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The author advocates testing, diagnosis and therapy of HIV infection as soon as possible after contracting the virus and whenever feasible. The arguments are deduced from the results of basic research. The following should be reduced, delayed or inhibited: (1) the viral load in blood plasma and semen; (2) rapid internal propagation of the virus, which is combined with integration of proviruses into cells of unknown life span and compartmentalisation (e.g. the brain may present a sanctuary site); (3) rapid individual formation of quasispecies out of initially homogeneous virus strains of suboptimal fitness, combined with the transition of NSI strains to the more aggressive SI strains and escape from the immune response and therapy; (4) irreversible damage to the immune system; later opportunistic infections; (5) unconscious transmission of possibly drug-resistant virus. Early diagnosis and therapy appear possible in many cases, involving major advantages for individuals and society.

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Translocation of baculovirus nucleocapsids (45 nm in diameter, approximate length of 280-300 nm) from nucleoplasm to cytoplasm was studied morphologically using cryofixation and gold labeled wheat germ agglutinin (WGA-gold) during recombinant Autographa californica nuclear polyhedrosis virus infection in Sf9 cells. Nucleocapsids formed in the nucleoplasm migrated into protrusions of the nuclear envelope, but not into nuclear pore complexes. We found cross-like membranous structures. Small pores seemed to be in the protruding nuclear double membranes. The middle piece of a nucleocapsid was located within the small pore whereas the upper part was in the cytoplasm. Other nucleocapsids were situated within pores without colocalization of WGA-gold in the nuclear envelope. These results suggest that baculovirus nucleocapsids use small pores in the nuclear-derived membranes or incomplete nuclear pores in the nuclear envelope to migrate from the nucleoplasm to the cytoplasm, but not complete nuclear pore complexes proper.

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We have analysed the temperature dependence of the transport of Semliki Forest virus (SFV) envelope proteins in mosquito cells, the natural host cells of alphaviruses. These cells are cultivated at a lower temperature (28 degrees C) and have a different lipid composition as compared to mammalian cells. When the incubation temperature was reduced at early times after infection, the onset of virus shedding was delayed and the maximal titers decreased correspondingly to the temperature. No virus was shed at 12 degrees C. No evidence was observed for a block of virus release due to a shift of the sites of virus maturation. When the incubation temperature was reduced at later times after infection a critical temperature of 12 degrees C was again observed. At this temperature no transport of viral proteins took place, p62 remained uncleaved, the glycan processing of E1 did not occur and the envelope proteins accumulated in a pre-Golgi compartment. We suggest a mathematical formula which allows the extrapolation of transport data to the temperature at which intracellular protein transport becomes blocked.

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Binding to the surface of susceptible cells, the initial step of viral infections, represents an yet poorly understood event in the case of alphaviruses. In intact cells, the binding per se can only be studied when endocytosis is inhibited, e.g., at low incubation temperatures. However, nonphysiologically low temperatures may give only an imperfect picture of the events taking place at the cell surface during binding. In this report we present the application of chemically induced plasma membrane vesicles (PMV) for binding studies. PMV produced overnight from Vero cells give high yields of homogeneous vesicles. PMV represent cellular plasma membranes as far as protein composition and enzyme activities are concerned, but are not competent for endocytosis. Binding experiments to PMV were performed using Semliki Forest virus (SFV), a prototype of alphaviruses. The results show that the binding sites at the PMV surface are saturable and not competed by bovine serum albumin. Binding appeared to be specific and biologically relevant since fusion between viral and PMV membranes could be induced by lowering the incubation pH. Our model is of general interest since many cell types that are susceptible to viral infection may be induced to release PMV after adapting the method.

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Three lines of observation demonstrate the role of arthropods in transmission and evolution of viruses. a) Recent outbreaks of viruses from their niches took place and insects have played a major role in propagating the viruses. b) Examination of the list of viral families and their hosts shows that many infect invertebrates (I) and vertebrates (V) or (I) and plants (P) or all kingdoms (VIPs). This notion holds true irrespective of the genome type. At first glance the argument seems to be weak in the case of enveloped and non-enveloped RNA viruses with single-stranded (ss) segmented or non-segmented genomes of positive (+) or negative polarity. Here, there are several families infecting V or P only; no systematic relation to arthropods is found. c) In the non-enveloped plant viruses with ss RNA genomes there is a strong tendency for segmentation and individual packaging of the genome pieces. This is in contrast to ss+ RNA animal viruses and can only be explained by massive transmission by seed or insects or both, because individual packaging necessitates a multihit infection. Comparisons demonstrate relationships in the nonstructural proteins of double-stranded and ss+ RNA viruses irrespective of host range, segmentation, and envelope. Similar conclusions apply for the negative-stranded RNA viruses. Thus, viral supergroups can be created that infect V or P and exploit arthropods for infection or transmission or both. Examples of such relationships and explanations for viral evolution are reviewed and the arthropod orders important for cell culture are given.

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The acylation of the envelope proteins of Semliki Forest virus by palmitic acid in infected mosquito (C6/36) cells was investigated. It is shown that in these cells palmitic acid was incorporated post-translationally via hydroxylamine-labile linkages onto cysteines in the inner domains of the viral envelope proteins. The kinetics of incorporation, however, differed considerably as compared to higher eukaryotic cells. (i) The precursor of the envelope proteins E2 and E3, p62, was weakly and incompletely palmitoylated irrespective of the duration of labeling. (ii) Under all conditions tested complete acylation of E2 was delayed as compared to E1. (iii) Heavy protein complexes were formed consisting of unacylated p62 and partially unacylated E1. From this data, we conclude that during the maturation of SFV glycoproteins in mosquito cells differently acylated intermediates of p62/E2 exist. Furthermore, acylation of p62/E2 and cleavage of p62 are coupled events, occurring in an early compartment and allowing the release of the envelope oligomers for transport.

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The structure of the N-linked oligosaccharides of Semliki Forest viral glycoproteins produced in infected mosquito cells (C6/36) was investigated by biosynthetic labeling, enzymic deglycosylation using endo-beta-N-acetylglucosaminidases H, D, F/glycopeptidase F, exoglycosidase and analysis of the sugars on Concanavalin A-Sepharose columns and by gel filtration chromatography. The results demonstrated that the glycoproteins decorating the virus shed from infected cells have N-linked glycans with a trimannosyl core similar to the core glycans produced by vertebrate and yeast cells. However, the E1 glycoprotein produced by infected C6/36 cells exhibited both a trimannosyl core and a modified trimannosyl core most probably with terminal N-acetylglucosamine. The carbohydrate side chains of Semliki Forest envelope proteins displayed two types of structural heterogeneities existing either at different N-glycosylation sites as in the case of E2, or at the same N-glycosylation site as in the case of E1. In the presence of 1-deoxymannojirimycin, no structural heterogeneities in the glycan chains were found. This strongly suggests that the glycosylation events that lead to the observed sugar heterogeneities occur in the Golgi membranes.

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Semliki Forest virus capsid (C) protein molecules (Mr, 33,000) can be introduced efficiently into the cytoplasm of various target cells by electroporation, liposome, and erythrocyte ghost-mediated delivery (M. Elgizoli, Y. Dai, C. Kempf, H. Koblet, and M.R. Michel, J. Virol. 63:2921-2928, 1989). Here, we show that the transferred C protein molecules partition rapidly from the cytosolic compartment into the nucleus. Transport of the C protein molecules into the nucleus was reversibly arrested by metabolic inhibitors, indicating that the transfer process is energy dependent. Fractionation of isolated nuclei revealed that the delivered C protein preferentially associates with the nucleoli. This finding was confirmed by morphological studies, showing that in an in vitro system containing ATP isolated nuclei rapidly accumulated rhodamine-labeled C protein in their nucleoli. Furthermore, in this assay system, the lectin wheat germ agglutinin prevented transfer of C protein through nuclear pores. These results are in agreement with our observation that nucleoli contain measurable amounts of newly synthesized C protein as early as 5 h after infection of cells with SFV. Thereafter, nucleolar-associated C protein increased progressively during the course of infection.

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The cleavage of p62 of Semliki Forest virus (SFV) in C6/36 (Aedes albopictus) cells was investigated by pulse-chase labeling experiments and analysis of the sugar side chain of E1 using endoglycosidases. Similar to vertebrates, E1, E2, and p62 are transported as complexes in C6/36 cells. This observation allows the use of E1 as a positional marker for the transport and processing of E2 and p62. The oligosaccharide on the viral spike E1 protein was modified first to an Endo-D-sensitive (35 min) and then to an Endo-H-resistant structure (55 min), whereas the oligosaccharides of p62 remained sensitive towards Endo-H the whole time. E2 could be detected already at 10-20 min post synthesis, suggesting that p62 cleavage starts early, probably before the protein has been transported to the Golgi apparatus. This is in contrast to the cleavage taking place later mainly near the plasma membrane of higher eukaryotes. The spike proteins finally appeared in extracellular virions after about 70-90 min post synthesis.

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The Semliki Forest virus capsid (C) protein was introduced into various target cells by electroporation-, liposome-, and erythrocyte-ghost-mediated delivery. Data are presented which show that the incorporated C protein is biologically active and, at low concentrations (10(3) to 10(4) molecules per cell), markedly induces host cellular protein synthesis (average value, up to 90%). On the other hand, high concentrations (10(5) to 10(6) molecules per cell) led to a significant inhibition (average value, up to 60%). The cellular response to C protein was found to be identical in P3X63Ag8 suspension cells, CV-1 cells, and GpBind4 cells. Following electroporation-mediated delivery of C-protein molecules, both induction and repression of cellular protein synthesis were immediate, whereas with liposome-mediated delivery these events were delayed by about 1 h. Maximum stimulation and repression occurred between 0 and 1 h after delivery of C protein and decreased thereafter to reach control values at about 4 h. The analysis of the proteins synthesized suggests that low amounts of microinjected C protein are responsible for the induction of classes with specific Mrs, whereas high amounts lead to an inhibition of overall protein synthesis.

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In this study we have used 35S-labeled sulfite to modify the disulfide bonds of the proteins at the cell surface of Semliki Forest-infected Aedes albopictus cells before and after low pH treatment. This reagent specifically cleaves disulfide bonds and concomitantly reacts with the newly formed cysteines, thereby labeling the respective protein. Treatment of the infected cells with sulfite led to inhibition of the fusion activity only when applied after low pH exposure. These cells exhibited substantial incorporation of the label into the viral E1 glycoprotein as compared to the E2 glycoprotein. These results provide direct evidence for the low pH-induced conformational change of E1 during the generation of its fusogenic potential.

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Hydrophobic interaction chromatography (phenyl- and octyl-Sepharose) was performed with Semliki Forest virus to investigate the effect of low pH on its hydrophobicity. At neutral pH, the virus could be bound to the column and completely eluted by the detergent NP-40. Low pH treatment of virus prior to application to the column resulted in stronger binding as reflected by the increased amount of detergent necessary to totally elute the virus. If, however, the low pH treatment was done after binding of the virus to the column, only 15% of the input virus could be eluted by the detergent, indicating a drastic increase in hydrophobicity. Thus binding of the virus to a hydrophobic environment potentiates the effect of low pH on viral hydrophobicity. Trypsin digestion of column-bound virus after low pH treatment resulted in complete digestion of E2 and E3; however, E1 was totally resistant. From this result, we conclude that E1 alone is responsible for the hydrophobic interaction. We have made use of these observations to produce viral particles which were devoid of E2 and E3 by trypsin digestion in the presence of octyl glucoside. These E1 viral particles were infectious and could induce membrane fusion. We conclude that only E1 is necessary and sufficient to mediate membrane fusion. Acid pH induces a drastic increase in the hydrophobicity of E1 which probably facilitates its interaction with the lipid bilayers during the fusion event in endosomes.

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In this short review, the impact of molecular biology on microbiology in general is described. Specifically, molecular biology is increasingly enlarging the available choice of methods for the diagnosis of microbial disease. In situ hybridization seems to be a particularly promising procedure. In epidemiology, an interesting facet is the high mutation rate of RNA viruses. In pathogenesis, molecular biology will help to elucidate pathways of infection and the targeting of pathogenic macromolecules within the cell and within an organism.

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The mechanism of the processes leading to membrane fusion is as yet unknown. In this report we demonstrate that changes in membrane potential and potassium fluxes correlate with Semliki Forest virus induced cell-cell fusion at mildly acidic pH. The changes observed occur only at pH's below 6.2 corresponding to values required to trigger the fusion process. A possible role of these alterations of the plasma membrane related to membrane fusion phenomena is discussed.

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Using the suspension cell line P3X63Ag8 we have studied the impact of the composition of the diffusion medium on cellular protein synthesis under standard electroporation conditions in TBS-Na. This buffer contains the high saline concentration usually present in electroporation-mediated DNA transfection. Electroporation in the presence of TBS-Na resulted in an immediate shut-off of protein synthesis, even though both FITC-dextran (Mr 40 kD) and Semliki Forest virus core protein (Mr 33 kD) were incorporated efficiently into the cytoplasm across the electropores at 0 degrees C. Subsequent resealing of the pores was completed after a 5-min incubation at 37 degrees C. When compared with control cells, overall protein synthesis of electroporated cells recovered slowly to resume a 30% activity after 1 h of incubation at 37 degrees C. We have determined optimal conditions for diffusion loading (which necessitates the presence of ATP, GTP, amino acids, K+, Mg2+, and Ca2+) and resealing (in the presence of K+, Mg2+, and Ca2+), leading to a full and lasting recovery of protein synthesis within 5 min after pore closure.

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The effects of N-linked-oligosaccharide-processing inhibitors on the formation of Semliki Forest virus (SFV) in C6/36 Aedes albopictus cells were investigated. The glycosidase inhibitors deoxynojirimycin, deoxymannojirimycin and swainsonine prevented the formation of Endo-H resistant structures, but had little effect on virus formation and on the biological activities of the virus. Tunicamycin greatly inhibited virus formation, but had little effect on cell-cell fusion from within and the cleavage of p 62. These results indicate that correct glycosylation is not a prerequisite for biological activities of SFV, whereas glycosylation per se is needed for virus production.

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The conditions needed to trigger Semliki Forest virus (SFV) induced cell-cell fusion from within (FFWI) of baby hamster kidney (BHK) cells differs from some other cells (e.g., Aedes albopictus cells). In contrast to such cells it is mandatory for BHK cells to be brought back to a neutral pH after a short exposure to mildly acidic pH for fusion to occur. This phenomenon can be explained by a sudden drop of the intracellular pH observed after clamping the extracellular pH below 6.2--the pH required to trigger fusion--and by the thereby implied increased expenditure of ATP. Since the fusion from within of SFV-infected cells is energy dependent [Kempf et al. (1987) Arch Virol 95: 111-122] the fusion process is blocked due to the ATP depletion.

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