Infection control in gene therapy.

Gene therapy is now being studied for the treatment of a wide variety of acquired and inherited diseases. Viruses used as vectors for gene transfer include retroviruses, adenoviruses, vaccinia viruses, adeno-associated viruses, and herpesviruses. These vectors, developed in the laboratory and in animal studies, are now being introduced into the clinical arena Infection control practitioners will be involved invariably in reviewing the use of these agents in their clinics and hospitals. This review summarizes key aspects of the more common vectors and makes recommendations for infection control.

Expression of potyvirus proteins in insect cells infected with a recombinant baculovirus.

The N-terminal portion (P1-HC-Pro-P3) of the tobacco vein mottling virus (TVMV) polyprotein was expressed in insect cells and larvae by a recombinant baculovirus. The proteases necessary to process this TVMV polyprotein fragment were active in insect cells, since mature P1, HC-Pro and P3 proteins were detected by specific antisera in Western blots. Antisera to P1, HC-Pro and P3 also recognized polypeptides with apparent M(r) values predicted for the intermediate processing products of the polyprotein fragment. The results of this study indicate that the autocatalytic processing of TVMV HC-Pro from the polyprotein is supported by insect cells. Helper component activity in extracts of cells infected with recombinant baculovirus was not detected by aphid transmission assay.

A simple and efficient procedure for the oral inoculation of Trichoplusia ni larvae with polyhedrin-negative recombinant baculovirus.

Current procedures for inoculating lepidopteran larvae with polyhedrin-negative recombinant baculovirus, i.e. intracoelomic injection or coinfection with wild type virus, are laborious and can compromise final yields of recombinant protein. Herein is described a simple and efficient method for oral inoculation. Up to 100% infection was obtained when individual early fifth instar Trichoplusia ni larvae were fed a small piece of a formaldehyde-free insect diet to which 4.2 x 10(5) PFU of a polyhedrin-negative recombinant Autographa californica nuclear polyhedrosis virus (AcNPV) containing the gene for beta-galactosidase was applied. Infected larvae were identified by assaying hemolymph for beta-galactosidase activity. The maximum levels of beta-galactosidase detected in these hemolymph samples were identical to those obtained for larvae infected by intracoelomic injection. The dose of polyhedrin-negative recombinant virus recommended for intracoelomic injection of T. ni was efficacious for the oral route of inoculation.

The L protein of vesicular stomatitis virus transcription complexes is specifically photolabelled by 5-azido-uridine 5'-triphosphate, an analogue of the RNA polymerase substrate uridine 5'-triphosphate.

A photoactive nucleotide analogue of UTP, 5-azido-uridine 5'-triphosphate (5-N3UTP), has been demonstrated to interact with the RNA polymerase of the vesicular stomatitis virus (VSV) transcription complex. Kinetic studies indicated that 5-N3UTP served as an efficient replacement for UTP in in vitro polymerase reactions. The Km for the azido analogue was 27 microM and that of the natural substrate, UTP, was 7 microM. Photolysis of [gamma-32P]5-N3UTP in the presence of VSV transcription complexes resulted in selective radio-labelling of the L protein. This photolabelling was saturable with an apparent Kd of 28 microM. The L protein was protected from [gamma-32P]5-N3UTP-mediated photolabelling by competing natural substrates (UTP, CTP, ATP, GTP). The stoichiometry of photoprobe incorporation into the transcription complex was close to unity with respect to the L protein. These data provide evidence that the nucleotide-binding domain of the VSV RNA polymerase contains amino acid residues of the L protein.

Identification and characterization of serine/threonine protein kinase activity intrinsic to the L protein of vesicular stomatitis virus New Jersey.

A photoaffinity analogue of ATP, 8-azido-adenosine 5'-triphosphate (8-N3ATP), was used to probe ATP-binding sites in native transcription complexes of vesicular stomatitis virus (VSV) (New Jersey serotype). The analogue was found to be a substrate for a serine/threonine protein kinase that phosphorylated both the NS and L proteins of native complexes. The analogue failed to interact with the RNA polymerase, another ATP-utilizing activity associated with the transcription complex. Kinetic analyses of both ATP and 8-N3ATP utilization by the protein kinase yielded biphasic saturation curves. Photolysis of 8-N3ATP in the presence of VSV transcription complexes resulted in selective labelling of the L protein. The photolabelling of L was saturable and apparently biphasic. Photolabelling of the L protein was significantly reduced by competition with ATP whereas other nucleoside triphosphates (GTP, UTP and CTP) were ineffective competitors. The stoichiometry of photolabelling was 0.2 at 10 microM-8N3ATP and 1.3 at 100 microM-ATP. These data provide chemical evidence for a virus-encoded serine/threonine protein kinase which resides on the L protein.

Nucleotide sequence of the L gene of vesicular stomatitis virus (New Jersey): identification of conserved domains in the New Jersey and Indiana L proteins.

The nucleotide sequence of the L gene of vesicular stomatitis virus, New Jersey serotype (Hazelhurst subtype), was determined. Primer extension dideoxy sequencing of genomic RNA using reverse transcriptase initiated within the adjacent G gene provided a consensus sequence of 6522 nucleotides. The G/L intergenic junction spanned 21 nucleotides and contained a pseudo transcription start signal as well as two sequences (10 and 6 nucleotides in length) which are reiterated within the L coding region. The predicted L mRNA was 6398 nucleotides long and contained a single open reading frame corresponding to an L protein encompassing 2109 amino acids with a MW of 241,546. Comparison of the amino acid sequence of this New Jersey serotype L protein to that previously reported for the L protein of the serologically and genetically distinct Indiana serotype (M. Schubert, G. G. Harmison, and E. Meier (1984). J. Virol. 51, 505-514.) revealed a high degree of functional homology. In addition, six regions (43 to 103 amino acids in length) which displayed a high percentage of identical amino acids (85 to 96%) were identified. Five of these regions were clustered within the amino-terminal half of the L protein. Two of these regions contained sequences, 41 amino acids in length, which were significantly similar to corresponding regions of the L proteins of the paramyxoviruses Sendai and Newcastle disease virus. These structurally conserved regions may correspond to functional domains of the multifunctional L protein.

Functional analysis of hypomethylation variants of the New Jersey serotype of vesicular stomatitis virus.

The ts mutant F1 of vesicular stomatitis virus, New Jersey serotype, directs the synthesis of undermethylated 5'-terminal cap structures in vitro. In order to determine the relationship between the ts and hypomethylation phenotypes, a spontaneous revertant rev(ts)F1 of the ts phenotype was analyzed. The revertant retained the hypomethylation phenotype. The four cap structures (GpppA, 7mGpppA, GpppAm, and 7mGpppAm) synthesized in mutant and revertant-directed reactions in the presence of low as well as high concentrations of AdoMet were resolved by HPLC. Quantitation of the data and analysis of cap substrate to product ratios revealed that despite apparent similarities between the two hypomethylation phenotypes, the functional lesions in F1 and rev(ts)F1 were different. F1 displayed an AdoMet concentration-dependent alteration in the GpppA----GpppAm reaction and an AdoMet concentration-independent alteration in the GpppA----7mGpppA reaction. In contrast, rev(ts)F1 displayed AdoMet concentration-dependent alterations in the reactions GpppA----7mGpppA and GpppAm----7mGpppAm.

The fates of undermethylated mRNA cap structures of vesicular stomatitis virus (New Jersey) during in vitro transcription.

A dual label pulse-chase analysis employing S-adenosyl-L-[methyl-3H]methionine and [beta-32P]GTP has been devised to identify precursors to the cap structure 7mG(5')ppp(5')Am present on the 5'-termini of vesicular stomatitis virus mRNAs. Both monomethylated cap structures, 7mG(5')ppp(5')A and G(5')ppp(5')Am, have been detected in vitro in New Jersey serotype reactions containing suboptimal concentrations of S-adenosyl-L-methionine. The simultaneous chasing of both radiolabeled substrates allowed the determination of the transcriptive fate of each pulse-labeled cap structure in the total RNA population. Ten percent of the 7mG(5')ppp(5')A cap structure and 27% of the G(5')ppp(5')Am cap structure generated during the pulse were chased into 7mG(5')ppp(5')Am. These results suggested that while there may have been a preferred order of 5'-cap methylation, the order was not compulsory. The dual label analysis also revealed that only 34% of the pulse-labeled G(5')ppp(5')A cap structure could be chased into methylated cap structures. A nascent RNA chain length-dependent "methylation window" is proposed.

The capsid polypeptides of the 190S virus of Helminthosporium victoriae.

SDS-PAGE of the 190S virus of Helminthosporium victoriae, using a discontinuous buffer system, revealed two major capsid polypeptides of mol. wt. 88K and 83K (p88 and p83) and a minor polypeptide, p78. Peptide mapping by both limited proteolysis and selective chemical cleavage showed p83 and p78 to be closely related to p88. The origin of p83/p78 could not be explained by proteolysis of p88 during virus preparation and storage. In rabbit reticulocyte lysates, denatured dsRNA directed the synthesis of a single major translation product which was identical to capsid polypeptide p88 on the basis of coelectrophoresis, immunoprecipitation and peptide mapping. No translation products comparable in size to p83 or p78 were detected in vitro. These data indicated that the capsid of the 190S virus is encoded by a single gene and verified the classification of the virus as a member of the family Totiviridae. Radioiodination of intact virus under conditions considered optimum for surface-specific iodination showed p88 to be more readily available for labelling than p83 or p78. Furthermore, when Western blots of capsid polypeptides were reacted with an antiserum to glutaraldehyde-stabilized virus (190S-G), p88 was more reactive to 190S-G antibodies than was p83/p78. These results suggest p88 is external to p83/p78 in the capsid.

Assignment of the temperature-sensitive lesion in the replication mutant A1 of vesicular stomatitis virus to the N gene.

The replication defect in the temperature-sensitive mutant A1 of the New Jersey serotype (Hazelhurst subtype) of vesicular stomatitis virus was confirmed by the absence of intracellular nucleocapsids in infected cells incubated at the restrictive temperature. After preamplification, the relative yield of the A1 N protein accumulated intracellularly after 1 h of incubation at the restrictive temperature was decreased by 50% that of the wild-type or revertant A1 N protein. This difference was not as apparent in pulse-chase experiments. The functional lesion in A1 was correlated with a structural alteration in the N protein on the basis of the thermolability of the template activity of the A1 N protein-RNA complex in in vitro transcription reactions and the covariance of this phenotype with the temperature-sensitive phenotype in a spontaneous A1 revertant. This correlation was consistent with a direct role of the N protein in replication and allowed the assignment of the N gene to complementation group A.

Functional relationships within the New Jersey serotype of vesicular stomatitis virus: genetic and physiological comparisons of the Hazelhurst and Concan subtypes.

Seventeen temperature-sensitive mutants of the Concan subtype of the New Jersey serotype of vesicular stomatitis virus have been isolated following mutagenesis and assigned to two complementation groups: CC/A, containing three mutants, and CC/B, containing 14 mutants. Prototype mutants of these two Concan groups efficiently complemented prototype mutants of the Hazelhurst complementation groups (with the exception of the corresponding group) which correspond to genes specifying the L, N, M and NS proteins. The pattern of intersubtypic complementation allowed the correlation of the Concan CC/B group with the Hazelhurst B group (L gene) and of the Concan CC/A group with the Hazelhurst A group (N gene). In contrast, the Concan prototype mutants failed to complement the prototype mutant of each of the five Indiana complementation groups for which genetic assignments have been made. The partitioning of intracellular nucleocapsids of the Concan and Hazelhurst subtypes during isolation was identical, and distinct from that of Indiana serotype intracellular nucleocapsids. The M protein of the Concan, but not of the Hazelhurst, subtype was observed to migrate as a doublet on SDS-polyacrylamide gels electrophoresed in a phosphate buffer.

Structural and functional characterization of the RNA-positive complementation groups, C and D, of the New Jersey serotype of vesicular stomatitis virus: assignment of the M gene to the C complementation group.

The structural and functional lesions in the RNA-positive complementation groups, C and D, of the New Jersey serotype (Hazelhurst subtype) of vesicular stomatitis virus have been characterized. The M protein of the temperature-sensitive mutant C1, the prototype of the C complementation group, was degraded at the restrictive temperature in vivo, and was resolved from the wild-type M protein by SDS-polyacrylamide gel electrophoresis and nonequilibrium pH gradient electrophoresis. Coreversion of these properties and the temperature-sensitive phenotype was observed in a spontaneous revertant. On the basis of these results, the M gene was assigned to the C complementation group. Intracellular nucleocapsids could not be isolated from New Jersey serotype infections by procedures developed for Indiana serotype infections. Therefore, in order to assess the ability of New Jersey ts mutants to accumulate nucleocapsids at the restrictive temperature, a procedure for their isolation was developed. Hypertranscription was observed in C1-infected cells incubated at the restrictive temperature, but was not accompanied by proportionate increases in intracellular viral nucleocapsids or protein synthesis. The G and N proteins of the temperature-sensitive mutant D1, the sole representative of the D complementation group, were electrophoretic variants. The relative yield of intracellular D1 N protein was lower at the restrictive than at the permissive temperature, and the D1 L protein was thermolabile. No intracellular viral nucleocapsids were detected in D1 infected cells incubated at the restrictive temperature; however, more 40 S and less message-sized RNA were synthesized at the restrictive than at the permissive temperature. These results suggested functional defects in both the N protein and polymerase of D1.

Cell-free translation of tobacco vein mottling virus RNA.

Tobacco vein mottling virus (TVMV), a member of the potyvirus group, was translated in a rabbit reticulocyte cell-free system. The RNA was approximately 5% as active as rabbit globin mRNA in directing protein synthesis. Translation was stimulated approximately 15% by the cap analog pppm7G, a phenomenon which has also been observed with uncapped viral RNAs. Treatment with NaIO4 and NaB(3H]4 under conditions which label the caps of globin and ovalbumin mRNA failed to produce labeled cap structures in TVMV RNA. Approximately 20 polypeptides, ranging from 20,000 to 100,000 daltons, were produced in the reticulocyte system programmed with TVMV RNA. The predominant species (P75) had a molecular weight of 75,000. The same major polypeptides were produced in the wheat germ system, but there was relatively greater synthesis of the smaller polypeptides. Incubation of the reticulocyte system in the presence of cycloheximide following an initial period of synthesis failed to cause breakdown of larger polypeptides into smaller polypeptides. Preincubation of TVMV RNA with the reticulocyte system before addition of labeled amino acids caused greater synthesis of the smaller polypeptides, suggesting that they are derived from fragments of the RNA produced during the incubation. Translation of TVMV RNA which had been bound to oligo(dT)-cellulose yielded nearly the same spectrum of polypeptides, but synthesis of polypeptides smaller than 52,000 daltons was reduced. At low ionic strength, only polypeptides of 52,000 daltons and smaller were synthesized. At high ionic strength, P75 was essentially the only product synthesized. Antibody to whole virus precipitated many of the polypeptides, including P75, suggesting that they contain the coat protein sequence. No polypeptide with the electrophoretic mobility of authentic coat protein, however, was precipitated. Comparison of partial proteolytic digests of P75 and authentic coat protein provided further evidence for sequence homology.

Proposed replicative role of the NS polypeptide of vesicular stomatitis virus: structural analysis of an electrophoretic variant.

The structural lesion in the temperature-sensitive mutant E1 of the New Jersey serotype of vesicular stomatitis virus has been assigned to the NS protein. Although the packaged wild-type and mutant NS proteins were similarly phosphorylated, the mutant NS protein migrated faster than the wild-type NS protein in polyacrylamide slab gels electrophoresed in the presence of sodium dodecyl sulfate. The resolution appears to be the result of conformational rather than size differences since the two proteins comigrated in polyacrylamide gels which contained 4 M urea in addition to sodium dodecyl sulfate. Peptide maps, obtained by limited proteolysis of 32P-labeled wild-type and mutant NS proteins with Staphylococcus aureus V8 protease and papain, revealed striking differences which suggested that the mutant alteration could involve an aspartic or glutamic acid residue. Since NS proteins obtained from naturally occurring revertants of E1 were indistinguishable from the wild-type protein in all of these analyses, the structural alteration in the mutant NS protein correlates with the functional lesion. Because E1 is defective in the RNA replication pathway at the restrictive temperature, a replicative role is proposed for the NS protein.

Physical properties of New Jersey serotype of vesicular stomatitis virus and its defective particles.

The wild-type New Jersey serotype of vesicular stomatitis virus generated two types of defective interfering T-particles. The physical properties of these particles and the wild-type virion were determined by laser light scattering spectroscopy, sedimentation measurements, and electron microscopy.

Molecular weights of vesicular stomatitis virus and its defective particles by laser light-scattering spectroscopy.

Laser light-scattering spectroscopy has been used to determine the diffusion coefficients of vesicular stomatitis virus and three of its defective particles in order to calculate their molecular weights.