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AIM: To describe the autofluorescence features of choroidal melanoma. DESIGN: Non-comparative case series. PARTICIPANTS: 51 consecutive patients. METHODS: Standard fundus photography and autofluorescence photography (580 nm excitation, 695 nm barrier filter) were performed on all patients. Clinical features were correlated with autofluorescence features. MAIN OUTCOME MEASURE: Autofluorescence features of choroidal melanoma and overlying retinal pigment epithelium (RPE). RESULTS: The mean patient age was 59 years. The choroidal melanoma was a mean of 3.6 mm from the optic disc and 2.6 mm from the foveola. The mean tumour basal dimension was 11 mm and the mean tumour thickness was 4 mm. The choroidal melanoma showed intrinsic hypoautofluorescence (39%), isoautofluorescence (6%) and hyperautofluorescence (55%). Slightly increased hyperautofluorescence of the melanoma was found in pigmented tumours (versus non-pigmented), those with greater thickness and basal dimensions, and those with overlying disrupted RPE. Related RPE hyperplasia and atrophy showed hypoautofluorescence, drusen, RPE detachment and subretinal fluid showed slight hyperautofluorescence, and orange pigment displayed the brightest hyperautofluorescence. CONCLUSIONS: Choroidal melanoma generally shows slight intrinsic hyperautofluorescence and the brightness increases with pigmented tumours, larger tumours, and those associated with disrupted RPE. Overlying orange pigment shows remarkably bright hyperautofluorescence.

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Hepatitis A virus (HAV) encodes a single polyprotein which is posttranslationally processed into the functional structural and nonstructural proteins. Only one protease, viral protease 3C, has been implicated in the nine protein scissions. Processing of the capsid protein precursor region generates a unique intermediate, PX (VP1-2A), which accumulates in infected cells and is assumed to serve as precursor to VP1 found in virions, although the details of this reaction have not been determined. Coexpression in transfected cells of a variety of P1 precursor proteins with viral protease 3C demonstrated efficient production of PX, as well as VP0 and VP3; however, no mature VP1 protein was detected. To identify the C-terminal amino acid residue of HAV VP1, we performed peptide sequence analysis by protease-catalyzed [18O]H2O incorporation followed by liquid chromatography ion-trap microspray tandem mass spectrometry of HAV VP1 isolated from purified virions. Two different cell culture-adapted isolates of HAV, strains HM175pE and HM175p35, were used for these analyses. VP1 preparations from both virus isolates contained heterogeneous C termini. The predominant C-terminal amino acid in both virus preparations was VP1-Ser274, which is located N terminal to a methionine residue in VP1-2A. In addition, the analysis of HM175pE recovered smaller amounts of amino acids VP1-Glu273 and VP1-Thr272. In the case of HM175p35, which contains valine at amino acid position VP1-273, VP1-Thr272 was found in addition to VP1-Ser274. The data suggest that HAV 3C is not the protease responsible for generation of the VP1 C terminus. We propose the involvement of host cell protease(s) in the production of HAV VP1.

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A full-length chicken growth hormone (cGH) cDNA was placed downstream from the Autograph californica nuclear polyhedron virus, AcNPV, polyhedron gene promoter and expressed in Sf9 insect cells. Secreted recombinant cGH levels averaged 2-10 micrograms/ml from day 5-10 postinfection. The recombinant cGH analyzed by SDS-PAGE gels and Western blotting consisted of a doublet with M(r) of 26.5 and 23.5 kDa. Analysis by 2-D electrophoresis of partially-purified recombinant cGH and purified native cGH revealed similar immunoreactive charge isoforms and M(r) variants. The recombinant hormone was biologically active in a homologous radioreceptor assay. The results show that cGH expressed in insect cells is biologically and immunologically active, and that a variety of isoforms are secreted which exhibit size and charge properties similar to those of pituitary-derived cGH.

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The 2A gene of hepatitis A virus (HAV) bears no obvious similarity to the corresponding genes of other picornaviruses and has no known function. In a preliminary effort to gain information about the HAV 2A gene product, we constructed several HAV cDNAs containing deletions of 30 or 45 nucleotides in the predicted central portion of the 2A gene. These deletions did not affect the sites of protein processing, although the rates or efficiencies of polyprotein cleavage at the surrounding cleavage junctions appeared slightly reduced. Transfection of FRhK-4 cells with RNA transcripts of the deleted HAV cDNAs generated small foci of infected cells and produced infectious virus that retained the deletion mutations. In contrast, a single amino acid insertion in the 2B coding region was lethal to virus replication despite normal protein processing. Another deletion, which included the predicted 2A/2B junction and extended into the 2B coding sequence, did not support polyprotein processing or generate viable virus. One of the viable internal 2A deletions was introduced into a wild-type HAV cDNA background, and transcripts were tested for infectivity by inoculation directly into the livers of two marmosets. Both animals seroconverted, displayed elevated serum liver enzymes, and excreted infectious virus. Thus, deletion of 10 or 15 amino acid residues from the predicted central portion of the 2A protein was tolerated with only relatively minor effects on the growth of HAV in cultured cells and in marmoset liver.

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To determine the P3 region protein-processing sites cleaved by the hepatitis A virus 3C protease, a nested set of constructs containing a portion of 3A (3A* [the asterisk denotes an incomplete protein]), 3B and 3C and various amounts of 3D, fused in frame to Escherichia coli TrpE-coding sequences under control of the tryptophan promoter, was made. Additional plasmids that encoded a portion of 2C (2C*) and the P3 proteins, including complete or incomplete 3D sequences, were constructed. After induction, E. coli containing these recombinant plasmids produced high levels of fusion proteins as insoluble aggregates. 3C-mediated cleavage products were identified by comparison of expression with a matching set of plasmids, containing an engineered mutation in 3C. Cleavage products were detected by immunoblot analyses by using antisera against the TrpE protein, against 3D*, and against 3CD*. Scissile bonds were determined by N-terminal amino acid sequencing of the proteins formed by cleavage. The results showed that when a portion of 2C was present, the primary cleavage by the 3C protease was between 2C and 3A, and the cleavage site was QG, as predicted by J. I. Cohen, J. R. Ticehurst, R. H. Purcell, A. Buckler-White, and B. M. Baroudy, J. Virol. 61:50-59, 1987. Very little further cleavage of the released P3 protein was detected. When the fusion protein contained no 2C and included only 3A*-to-3D sequences, efficient cleavage occurred between 3B and 3C, at the QS pair, also as predicted by Cohen et al. (J. Virol. 61:50-59, 1987). The latter proteins were also cleaved between 3C and 3D, but less efficiently than between 3B and 3C. Extracts of bacteria expressing proteins from 3A* to 3D also cleaved a radiolabelled hepatitis A virus substrate containing VP1*2ABC* sequences in trans.

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The genomic RNA of hepatitis A virus has two potential translation initiation sites for synthesis of a 251-kDa polyprotein. It is not known which of these AUG codons, located at positions 735-737 and 741-743, is used in vitro or in vivo. Site-directed mutagenesis was carried out to eliminate each start codon independently. Transcripts from the unmodified and modified cDNA clones were used either to program an in vitro translation system or for transfection of BS-C-1 cells. In vitro and in vivo translation data revealed preferential usage of the downstream AUG located at position 741 to 743, although either site could be utilized in the absence of the other. Both modified RNAs were able to induce productive infections in BS-C-1 cells. Deletion of almost all of the 5'-untranslated region (5'UTR) of the RNA, however, stimulated selection of AUG 735-737 in vitro resulting in equal utilization of both sites, suggesting a strong influence of the 5'UTR for directing the ribosome to a specific internal initiation site.

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A series of plasmids containing hepatitis A virus (HAV) cDNA was constructed such that positive-strand HAV RNA could be transcribed with T7 RNA polymerase. The plasmids differed in the number of 5'-terminal nucleotides representing the junctions between vectors and HAV sequences that were present in the transcripts. When these transcripts were used to transfect cultured BS-C-1 cells, it was found that only those transcripts that contained all of the 5'-terminal HAV nucleotides, in addition to one or more nucleotides from the vector, were capable of initiating an infectious cycle leading to production of progeny virus. Transcripts that contained one 5'-terminal nucleotide from the vector sequence but were missing two uridylate residues corresponding to the first two nucleotides of HAV sequences, or were missing U and C residues corresponding to nucleotides 2 and 3 of the HAV sequence, were not infectious. A similar plasmid containing poliovirus cDNA was engineered to produce transcripts similarly lacking the first two uridylate residues of the poliovirus RNA sequence. These transcripts were infectious.

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The replication of hepatitis A virus (HAV) RNA and the production of HAV VP1 protein were examined in cultures of BS-C-1 cells under one-step growth conditions by in situ hybridization and immunofluorescence. Individual cells that had undergone active viral RNA replication were detectable at 24 h post-infection. During subsequent days, increasing numbers of cells began replicating viral RNA, so that by seven days post-infection, all cells had accumulated significant amounts of viral RNA. The results show that the protracted replication cycle of HAV in cultured cells represents a slow recruitment of infected cells into a replication mode, rather than an inherently slow virus reproduction in all cells. With the reagents utilized in this study, nucleic acid hybridization was more sensitive than antigen detection by immunofluorescence or immunoblot analysis.

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We have constructed a recombinant plasmid producing, in bacteria, a hepatitis A virus (HAV) capsid protein that may be useful as a subunit vaccine. HAV VP1 coding sequences were fused in-frame to NH2-terminal Escherichia coli TrpE coding sequences under the control of the tryptophan promoter. In the absence of exogenous tryptophan, E. coli containing this recombinant plasmid produced high levels of an 88-kilodalton fusion protein that was recognized in immunoblots by antibodies to TrpE and HAV. The TrpE/HAV VP1 protein was gel-purified and used to immunize rabbits. The resulting antiserum reacted with denatured HAV VP1 in immunoblots but did not react with intact virus. However, subsequent inoculation of an immunized animal with a subimmunogenic dose of inactivated, whole HAV resulted in the rapid appearance of a stable, virus-neutralizing antibody response.

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A cDNA coding for hepatitis A virus (HAV) VP1 and portions of the flanking VP3 and P2 sequences was inserted into the genome of Autographa californica nuclear polyhedrosis virus under the control of the polyhedrin promoter and translational start codon. Cells infected with recombinant virus produced high levels of a 55 kDa protein, identified as containing HAV VP1 by reactivity with anti-VP1 serum. The expressed protein also reacted on immunoblots with human HAV convalescent sera as well as sera from rabbits immunized with intact HAV. This protein was found predominantly in the cytoplasm of infected insect cells, probably as an insoluble aggregate.

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Microtubule-associated proteins purified from bovine brains stimulated the in vitro transcription and replication reactions of vesicular stomatitis virus. The products of these reactions were intact messenger or genome-sized RNA species. A preparation from HeLa cells containing tubulin and microtubule-associated proteins also stimulated vesicular stomatitis virus transcription in vitro. This observation is in accord with previous studies, which suggested that a host cell factor was involved with the function of the vesicular stomatitis virus RNA polymerase, and others that indicated that several animal viruses displayed an association with host cell cytoskeletal elements during their replication cycles. We show evidence in this report of a host cell protein that seems to have a functional role in interacting with the virion polymerase.

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Colloidal gold spheres were coated with staphylococcal protein A and were used to determine the location of NS and L proteins on vesicular stomatitis virus (VSV) ribonucleoprotein (RNP) complexes using monospecific anti-NS and anti-L IgG preparations. Conjugates using either anti-NS or anti-L demonstrated that these enzyme subunits were uniformly distributed along the entire length of the RNP complex. Under saturating conditions of IgG concentrations, it was observed that there were at least 60-70 molecules of NS protein and 30-35 molecules of L protein labeled per RNP complex.

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Avian myeloblastosis virus (AMV) genomic RNA was translated in vitro to identify the AMV transforming gene (myb) product(s). Full-length RNA yielded, in addition to the expected Pr76gag, a gag-related product of 92,000 daltons which did not appear to be a gag-oncogene fusion protein. Subgenomic AMV RNA in vitro translation, nonstructural products (specific for AMV) were of 54,000, 49,000 and 34,000 daltons. Based on their sizes and similar peptide maps for the smaller two proteins, it is proposed that they derive from the myb region of AMV.

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Ribonucleoprotein particles (RNPs) of vesicular stomatitis virus (VSV) were fractionated by column chromatography through Fractogel TSK HW-55F and by centrifugation through KCl sucrose. Analyses of fractions for protein content and for protein kinase activity indicated that the major peak of kinase activity did not correspond exactly with any of the VSV-specific proteins. Neither anti-NS nor anti-M IgG preparations inhibited protein kinase activity, and IgG did not act as an exogenous phosphate acceptor. Reconstitution of an RNP-enzyme complex did not result in a restoration of protein kinase activity. In vitro translation of VSV-specific poly(A)-containing RNA did not result in any detectable production of kinase activity. Thus, the major RNP-associated kinase is a host cell protein which is tightly bound to the RNP particle.

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Spontaneous mutants of Staphylococcus aureus resistant to rifampin, rifamycin SV, streptovaricin, or streptolydigin were isolated and shown to be resistant due to chromosomal rather than plasmid mutations. Based on data concerning spontaneous mutation rates, genetic cotransduction rates, and in vitro sensitivity studies, four major antibiotic cross-resistance patterns were found. The genetic markers responsible for these cross-resistance patterns were shown to be separable by transduction. Nonpurified RNA polymerase activity in lysates of mutants showed the same sensitivity to these antibiotics as shown by the mutants on solid media. A model is proposed explaining possible structure-function relationships involved in the binding of these antibiotics to the RNA polymerase molecule and the mutations resulting in resistance to these antibiotics. This model includes generally overlapping but different-sized binding sites on the RNA polymerase protein coded for by similarly arranged mutable sites on the DNA.

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