Genetic characterization of CHO production host DG44 and derivative recombinant cell lines.

The dihydrofolate reductase-deficient Chinese hamster ovary (CHO) cell line DG44 is the dominant mammalian host for recombinant protein manufacturing, in large part because of the availability of a well-characterized genetic selection and amplification system. However, this cell line has not been studied at the cytogenetic level. Here, the first detailed karyotype analysis of DG44 and several recombinant derivative cell lines is described. In contrast to the 22 chromosomes in diploid Chinese hamster cells, DG44 has 20 chromosomes, only seven of which are normal. In addition, four Z group chromosomes, seven derivative chromosomes, and 2 marker chromosomes were identified. For all but one of the 16 DG44-derived recombinant cell lines analyzed, a single integration site was detected by fluorescence in situ hybridization regardless of the gene delivery method (calcium phosphate-DNA coprecipitation or microinjection), the topology of the DNA (circular or linear), or the integrated plasmid copy number (between 1 and 51). Chromosomal aberrations, observed in more than half of the cell lines studied, were mostly unbalanced with examples of aneuploidy, deletions, and complex rearrangements. The results demonstrate that chromosomal aberrations are frequently associated with the establishment of recombinant CHO DG44 cell lines. Noteworthy, there was no direct correlation between the stability of the genome and the stability of recombinant protein expression.

In vitro analysis of an RNA binding site within the N-terminal 30 amino acids of the southern cowpea mosaic virus coat protein.

Southern cowpea mosaic virus (SCPMV) is a positive-sense RNA virus with T = 3 icosahedral symmetry. The coat protein (CP) has two domains, the random (R) domain and the shell (S) domain. The R domain is formed by the N-terminal 64 amino acids (aa) and is localized to the interior of the particle where it is expected to interact with the viral RNA. The R domain (aa 1--57) was expressed in Escherichia coli as a recombinant protein (rWTR) containing a nonviral C-terminal extension with two histidine tags. The RNA binding site of the R domain was identified by Northwestern blotting and electrophoretic mobility shift assay (EMSA) using recombinant wild-type and mutant R domain proteins. Deletions within the R domain revealed that the RNA binding site is localized to its N-terminal 30 aa. RNA binding by this element was found to be nonspecific with regard to RNA sequence and was sensitive to high salt concentrations, suggesting that electrostatic interactions are important for RNA binding by the R domain. The RNA binding site includes 11 basic residues, eight of which are located in the arginine-rich region between aa 22 and 30. It was demonstrated using alanine substitution mutants that the basic residues of the arginine-rich region but not those present at positions 3, 4, and 7 are necessary for RNA binding. None of the basic residues within the arginine-rich region are specifically required for RNA binding, but the overall charge of the N-terminal 30 aa is important. Proline substitution mutations within the N-terminal 30 aa, and alanine substitutions for prolines at positions 18, 20, and 21, did not affect the RNA binding activity of the R domain. However, it was demonstrated by circular dichroism (CD) that the conformation of the N-terminal 30 aa of the R domain changes from a random coil to an alpha-helix in the presence of 50% trifluoroethanol (TFE). The possible role for this structural change in RNA binding by the R domain is discussed.

Membrane activity of the southern cowpea mosaic virus coat protein: the role of basic amino acids, helix-forming potential, and lipid composition.

Southern cowpea mosaic virus (SCPMV) is a spherical RNA virus with T = 3 icosahedral symmetry. The particle is composed of 180 subunits of the coat protein (CP) and one copy of the positive-sense viral RNA. The CP has two domains, the random (R) domain formed by the N-terminal 64 aa and the shell (S) domain (aa 65--260). The R domain is highly charged, with 11 of the N-terminal 30 residues being basic. It is localized to the interior of the native particle where it may interact with the viral RNA, but under certain pH and salt conditions the topology of the particle changes to externalize the R domain. Since the CPs of several spherical RNA viruses have been shown to interact with host membranes during infection, we have begun investigating the membrane interactions of the SCPMV CP using the artificial liposome membranes. Both the native CP and the R domain overexpressed in Escherichia coli were observed to interact with liposomes. The interaction between the R domain and liposomes required either anionic phospholipids or non-bilayer-forming lipids and involved electrostatic interactions since it was shown to be both pH and ionic strength dependent. The analysis of four different deletion and six different site-directed substitution mutations partially mapped the region responsible for this interaction to residues 1--30. Analysis of this region of the R domain by circular dichroism indicated that it assumes an alpha-helical structure when exposed to liposomes composed of anionic lipids. Mutations, which extend the helical nature of this region, promoted an increased interaction. The possible role of the CP/lipid interaction in the SCPMV infection is discussed.

Complementation of the host range restriction of southern cowpea mosaic virus in bean by southern bean mosaic virus.

Vigna unguiculata (cowpea) and Phaseolus vulgaris (common bean) are permissive hosts for southern cowpea mosaic virus (SCPMV) and southern bean mosaic virus (SBMV), respectively. Neither of these two sobemoviruses systemically infects the permissive host of the other. Although bean cells are permissive for SCPMV RNA synthesis, they do not support the assembly of this virus. Thus, the host range restriction of SCPMV in bean may occur at the level of movement and may involve the inability of SCPMV to assemble in this host. In this study, it was demonstrated that SCPMV accumulates in an encapsidated form in the inoculated and systemic leaves of bean plants following coinoculation with SBMV. No evidence was observed that the SCPMV that accumulated in coinoculated bean plants had an altered host range relative to wild-type SCPMV. These results suggested that SBMV complemented the host range restriction of SCPMV in bean. Additional experiments demonstrated that cowpea protoplasts are permissive for SBMV RNA synthesis and assembly. It was concluded from these results that the host range restriction of SBMV in cowpea occurs at the level of movement. In mixed infections of cowpea with SCPMV and SBMV, the latter was recovered from the inoculated but not the systemic leaves. Its recovery from the inoculated leaves, however, was not dependent on the presence of SCPMV in the inoculum. From these results, it was concluded that SCPMV did not complement the host range restriction of SBMV in cowpea.

The 105-kDa polyprotein of southern bean mosaic virus is translated by scanning ribosomes.

The cowpea strain of southern bean mosaic virus (SBMV-C) is a positive-sense RNA virus. Three open reading frames (ORF-1, ORF2, and ORF3) are expressed from the genomic RNA. The ORF1 and ORF2 initiation codons are located at nucleotide (nt) positions 49 and 570, respectively. ORF1 is expressed by a 5' end-dependent scanning mechanism, but it is not known how ribosomes gain access to the ORF2 initiation codon. In experiments described here, it was demonstrated that the translation of ORF2 was sensitive to cap analog in a cell-free extract. In vitro and in vivo studies showed that the addition of one or more AUG codons between the 5' end of the SBMV-C RNA and the ORF2 initiation codon reduced ORF2 expression and that elimination of the ORF1 initiation codon increased ORF2 expression. Altering the sequence context of the ORF1 initiation codon to one more favorable for translation initiation also reduced ORF2 expression in vivo. Nucleotide deletions and insertions between SBMV-C nt 218-520 did not abolish ORF2 expression. In most cases, these mutations resulted in reduced expression of both ORF1 and ORF2. These results are consistent with translation of ORF2 by leaky scanning.

Identification of viral genes required for cell-to-cell movement of southern bean mosaic virus.

Inoculation of Vigna unguiculata (cowpea) with transcripts synthesized in vitro from a genome-length cDNA clone of the cowpea strain of southern bean mosaic virus (SBMV-C) resulted in a systemic SBMV-C infection of this host. Capped RNA was about five times more infectious than uncapped RNA as determined by a local lesion assay. The SBMV-C cDNA clone was also used for mutagenesis of the four SBMV-C open reading frames (ORFs). ORF1, ORF3, and coat protein (CP) mutants were not infectious in cowpea. Electroporation of cowpea protoplasts with mutant transcripts demonstrated that the ORF1, ORF3, and CP gene products were not required for SBMV-C RNA synthesis, and the ORF1 and ORF3 gene products were not required for SBMV-C assembly. From these results, it was concluded that the ORF1 and ORF3 proteins and the CP are required for SBMV-C cell-to-cell movement. One of the ORF3 mutants pSBMV2-UAA1833 contained a nonsense codon between the predicted -1 ribosomal frameshift site (SBMV-C nucleotides 1796-1802) and a potential ORF3 translation initiation codon at SBMV-C nucleotide 1895. The lack of infectivity of this mutant suggested that ORF3 was expressed by a -1 ribosomal frameshift in ORF2 rather than by initiation of translation at nucleotide 1895.

Mapping and expression of southern bean mosaic virus genomic and subgenomic RNAs.

The coat protein of the cowpea strain of southern bean mosaic sobemovirus (SBMV-C) is translated from a subgenomic RNA (sgRNA) that is synthesized in the virus-infected cell. Like the SBMV-C genomic RNA, the sgRNA has a viral protein (VPg) covalently bound to its 5' end. The mechanism(s) by which ribosomes initiate translation on the SBMV-C RNAs is not known. To begin to characterize the translation of the sgRNA it was first necessary to precisely map its 5' end. Primer extension was used to identify SBMV-C nucleotide (nt) 3241 as the transcription start site. As a control, the 5' end of the genomic RNA was also mapped. Surprisingly, the 5' terminal nt of this RNA was identified as SBMV-C nt 2. The primary structure of the 5' ends of these two RNAs is therefore expected to be VPg-ACAAAA. Precise mapping of the 5' end of the sgRNA of the bean strain of SBMV (SBMV-B) demonstrated that it has these same elements. Translation of coat protein from the SBMV-C sgRNA and p21 from the SBMV-C genomic RNA was compared using a cell-free system. The results of these experiments were consistent with translation of these proteins by a 5' end-dependent scanning mechanism rather than by internal ribosome binding.

Determination and comparative analysis of the small RNA genomic sequences of California encephalitis, Jamestown Canyon, Jerry Slough, Melao, Keystone and Trivittatus viruses (Bunyaviridae, genus Bunyavirus, California serogroup).

The nucleotide sequences of the small (S) genomic RNAs of six California (CAL) serogroup bunyaviruses (Bunyaviridae: genus Bunyavirus) were determined. The S RNAs of two California encephalitis virus strains, two Jamestown Canyon virus strains, Jerry Slough virus, Melao virus, Keystone virus and Trivittatus virus contained the overlapping nucleocapsid (N) and non-structural (NSs) protein open reading frames (ORFs) as described previously for the S RNAs of other CAL serogroup viruses. All N protein ORFs were 708 nucleotides in length and encoded a putative 235 amino acid gene product. The NSs ORFs were found to be of two lengths, 279 and 294 nucleotides, which potentially encode 92 and 97 amino acid proteins, respectively. The complementary termini and a purine-rich sequence in the 3' non-coding region (genome-complementary sense) were highly conserved amongst CAL serogroup bunyavirus S RNAs. Phylogenetic analyses of N ORF sequences indicate that the CAL serogroup bunyaviruses can be divided into three monophyletic lineages corresponding to three of the complexes previously derived by serological classification. The truncated version of the NSs protein, which is found in five CAL serogroup bunyaviruses, appears to have arisen twice during virus evolution.

Identification of a coat protein binding site on southern bean mosaic virus RNA.

The cowpea strain of Southern bean mosaic virus (SBMV-C), a T = 3 icosahedral RNA virus, was dissociated to yield a ribonucleoprotein complex (RNPC) composed of the viral RNA and coat protein subunits. To determine if the coat protein subunits were bound to a specific site on the viral RNA, the RNPC was treated with ribonuclease, and the remaining coat protein--RNA complexes were recovered by filter binding. A single species of RNA was isolated by this procedure and further characterized by sequencing. The RNA was mapped to nucleotides 1410-1436 of the SBMV-C genome. This region of the viral RNA was predicted to fold into a hairpin with a 4-base loop and a duplex stem of 24 nucleotides. The stability and specificity of the coat protein-RNA complex isolated from dissociated virus suggest a possible role for this interaction in the selective encapsidation of the viral RNA.

Characterization of an internal element in turnip crinkle virus RNA involved in both coat protein binding and replication.

The major coat-protein-binding element of turnip crinkle virus RNA was previously mapped in the region of the UAG termination codon in the viral polymerase gene. This region encompasses two of the high-affinity coat-protein-binding sites (Fa and Ff) that we suggested were physically associated in a stem-loop in a ribonucleoprotein complex involved in assembly initiation (Wei, Heaton, Morris, and Harrison, J. Mol. Biol. 214, 85-95, 1990). We have also demonstrated that this RNA element was capable of specific coat protein binding in vitro (Wei and Morris, J. Mol. Biol. 222, 437-443, 1991). We now provide physical evidence, by in vitro chemical and enzymatic probing of the viral RNA, that support the suggestion that the two coat-protein-binding sites base pair to form a stem structure (A/F stem) surrounding the UAG terminator in wild-type RNA. We have shown here that a mutant with seven conservative nucleotide substitutions in Fa does not accumulate to detectable levels in plants or protoplasts and that the A/F stem structure is drastically altered in this mutant. We suggest that the primary effect of this mutation is on replication rather than on a reduction in RNA stability resulting from a defect in encapsidation of the virion RNA because previous results have shown that encapsidation-deficient mutants have little or no effect on viral RNA replication (Hacker, Petty, Wei, and Morris, Virology 186, 1-8, 1992). The analysis of the A/F stem was extended by construction and characterization of a series of mutants and revertants that displayed variable levels of replication deficiency but minimal concomitant defect in encapsidation efficiency. The extent of the replication defect correlated with the predicted destabilization of the A/F stem structure. We conclude from these results that this RNA element is involved in viral replication, and we tentatively suggest that the A/F stem structure may be functionally involved in the readthrough translation of the viral polymerase.

Turnip crinkle virus genes required for RNA replication and virus movement.

We have used infectious in vitro transcripts from mutagenized turnip crinkle virus (TCV) cDNA clones to identify the gene products required for viral RNA replication, virion assembly, and intercellular movement. Previous sequence analysis of the TCV genome revealed the presence of five open reading frames which had the potential to encode gene products of 88, 38, 28, 9, and 8 kDa. Inoculation of protoplasts with infectious RNA revealed that only the p28 and p88 gene products are required for viral RNA synthesis. Although the p8 and p9 gene products were dispensable for RNA replication and virion assembly in protoplasts, mutations in the p8 and p9 genes prevented the production of systemic infections in plants. No viral RNA or protein was observed in the inoculated or systemic leaves of plants inoculated with transcripts synthesized from p8 or p9 mutant cDNAs. In contrast to these results, viral RNA was recovered from the inoculated, but not the systemic leaves, of plants inoculated with an RNA lacking the coat protein (CP) gene. With the CP mutant, no symptoms were observed on normally systemic hosts, but small local lesions were induced on Chenopodium amaranticolor. These results indicate that p8, p9, and CP are required for viral movement.

A nonlethal mutation in large T antigen of polyomavirus which affects viral DNA synthesis.

A mutation in polyomavirus large T antigen which affects viral DNA synthesis was discovered in strain NG59RA (RA). The effect was most visible in nonpermissive cells. Although a substantial yield in DNA synthesis is normally observed in infections of Fischer rat cells when these are maintained at 33 degrees C (D.L. Hacker, K.H. Friderici, C. Priehs, S. Kalvonjian, and M.M. Fluck, p. 173-181, in R.E. Moses and W.C. Summers, ed., DNA Replication and Mutagenesis, 1988; D.L. Hacker and M.M. Fluck, Mol. Cell. Biol., in press), a 10- to 20-fold decrease in yield was obtained in infections with RA. The yield of free viral DNA in RA transformants was also strongly diminished, whether the transformants were maintained at 37 or 33 degrees C. A large reduction in the apparent number of integration sites, as well as a small reduction in the incidence of tandem integration of the viral genome, was observed in F-111 or FR-3T3 cells transformed by the mutant strain. This appears not to be directly related to the number of integration templates. A DNA fragment was identified which rescues these phenotypes. The fragment is located between the HindIII and NsiI restriction sites (nucleotides 1656 to 1910), a region which encodes only large T antigen. Sequence analysis of this region reveals a C-to-G transition at nucleotide 1791 which causes a proline-to-alanine change in the amino acid sequence of large T antigen. No other mutations have been previously reported in this region of large T antigen.

Viral DNA synthesis in nonpermissive rat F-111 cells and its role in neoplastic transformation by polyomavirus.

We have investigated the occurrence and role of polyomavirus DNA synthesis in neoplastic transformation by this virus. We show that after infection of Fischer rat F-111 cells at 37 degrees C, there is two- to threefold increase in the level of viral DNA as compared with the input signal, with a peak observed between 5 and 7 days postinfection. Viral DNA synthesis is about 10 times higher at 33 degrees C and increases up to 15 days postinfection. Most of the viral DNA produced is supercoiled (form I DNA). On the basis of in situ hybridization, it appears that viral replication is restricted to a small fraction of the population. At the lower temperature, more cells are permissive for viral DNA synthesis and the level of synthesis per permissive cell is higher. The DNA synthesis observed is large T-antigen dependent, and the increase in viral DNA synthesis at 33 degrees C is paralleled by an increase in the expression of this viral protein. When large T antigen is inactivated, the half-life of de novo-synthesized viral DNA is less than 12 h, suggesting that large T antigen may be responsible for the stability of the viral genomes as well as their synthesis. Surprisingly, at early times postinfection (0 to 48 h), when the essential function of large T antigen in transformation is expressed (as demonstrated in shift-up experiments with tsa mutants), the level of large T antigen is below the detection level and is at least 10-fold lower than the levels observed in permissive infections at the start of viral DNA synthesis. The difference in viral DNA at 37 and 33 degrees C allowed us to study its effect on transformation. Although an increase in transformation frequency is observed in wild-type A2 infections carried at 33 degrees C (frequencies two to three times higher than at 37 degrees C), this increase appears to be unrelated to the increase in viral DNA synthesis. Furthermore, the overall level of viral DNA and large T antigen in F-111 cells may not affect the integration of the viral genome, since the patterns of integration in cells transformed by wild-type A2 at 33 and 37 degrees C appear similar. The results are compatible with a role for large T antigen in integration-transformation which is not simply to amplify the viral genome to enhance the probability of its integration.