A comparison of methods for the estimation of retroviral burden.

The presence of retroviral contamination is of vital concern in the manufacture of cell culture-derived biopharmaceuticals. These cell lines usually have A- or C-type retrovirus-like particles which are visible by transmission electron microscopy (TEM) even when infectivity (IF) or reverse transcriptase activity (RTA) cannot be demonstrated. The supernatant of the post-production cell cultures, therefore, also needs to be evaluated by TEM for viral burden. A major question, however, is how to establish a quantitative viral load estimate for the evaluation of a purification process. The FDA recommends that a purification process for viral contaminants remove or inactivate 3-5 logs over the estimated viral burden. Viral particles are difficult to identify and quantify, however, by conventional negative staining. We present a comparison of infectivity assay, reverse transcriptase assay, negative staining, and thin sectioned TEM. These assays were performed on four samples. Ultracentrifuged sediments of cleared cell-culture media were measured, fixed and processed. Thin sections were evaluated by TEM and the number of viral particles estimated by morphometric derived quantification. Retrovirus particles were easily identified and quantified when examined by TEM as compared to negative staining and correlated with the other viral assays (IF, RTA). These results demonstrate that the TEM thin section method was a superior technique to negative staining for estimating viral particle load in cell-culture supernatant. To validate further the plastic embedding with thin sectioning, we evaluated cell culture supernatants (pellets) for retroviral burden at various dilutions, from two cell lines. Morphometric determinations were made of the number of viral particles present per unit volume and compared to results obtained by infectivity assay. Since the morphometric calculation for viral density assumes even distribution of viral particles, we also evaluated and calculated viral counts on multiple thin sections taken throughout selected pellets.

Cell line issues related to specific expression vectors: retrovirus vectors.

Retroviruses have received attention as vectors for gene transfer because they can infect a variety of cell types, they integrate stably into the genome of the target cell and they have simple, well-defined structures. Insertion of the gene of interest into a retroviral vector results in the formation of replication-defective particles that can be produced in "packaging" cell lines. Due to the nature of the present generation of vectors and packaging cell lines, multiple recombination events would be needed to produce replication-competent particles. However, the concern still exists that replication-competent virus could arise either in the production of retroviral vector stocks or following their introduction into target cells. The current use of retrovirus vectors and the challenge they present to testing programmes are discussed.

Two noncontiguous regions of Sendai virus P protein combine to form a single nucleocapsid binding domain.

Binding of Sendai virus P protein to viral nucleocapsids requires amino acids in two separate regions of P protein. Both required regions are near the carboxyl terminus, and they are separated by a region which is expendable for binding (K. W. Ryan and A. Portner, 1990, Virology 174, 515-521). To examine the topography of these regions in the folded P protein molecule we mapped the epitopes present in several undenatured P proteins with overlaping deletions near their carboxyl termini. The epitopes recognized by two monoclonal antibodies were each composed of both protein regions necessary for binding, indicating that these two regions are each required at some point during the folding of P protein. To determine if these protein regions interact directly in forming the nucleocapsid binding domain, we constructed a deleted P gene which encodes a protein comprising only these two regions with all other P protein sequences deleted. This protein was able to bind to nucleocapsids, demonstrating that these two regions alone are sufficient to form the nucleocapsid-binding domain. In addition, this protein formed the folded epitopes comprising the two nucleocapsid-binding regions, indicating that the two regions interact directly with each other to form a single folded structure. The involvement of this binding domain in viral mRNA synthesis was examined by testing the ability of each monoclonal antibody to inhibit the in vitro transcription activity of full-size P protein. Several antibodies to epitopes near the binding domain were found to be potent inhibitors of viral transcription, showing that these regions contribute to P protein's role in mRNA synthesis.

Evaluation of Dacron-covered and plain bovine xenografts as replacements for the anterior cruciate ligament.

Surgical repair of the anterior cruciate ligament often involves the use of a suitable autograft. As alternatives to sacrificing these normal structures, various allografts, xenografts, and synthetic materials have been investigated as ligament replacement materials. This study investigates Dacron fabric-covered and plain bovine xenograft tendon as such materials in the canine knee. The implants were tested to failure in an MTS machine following 13 weeks of implantation in a canine knee. Dacron woven fabric-covered implants became more firmly attached than those covered by Dacron mesh fabric or plain xenografts. The implants were also analyzed according to their method of attachment (fixation staples or sutures). Overall, the sutured implants failed at slightly higher forces than did the stapled ones. Histologically, limited vascular invasion of the xenograft was observed. No host fibrous or osseous tissue could be identified within the graft. Fibrous tissues did form between the bone and xenograft. The implants exhibited extreme intraarticular wear, which suggests a low potential for intraarticular ligament replacement.

Antibodies against Sendai virus L protein: distribution of the protein in nucleocapsids revealed by immunoelectron microscopy.

Antibodies against the L protein of Sendai virus were made by immunizing rabbits with a synthetic peptide representing a carboxyl-terminal region of the protein predicted from the base sequence of its gene. These antibodies were used to localize the L protein in viral nucleocapsids by electron microscopy. Immunogold labeling revealed that L protein molecules were distributed in clusters along nucleocapsids, suggesting that L molecules act cooperatively in viral RNA synthesis. Immunogold double-labeling showed that all L clusters were associated with clusters of P molecules. We believe that this morphological association reflects the functional cooperation of the L and P proteins in viral RNA synthesis.

Sequence of the Sendai virus L gene: open reading frames upstream of the main coding region suggest that the gene may be polycistronic.

The sequence of the L gene of Sendai virus, encompassing 6799 nucleotides, has been determined, completing the primary sequence of the entire virus genome. An open reading frame beginning at position 569 codes for a basic protein of 2048 amino acids with an estimated Mr of 231,608. No nucleotide sequence similarities with the analogous L gene of vesicular stomatitis virus were observed. However, comparison of the deduced amino acid sequences of both proteins revealed a conserved 18 amino acid sequence that may have functional significance. Two additional overlapping reading frames which precede the L protein sequence could encode proteins with MrS of 6474 and 14,026, suggesting that the gene is polycistronic.

Nucleotide sequences responsible for generation of internally deleted Sendai virus defective interfering genomes.

The deletion points of four internally deleted defective interfering (DI) RNA species (7a, 7b, 7c, and 7d) that reside in a single Sendai virus strain were defined by nucleotide sequencing. DI RNA 7a (Mr 1.24 x 10(6)) retained the entire NP gene with the complete NP protein-coding sequence, except for the last two U residues of the polyadenylation signal, fused to an 1800-nucleotide sequence comprising 5'-terminal genome and adjacent L gene sequences. DI RNA 7b (Mr, 0.70 x 10(6)) consisted of 100 3'-terminal nucleotides fused to 1900 5'-terminal bases; the deletion point in the NP gene precedes the NP protein initiation codon. DI RNA 7c (Mr 0.55 x 10(6)) retained 420 3'-terminal and 1150 5'-terminal nucleotides. The sequence just downstream of the sequenced deletion site is M gene specific, indicating that 7c arose from at least two deletion events and that it comprises NP, M, and L gene fragments. Transcription of RNA 7c could yield an MRNA encoding a fusion protein with a 14,000 Mr (N-terminal NP sequence fused to out of frame M-specific amino acids). DI RNA 7d (Mr 0.92 x 10(6)) retained 1027 3'-terminal nucleotides fused to 1600 bases from the 5'-terminus. It has an open reading frame for a 33,000 Mr N-terminal NP protein fragment. Nucleotide sequences flanking each deletion and just downstream of the NP gene deletion site suggested that these DI genomes were generated by a copy-choice mechanism, involving polymerase jumping during replication of negative polarity virus genome templates. In this process, the termination and reinitiation of RNA synthesis would involve recognition of sequences that regulate virus genome transcription and replication.

Complete sequence of the Sendai virus NP gene from a cloned insert.

A DNA molecule representing all but the three terminal bases of the Sendai virus nucleoprotein (NP) gene, copied from viral mRNA, was inserted into pBR322. The NP insert comprised 1673 bases. The first AUG protein initiation codon, at position 65, began an open reading frame of 1551 bases, encoding a protein of 517 amino acids with an amino acid composition corresponding to previously published data. The NP gene sequence determined in the present work is similar to that described by Shioda et al. [ Nucl . Acids Res. 11, 7317 (1983)], but there are 14 amino acid differences that probably reflect differences in virus strains. The predicted secondary structure of the NP molecule and the locations within that structure of potential protease cleavage sites are in accord with structural domains previously defined by controlled protease digestion.

Molecular clones representing Sendai virus genes P, NP and M.

DNA copies of segments of Sendai virus genes P, NP and M, obtained by reverse transcription of virus mRNA species extracted from infected cells, were cloned in plasmid pBR322. Genes were identified by hybrid-arrested translation of viral mRNAs in vitro. Hybrid selection of NP mRNA confirmed the identity of an NP gene clone. Partial sequencing of this insert showed that it represents the 5'-terminal region of the gene, containing transcription termination signals. Hybridization of DNA inserts to blots of electrophoretically separated, denatured mRNA species indicated that the P, NP and M messages had sizes of 2400, 2100 and 1500 nucleotides, respectively. Specific T1 ribonuclease-resistant oligonucleotides, previously identified in the NP and M genes, were selected by hybridization to the respective inserts. Although most of the inserts are smaller than 500 base pairs, representing no more than 16% of the P gene and 24% of the NP gene, one of the M gene inserts, comprising 700 base pairs, represents almost half of that gene. These cloned virus gene segments will assist further investigations of the molecular biology of this model paramyxovirus.

Site-specific phosphorylation regulates the transcriptive activity of vesicular stomatitis virus NS protein.

In vitro transcription by vesicular stomatitis virus nucleocapsids is inhibited by enzymatic dephosphorylation of the NS protein. We provide evidence that specific, partial dephosphorylation of NS molecules is the only detectable change in nucleocapsids treated with bacterial alkaline phosphatase under conditions that prevent the action of adventitious protease. Dephosphorylation appeared to affect only the rate of transcription; there were no changes in sedimentation rates of transcripts. To identify the sites of phosphorylation required for NS activity in transcription, we examined phosphopeptides produced by chymotrypsin digestion of the two electrophoretic classes of NS molecules found in virions and infected cells. The electrophoretically slower class, NS1, abundant in the intracellular soluble pool, has a lower activity in transcription; it contained six chymotryptic phosphopeptides. Five of these peptides contained both phosphoserine and phosphothreonine, indicating that this peptide cluster represents at least 11 separate sites of phosphorylation. In the electrophoretically faster nucleocapsid-associated NS2 class of molecules, which support a higher rate of transcription, another group of eight phosphopeptides was superimposed on this pattern. Two of these peptides contained both phosphoserine and phosphothreonine, so this cluster of peptides represents at least 10 additional phosphorylation sites. These sites were especially sensitive to dephosphorylation by bacterial alkaline phosphatase. One or more of them appears to be responsible for the higher transcription rates medicated by NS2 molecules.

Pyridoxal phosphate as a probe of reovirus transcriptase.

The ribonucleoprotein core of reovirus is a multienzyme complex that transcribes messenger ribonucleic acid (mRNA) from double-stranded RNA templates. So far, the core has resisted attempts to disassemble it and identify the polypeptide species responsible for RNA polymerase activity. As an alternative approach, we tested pyridoxal 5-phosphate (PLP) as a potential affinity labeling reagent for reovirus transcriptase in vitro; PLP has been used as an affinity reagent for cellular and viral nucleic acid polymerases. We found that PLP inhibited reovirus transcriptase reversibly (apparent Ki = 0.2 mM), but the inhibition was noncompetitive with respect to each of the four ribonucleoside triphosphates. This interaction required both the aldehyde and phosphate moieties in PLP, since pyridoxamine and pyridoxal were relatively inactive. To identify the polypeptides involved, we labeled the PLP--core complex by reductive alkylation with [3H]borohydride. At PLP concentrations close to the apparent Ki, labeling was selective for the two largest virion polypeptides, lambda 1 and lambda 2. At saturation, there were only 10 high-affinity PLP binding sites per core in each of the lambda polypeptide species. These findings implicate either or both lambda polypeptide species in viral transcription and they indicate that a special population, representing no more than 10% of the total lambda molecules in each core, participates in RNA synthesis.

SSPE-BIKEN: a naturally arising hemagglutination-defective mutant of measles virus.

Biochemical and genetic methods have been used to investigate a defective variant of measles virus previously isolated from a patient with subacute sclerosing panencephalitis (SSPE). Since its isolation, this syncytiogenic strain (SSPE-BIKEN) has remained cell-associated; infected cells do not hemadsorb and do not release infectious virus. Immune precipitation and polyacrylamide gel electrophoresis were used to study the synthesis of measles virion proteins in SSPE-BIKEN-infected cells. All of the virion proteins were detected in immune precipitated whole cell extracts. However, the hemagglutinin (HA) protein was not detected on the cell surface by lactoperoxidase iodination. These results suggest that the failure of the HA protein to insert into the cell membrane accounts for the block in the release of infections virus. Radioactively labeled, noninfectious, virus-like particles have been purified from the media of SSPE-BIKEN-infected cells. These particles contain virus nucleocapsid, nucleocapsid-associated, and membrane proteins, but very little HA and hemolysin proteins. Genetic complementation between SSPE-BIKEN and a temperature-sensitive mutant of measles virus was observed and suggests that the SSPE isolate defect is due to a mutation. Additional evidence of a mutation is provided by the detection of low frequency revertant progeny in SSPE-BIKEN stocks. Our results support the hypothesis that genetic variants of measles virus are involved in the etiology of SSPE.