Amplification mediated by polyomavirus large T antigen defective in replication.

The polyomavirus large T antigen promotes homologous recombination at high rates when expressed in rat cells carrying the viral replication origin and two repeats of viral DNA sequences stably integrated into the cellular genome. Recombination consists of both reciprocal and nonreciprocal events and is promoted by mutants defective in the initiation of viral DNA synthesis (L. St-Onge, L. Bouchard, and M. Bastin, J. Virol. 67:1788-1795, 1993). We have extended our studies to a rat cell line undergoing amplification of the viral insert. We show that large T antigen promotes amplification independently of its replicative function but that its origin-specific DNA binding activity is not sufficient to promote homologous recombination.     

High-frequency recombination mediated by polyomavirus large T antigen defective in replication.

We investigated the mechanism by which the large T antigen (T-Ag) of both polyomavirus and simian virus 40 (SV40) promotes homologous recombination in mammalian cells. To this end, we constructed a rat cell line, designated Hy5, that carries two mutated copies of the polyomavirus middle-T-Ag (pmt) oncogene lying as direct repeats on the same chromosome. The structure of the viral insert was devised so that intrachromosomal recombination between the pmt repeats reconstitutes wild-type pmt and yields cell populations amenable to selection for the transformed phenotype. Correction of pmt by gene conversion occurred spontaneously at a rate of ca. 1.7 x 10(-7) per cell generation and was masked by another recombination event that also led to the transformation of the Hy5 cell line. This event was identified as chromosomal inversion and overexpression of the upstream pmt copy as a result of homologous recombination between adjacent pBR322 sequences. Both events were promoted by the polyomavirus large T-Ag by several orders of magnitude, as well as by mutants defective in the initiation of viral DNA synthesis. Large T-Ag also promoted reconstitution of wild-type pmt by unequal exchange between sister chromatids, yielding structures compatible with some of the chromosomal aberrations commonly observed in transformed cells. Our data indicate that large T-Ag has a recombination-promoting activity that can be dissociated from its replicative function.     

Inhibition of pseudorabies virus replication by vesicular stomatitis virus. II Activity of defective interfering particles.

Purified defective interfering (DI) particles of vesicular stomatitis virus (VSV) inhibit the replication of a heterologous virus, pseudorabies virus (PSR), in hamster (BHK-21) and rabbit (RC-60) cell lines. In contrast to infectious B particles of VSV, UV irradiation of DI particles does not reduce their ability to inhibit PSR replication. However, UV irradiation progressively reduces the ability of DI particles to cause homologous interference with B particle replication. Pretreatment with interferon does not affect the ability of DI particles to inhibit PSR replication in a rabbit cell line (RC-60) in which RNA, but not DNA, viruses are sensitive to the action of interferon. Under similar conditions of interferon pretreatment, the inhibition of PSR by B particles is blocked. These data suggest that de novo VSV RNA or protein synthesis is not required for the inhibition of PSR replication by DI particles. DI particles that inhibit PSR replication also inhibit host RNA and protein synthesis in BHK-21 and RC-60 cells. Based on the results described and data in the literature, it is proposed that the same component of VSV B and DI particles is responsible for most, if not all, of the inhibitory activities of VSV, except homologous interference.     

Molecular basis for interference of defective interfering particles of pseudorabies virus with replication of standard virus.

Serial passage of pseudorabies virus (PrV) at high multiplicity yields defective interfering particles (DIPs), but the sharp cyclical increases and decreases in titer of infectious virus that are observed upon continued passage at high multiplicity of most DIPs of other viruses are not observed with DIPs of PrV (T. Ben-Porat and A. S. Kaplan, Virology 72:471-479). We have studied the dynamics of the interactions of the virions present in a population of DIPs to assess the cis functions for which the genomes of the DIPs are enriched. The defective genomes present in one population of DIPs, [PrV(1)42], replicate preferentially over the nondefective genomes present in that virion population at early stages of infection, indicating that the DIP DNA is enriched for sequences that can serve as origins of replication at early stages of infection. This replicative advantage of the DIP DNA is transient and disappears at later stages of infection. The defective DNA does not appear to be encapsidated preferentially over the nondefective DNA present in this virion population, which might indicate that it is not enriched for cleavage-encapsidation sites. However, the nondefective DNA in the DIP virion population has become modified and has acquired reiterations of sequences originating from the end of the unique long (UL) region of the genome. Furthermore, both the infectious and defective genomes present in the DIP population compete for encapsidation more effectively than do the genomes of standard PrV. These results indicate that the defective genomes in the population of virions studied are enriched not only for an origin of replication but probably also for sequences necessary for efficient cleavage-encapsidation. Furthermore, the nondefective genomes present in this population of DIPs have also been modified and have acquired the ability to compete with the defective genomes for cleavage-encapsidation.     

Guanidine-resistant defective interfering particles of poliovirus.

A mixture containing standard poliovirus and D3 particles (mutants with deletions in the capsid locus) was serially passaged in the presence of guanidine. Within five growth cycles, the standard virus was guanidine resistant, but the D3 particles were guanidine sensitive, even after 21 passages with the inhibitor. By passage 40 with guanidine, D3 particles were eliminated, and a new deletion mutant (DX) appeared in the virus population. D3 particles contained a 15% deletion, and DX particles contained a 6% deletion in the capsid locus. Although neither mutant induced the synthesis of NCVP1a or a complete complement of capsid proteins after infection, cells infected with DX particles produced two novel proteins, which had molecular weights of approximately 68,000 and 25,000.     

A new pathway in the generation of defective retrovirus DNA.

We used a retrovirus shuttle vector to make molecular clones of circular viral DNA from infected cells. One-third of the molecules examined had deletions that started within or near the U5 domain of the long terminal repeat (LTR) region and extended a variable distance toward the gag gene. We present evidence that some of these deletions arose by cleavage of a single LTR unit, in contrast to the cleavage of tandem LTR units associated with the integration reaction. These results suggest that in the formation of defective circular DNA, the U5 domain can be recognized and cleaved in the absence of an adjacent U3 domain. The cleavage of isolated U5 domains may represent an important mechanism responsible for the generation of certain forms of both defective circular DNA and defective integrated DNA.     

An analysis of replication errors made by a defective T4 DNA polymerase

Summary An amber mutant in the head protein of bacteriophage T4D, amH36 has been induced to revert by a mutator, tsL56 in gene 43 (the structural gene for DNA polymerase, de Waard, Paul and Lehman, 1965) which is known to cause errors in replication. As a consequence the known am base triplet is converted to other triplets which assign certain amino acids. The nature of the replication errors has been analyzed by looking at the insertion of amino acids in a peptide from the head protein of 60 independent am ⁺ revertants. Of these, 38 had incorporated tyrosine (like spontaneous revertants also do) while in 21 cases glutamine was inserted and in one case glutamic acid. With the help of the codon catalogue it could be shown that the L56 polymerase promotes an A:TG:C transition as well as more than one type of transversion. The single revertant which had incorporated glutamic acid clearly represents an A:TC:G transversion. The other transversions leading to the insertion of tyrosine indicate that a C:G pair has been converted. In this case the degeneracy of the code does not allow to differentiate between the transversion C:GG:C and C:GA:T. These findings and the absence of certain amino acids as permissible substituents are discussed with regard to the specificity of the errors in replication made by L56 polymerase.     

Characterization of the la antigens involved in suppressor T cell generation in the rat

The WRC rat, an intra-class II recombinant strain (RT1.B ⁿ B ^a D _, ^a ), was used to study the relative roles of the two class II loci in mixed lymphocyte reaction (MLR) proliferation and suppressor T cell (Ts) generation. Both MLR proliferation and Ts generation were noted in cultures of WRC with DA (RTI ^{a) stimulator cells. In contrast, cultures of WRC with BN (RT1sun} ) stimulator cells proliferate but do not generate significant amounts of Ts. The data suggest that RT1.B incompatibility is important in the generation of Ts in the WRC rat. Suppressor cells generated in cultures of WF (RT1 ^u ) with WRC stimulator cells potently suppressed a WF+WRC_x test MLR, with less suppression when tested against either the WF+DA_x or WF+BN_x MLRs. The latter experiments suggest that Ts clones may be produced to either class II subregion, and therefore that MLR proliferation and Ts induction are not necessarily linked, but vary with particular genotypes. The current lack of other rat intra-class II recombinant strains precludes assignment of suppressor induction/activation to a single locus.     

Amber-mutants of plasmid mini-F defective in replication

Summary Amber mutants of the mini-F plasmid pML31 were isolated with the mutagen hydroxylamine. Under non-permissive conditions amber mutants segregate and show no incorporation of label into supercoiled plasmid DNA in double-label experiments. Wild-type and one mutant of mini-F were integrated by recombinant DNA techniques into the single EcoRI site of plasmid vector pBR322. Plasmid specific proteins were labeled in minicells and analysed by SDS-PAGE. A 34,000 dalton molecular weight protein was identified to be missing in the amber mutant of plasmid mini-F.     

Impact of defective interfering particles on virus replication and antiviral host response in cell culture-based influenza vaccine production

During the replication of influenza viruses, defective interfering particles (DIPs) can be generated. These are noninfectious deletion mutants that require coinfection with a wild-type virus but interfere with its helper virus replication. Consequently, coinfected cells mainly produce DIPs. Little is known about how such noninfectious virus particles affect the virus yield of cell culture-based influenza vaccine production. We compared infections of Madin-Darby canine kidney cells with two seed virus preparations of the influenza virus strain A/Puerto Rico/8/34 that contain different amounts of DIPs. A combination of conventional RT-PCR, RT-qPCR, and flow cytometry revealed that DI genomes indeed strongly accumulate in coinfected cells and impede the viral RNA synthesis. Additionally, cells infected at the higher DIP concentration showed a stronger antiviral response characterized by increased interferon-β expression and apoptosis induction. Furthermore, in the presence of DIPs, a significant fraction of cells did not show any productive accumulation of viral proteins at all. Together, these effects of DIPs significantly reduce the virus yield. Therefore, the accumulation of DIPs should be avoided during influenza vaccine production which can be achieved by quality controls of working seed viruses based on conventional RT-PCR. The strategy for the depletion of DIPs presented here can help to make cell culture-based vaccine production more reliable and robust.     

Structure of the intracellular defective viral RNAs of defective interfering particles of mouse hepatitis virus.

The intracellular defective RNAs generated during high-multiplicity serial passages of mouse hepatitis virus JHM strain on DBT cells were examined. Seven novel species of single-stranded polyadenylic acid-containing defective RNAs were identified from passages 3 through 22. The largest of these RNAs, DIssA (molecular weight [mw], 5.2 X 10(6)), is identical to the genomic RNA packaged in the defective interfering particles produced from these cells. Other RNA species, DIssB1 (mw, 1.9 X 10(6) to 1.6 X 10(6)), DIssB2 (mw, 1.6 X 10(6)), DIssC (mw, 2.8 X 10(6)) DIssD (mw, 0.82 X 10(6)), DIssE (mw, 0.78 X 10(6)), and DIssF (mw, 1.3 X 10(6)) were detected at different passage levels. RNase T1-resistant oligonucleotide fingerprinting demonstrated that all these RNAs were related and had multiple deletions of the genomic sequences. They contained different subsets of the genomic sequences from those of the standard intracellular mRNAs of nondefective mouse hepatitis virus JHM strain. Thus these novel intracellular viral RNAs were identified as defective interfering RNAs of mouse hepatitis virus JHM strain. The synthesis of six of the seven normal mRNA species specific to mouse hepatitis virus JHM strain was completely inhibited when cells were infected with viruses of late-passage levels. However, the synthesis of RNA7 and its product, viral nucleoprotein, was not significantly altered in late passages. The possible mechanism for the generation of defective interfering RNAs was discussed.     

A specific internal RNA polymerase recognition site of VSV RNA is involved in the generation of DI particles.

The 3′ terminal sequences of four different DI particle RNAs ranging in size from 10S to 30S have been determined directly using rapid RNA sequencing methods or deduced, in the case of the fourth DI RNA, from the complementary sequence of a small RNA transcribed from this part of the genome (Schubert et al., 1978). One DI particle (DI 011) contains covalently linked genomic and antigenomic RNA. The 5′ end of this RNA is identical to that of VSV RNA, as determined by annealing for at least 1 kb, as well as to the other DI particle RNAs used in this study. The 3′ ends of the other three DI particle RNAs are exact copies of the common 5′ terminal sequence for 48 nucleotides in two cases and 45 nucleotides in the third. Beyond these complementary regions the sequences are different for each DI RNA. The fact that these regions differ in length by only three nucleotides, despite the wide differences in the overall size of the DI particle RNAs, indicates that if these DIs were formed by the copy-back mechanisms similar to those proposed by Leppert, Kort and Kolakofsky (1977) and Huang (1977), a specific recognition site for the RNA polymerase must be involved in copying the 5′ terminus. We determined the 5′ terminal sequence from position 43–48 at the end of the complementary region and found it to be 5′-GGUCUU-3′. This hexamer is also part of other highly conserved terminal RNA polymerase initiation sites (Keene et al., 1978; Keene, Schubert and Lazzarini, 1979) and may be a specific internal RNA polymerase recognition site. We conclude that this sequence is one of the elements involved in the genesis of DI particle chromosomes containing short complementary sequences at their termini. The ability of the polymerase to resume synthesis at or near a specific recognition site is discussed.     

A cytoplasmic activator of DNA replication is involved in signal transduction in antigen-specific T cell lines.

Cytoplasmic extracts prepared from T cell lines undergoing antigen-specific, interleukin-2 (IL-2)-dependent proliferation were tested for their ability to induce DNA synthesis in isolated, quiescent nuclei. A tetanus toxoid (TET)-specific T cell line, established from peripheral blood of a normal human volunteer, was stimulated in the presence of relevant antigen and 1 unit/ml IL-2. Cytoplasmic extracts prepared from these cells were capable of inducing DNA synthesis in isolated, quiescent nuclei. The ability of cytoplasmic extracts to induce DNA synthesis in isolated, quiescent nuclei. The ability of cytoplasmic extracts to induce DNA synthesis in isolated nuclei correlated positively with the degree of proliferation induced in these cells. In contrast, incubation of this T cell line in the absence of antigen failed to induce proliferation and cytoplasmic extracts prepared from these cells induced little to no DNA synthesis in isolated, quiescent nuclei. The factor present in the cytoplasm of T cells stimulated with relevant antigen in the presence of IL-2 is similar, if not identical, to a factor which we have previously demonstrated in cytoplasmic extracts prepared from transformed lymphoblastoid cell lines and from mitogenically stimulated normal human peripheral blood mononuclear cells. This factor, which we have called activator of DNA replication (ADR) is a heat-labile protein, and is inactivated by treatment with protease inhibitors, including aprotinin. The ability of cytoplasmic extracts from T cells undergoing antigen-specific, IL-2-dependent proliferation to induce DNA synthesis in isolated, quiescent nuclei was markedly inhibited in the presence of aprotinin, providing strong evidence that a cytoplasmic activator of DNA replication, ADR, is involved in the signal transduction process for antigen-specific, IL-2-dependent T cell proliferation. ADR may represent a common intracellular mediator of DNA synthesis in activated and transformed lymphocytes.     

Impaired immunoregulation in systemic lupus erythematosus: defective adenosine-induced suppressor T lymphocyte generation

It is currently unclear whether the suppressor cell dysfunction observed during active systemic lupus erythematosus (SLE) reflects a primary T cell disorder or one that results from immunologic modulation of suppressor T cell activity by autoantibodies. To determine whether the suppressor T cell dysfunction of active SLE is the result of a primary T cell disorder, the model of adenosine-induced immunosuppression was utilized to study the suppressor T cell functions of 12 patients with SLE (seven active SLE, five inactive SLE) and 12 matched healthy controls. T lymphocyte phenotyping was performed by utilizing monoclonal antibodies directed against T cell-specific determinants. Suppressor T cell functions were assessed by two assays in parallel. The first technique tested the capacity of two suppressor T cell subsets (spontaneous suppressors, Ts; adenosine-inducible suppressors, TRA) to inhibit pokeweed mitogen- (PWM) induced B cell differentiation. In the second technique, the ability of enriched T cell preparations to suppress mitogen- and alloantigen-induced proliferation was assayed. It was demonstrated that brief treatment of the control theophylline-resistant T lymphocyte (TR) subset possessing inducer/helper activity with adenosine (10(-5) M, 30 min, 37 degrees C) triggered a rapid shift in phenotype (RFC gamma -, T-4+ leads to RFC gamma +, T-8+) in a proportion of the subset, and the development of radioresistant suppressor function. By contrast, exposure of active SLE TR to adenosine failed to induce either the switch of phenotype or suppressor activity. When compared to controls, both the TS and TRA suppressors failed to inhibit B cell differentiation (TS, p less than 0.001; TRA, p less than 0.001). Moreover, enriched T cell preparations incompletely suppressed the proliferative responses to phytohemagglutinin (p less than 0.003), PWM (p less than 0.0003), or alloantigens (p less than 0.01). During inactive SLE, the T cell responses were usually restored. Treatment of the TR subsets with adenosine induced a switch of phenotype in four of five patients and the subsequent expression of effective suppressor function. We conclude that a) during active SLE, there is impaired suppression of proliferation and B cell differentiation; b) the impaired suppression of B cell differentiation results from abnormal spontaneous (TS) and adenosine-inducible (TRA) suppressor functions; c) the defective generation of suppressor T cell function during active disease results, in part, from a block in the transition from inducer/helper to suppressor cell; and, d) the suppressor T cell dysfunction is reversible with disease remission. The investigation of immunopharmacologic events by using the adenosine-induced immuno-suppression model in T cells from normal donors and SLE patients may provide insights into the molecular basis of disordered immunoregulation in SLE.