Growth of the parvovirus minute virus of mice MVMp3 in EL4 lymphocytes is restricted after cell entry and before viral DNA amplification: cell-specific differences in virus uncoating in vitro

Two murine parvoviruses with genomic sequences differing only in 33 nucleotides (8 amino acids) in the region coding for the capsid proteins show different host cell specificities: MVMi grows in EL4 T lymphocytes and MVMp3 grows in A9 fibroblasts. In this study we compared the courses of infections with these two viruses in EL4 cells in order to investigate at which step(s) the infection process of MVMp3 is interrupted. The two viruses bound equally well to EL4 cells, and similar amounts of MVMi and MVMp3 input virion DNA appeared in the nuclear fractions of EL4 cells 1 h after infection. However, double-stranded replicative-form (RF) DNA of the two viruses appeared at different times, at 10 h postinfection with MVMi and at 24 h postinfection with MVMp3. The amount of MVMp3 RF DNA detected at 24 h was very small because it was produced only in a tiny subset of the population of EL4 cells that proved to be permissive for MVMp3. Replication of double-stranded viral DNA in EL4 cells was measured after transfection of purified RF DNA, cloned viral DNA, and cloned viral DNA with a mutation preventing synthesis of the capsid proteins. In each of these cases, DNA replication was comparable for MVMi and MVMp3. Production of virus particles also appeared to be similar after transfection of the two types of RF DNA into EL4 cells. Conversion of incoming 32P-labeled single-stranded MVM DNA to 32P-labeled double-stranded RF DNA was detected only after RF DNA amplification, indicating that few molecules serve as templates for viral DNA amplification. We showed that extracts of EL4 cells contain a factor which can destabilize MVMi virions but not MVMp3 by testing the sensitivity of viral DNA to DNase and by CsCl gradient analyses of viral particles. We therefore conclude that the MVMp3 life cycle is arrested after the transport of virions to the nucleus and prior to the replication of RF DNA, most likely at the stage of viral decapsidation.

Two segments in the genome of the immunosuppressive minute virus of mice determine the host-cell specificity, control viral DNA replication and affect viral RNA metabolism

Peter Martin Beard

Two strains of minute virus of mice (MVM) show different host-cell specificities. MVM(i) grows in T lymphocytes whereas MVM(p) is fibroblast-specific. By constructing recombinant viral DNAs between the genomes of the two strains, we have shown that two segments of the MVM(i) genome are required for lytic viral growth in T lymphocytic EL4 cells. One segment (iE) was found between nucleotides 1084 and 2070, in a region encoding the early viral proteins and containing mRNA splice signals and the late P39 promoter. The other (iL) was between nucleotides 3523 and 4339 in the region coding for capsid protein. The P39 promoters within the E segment from MVM(i) or MVM(p) were equally active in transfected EL4 cells. However, pE-containing MVM DNA produced more NS2 mRNA than iE-containing DNA, apparently the result of virus-strain-specific differences in the regulation of splicing.

1998

The mismatched nucleotides in the 5'-terminal hairpin of minute virus of mice are required for efficient viral DNA replication

Peter Martin Beard

The 5'-terminal sequence in the DNA of the parvovirus minute virus of mice (MVM) is a palindrome. It can form a hairpin, the stem of which is entirely base-paired except for three consecutive unpaired nucleotides which form a bubble. Since this structure is well conserved among different parvoviruses, we examined its importance for viral replication by generating MVM mutants with alterations in this region. A clone of MVMp DNA which contained the entire 3' end and more than half of the 5' palindrome was made. Although it lacked the sequence information to form a wild-type bubble, this DNA was infectious. On transfection into A9 fibroblasts, it gave rise to a virus (MVMs) which had a bubble in its 5' palindrome. The bubble consisted of four mismatched nucleotides in the same location as the unpaired nucleotides of the wild-type palindrome. Apparently, neighboring plasmid sequences were incorporated into the viral DNA, enabling formation of the mismatch. This observation suggested that a bubble is critical for growth of MVM but that its sequence is not. To find out whether MVM lacking a bubble in the 5' palindrome is viable, we made a second clone in which the plasmid sequences incorporated in MVMs were removed. Transfection of this DNA gave rise to a virus (MVMx) in which the nucleotides unpaired in the wild-type hairpin are now fully base-paired. Although MVMx can be propagated, it is defective in comparison with wild-type MVMp; it exhibited about a 50-fold-lower ratio of plaque-forming units to DNA content. In mixed infections, MVMp consistently outgrew the bubbleless MVMx. The rate of accumulation of DNA replication intermediates was lower for MVMx than for the wild-type virus. Quantitative analysis of the 5' termini of replicative form DNA suggested that the ability of MVMx to convert hairpin 5' termini to extended termini is impaired. In contrast, the virus with the altered bubble, MVMs, behaved like the wild-type MVMp in all the assays. We conclude that MVM lacking a bubble in its 5'-terminal DNA hairpin is less infectious than and has a selective disadvantage compared with wild-type MVM. The nucleotide sequence of the bubble is not critical. We provide evidence that the presence of a bubble is necessary for efficient viral DNA replication.

1995

The use of RNA interference to increase PEI-mediated transient gene expression in mammalian cells

Martin Bertschinger

DNA uptake by polyethylenimine (PEI)-mediated transfection was investigated in Chinese hamster ovary (CHO DG44) cells. Rapid DNA uptake was observed with the maximum occurring during the first 45-60 min after addition of PEI-DNA complexes to cells. With longer incubation times, the aggregation of PEI-DNA particles reduced the rate of DNA uptake. At early times after transfection, the kinetics of DNA uptake were constant, suggesting that the limiting factor to PEI transfection was the rate of endocytosis. The most efficient transfection with PEI was observed at a density of 2 x 106 cells/ml and at a PEI:DNA ratio of 2:1 (w/w). DNA uptake at higher PEI:DNA ratios was also efficient, but the toxicity of PEI decreased the recombinant protein yield. The optimal PEI:DNA ratio was found to be related to the number of cells in the culture. Once in the cell, PEI:DNA particles must be disassembled to allow transcription of the recombinant gene. Here their disassembly was investigated using a simple in vitro assay. The particles were formed with either a linear (25 kDa or JetPEITM) or a branched PEI (2, 25, or 750 kDa) and then mixed with varying amounts of a competitor (RNA, DNA, or heparin). The amount of plasmid DNA released from particles was determined by agarose gel electrophoresis or spectroscopy. The stability of the particles to changes in pH, osmolarity, and the PEI:DNA ratio was also determined. Either the presence of heparin or high salt concentrations (1-2 M) yielded complete particle disassembly for all PEIs tested. The addition of RNA to particles formed with linear PEIs or branched 2 kDa PEI resulted in the rapid release of all the plasmid DNA, even at an RNA concentration of 50 µg/ml. However, the presence of RNA at concentrations up to 400 µg/ml induced only partial disassembly of particles formed with branched PEIs of 25 or 750 kDa. In the presence of competitor DNA, slow particle disassembly was observed with particles made with linear 25 kDa PEI, JetPEI, and branched 2 kDa PEI, but DNA release was not seen for particles made with branched PEIs of higher molecular weight. The presence of BSA only resulted in partial disassembly of PEI-DNA particles, and DNA release was not observed after the exposure of particles to acidic pH as low as 3. For all the PEIs tested, their stability increased as the PEI:DNA ratio increased. It was concluded from these studies that branched PEIs have a higher affinity for DNA than linear PEIs and that the intracellular disassembly of PEI-DNA particles may involve interactions between PEI and cellular RNA. In the second part of the work, RNA interference (RNAi) was used to enhance transient gene expression by knockdown of two different mRNAs, the E1A mRNA in human embryonic kidney 293 EBNA (HEK293E) cells and the ube2i mRNA in CHO DG44 cells and HEK293 cells. HEK293 cells are transformed with the adenovirus E1A and E1B genes. Because the E1A proteins function as transcriptional activators or repressors, they may have a positive or negative effect on transient transgene expression in this cell line. Suspension cultures of HEK293 cells were co-transfected with a reporter plasmid expressing the GFP gene and a plasmid expressing a short hairpin RNA (shRNA) targeting the E1A mRNAs for degradation by RNAi. The presence of the shRNA in HEK293E cells reduced the steady state level of E1A mRNA up to 75% and increased transient GFP expression from either the elongation factor-1α (EF-1α) promoter or the human cytomegalovirus (HCMV) immediate early promoter up to 3-fold. E1A mRNA depletion also resulted in a 2-fold increase in transient expression of a recombinant IgG in suspension cultures when the IgG light and heavy chain genes were driven by the EF-1α promoter. These results demonstrated that E1A has a negative effect on transient gene expression in HEK293E cells. Efficient RNAi-mediated depletion of ube2i mRNA in CHO DG44 cells was accomplished by co-expression of 2 shRNAs targeting two distinct sides in the ube2i mRNA, whereas knockdown with only one of the shRNAs at a time was insufficient for reducing ube2i mRNA levels. The reduction of the ube2i mRNA level in CHO DG44 cells was shown to enhance transient GFP expression 2-fold and IgG expression 2.5-fold. For these experiments, the GFP gene was under the control of the CMV IE promoter and the IgG light and heavy chain genes were under the control of the human EF-1α promoter. Thus, the enhancement of transient gene expression resulting from ube2i mRNA depletion was not promoter-dependent. These results demonstrated that sumoylation has a negative effect on transient gene expression in CHO DG44 and HEK293E cells.

EPFL2006

The mismatched nucleotides in the 5'-terminal hairpin of minute virus of mice are required for efficient viral DNA replication

Peter Martin Beard

1995

The use of RNA interference to increase PEI-mediated transient gene expression in mammalian cells

Martin Bertschinger

EPFL2006