresponds to the sum of VP1 and VP3. By cleavage with ammonia and electrophoresis on polyacrylamide gels in SDS, the proteins are identified as VP1 and VP3. This result gives evidence for a direct neighbourhood of VP1 and VP3 in the virus capsid. Treatment of the virus with the mono- and bifunctional reagents has no influence on the stability of the particle. The infectivity is reduced only by the bifunctional reagent.

Low doses of u.v. radiation rapidly inactivate poliovirus, and the virus is progressively converted into dense particles (DPs) of buoyant density 1.44 g/ml in CsCl. The DPs are structurally and antigenically related to standard virus (N-antigen), i.e. they are indistinguishable from virus in their RNA and protein content and in their sedimentation properties. Furthermore, there is no difference in reactivity of the structural proteins of virus and DPs with the monofunctional reagent [3H]N-succinimidyl propionate (3H-NSP). However, DPs differ from virus in that their capsids are permeable to several ions, and they can be degraded by RNase and protease. Increasing the radiation dose causes a successive transformation of DPs into 105S slow-sedimenting particles (SSPs). The SSPs are antigenically related to 76S artificial empty capsids (AECs) or H-antigen, but they differ physically and structurally from them. The SSPs have a higher S value than AECs and contain all the capsid proteins, including VP4, and the RNA, both of these macromolecules being absent from AECs. It is concluded, therefore, that transformation from N- to H-antigenicity by u.v. radiation does not require release of RNA and VP4. Conversion of virus particles to SSPs correlates with altered reactivity of VP2 and to a lesser extent VP1 and VP3, with the monofunctional reagent 3H-NSP.

Disoxaril [WIN 51711, 5-[7-[4(4,5-dihydro-2-oxazolyl)phenoxy]heptyl]-3- methylisoxazole] inhibits the replication of polioviruses types 1 and 2 in HeLa cells by stabilizing the virus capsid, which results in the inhibition of the pH-dependent viral uncoating in endosomes and/or lysosomes. As shown by electron microscopy the virus entered into the cell by receptor-mediated endocytosis via coated pits and coated vesicles into endosomes irrespective of the presence or absence of the compound. Measurements of viral RNA synthesis showed that disoxaril completely inhibited the arrival of viral RNA in the cytoplasm for new RNA synthesis only when the inocula were preincubated with disoxaril for 15 min at 37 degrees at 0.3 microgram disoxaril/ml for poliovirus type 1 and 0.03 microgram disoxaril/ml for poliovirus type 2. Simultaneous addition of the compound and virus resulted in reduced inhibition of viral RNA synthesis. The inhibitory effect of the compound could be partially reversed up to 25 min p.i. if the compound was eluted from the cells.

We have evaluated the autopsies of 11 patients with HTLV III/LAV-infection. The clinical diagnosis was AIDS in 10 cases and AIDS related complex (ARC) in one case. The most common infectious disease was pneumocystis carinii pneumonia, occurring in 5 cases. 3 patients showed evidence of mycobacterial infections and another three showed cytomegalovirus infections. Kaposi's sarcoma was found in 4 and other malignancies in 3 cases. Our results are in agreement with the findings of other authors.

Poliovirus type 1 enters HeLa cells by receptor-mediated endocytosis as an intact virus. Up to 30 min after adsorption complete virus particles still containing VP4 and sedimenting with 156 S could be recovered from the cells. These virus particles were N-antigenic and infectious. Thirty minutes after adsorption the recovery of intact and infectious virus decreased. This decrease presumably reflects viral uncoating in the acidic endosomes and/or lysosomes because virus particles could be localized in endosomes at this time. The direct involvement of clathrin-coated structures in the endocytosis of poliovirus has been deduced from the enclosure of poliovirus in coated vesicles at 10 min after adsorption. At this time intact and infectious virus could be recovered only after the coated vesicles were disrupted by treatment with 0.5 M Tris at pH 7.0.