P Beard

The genome of the human parvovirus B19 contains a transcriptional promoter (BP06) at map position 6, upstream from the nonstructural protein genes. By cotransfecting HeLa cells with this promoter cloned before the chloramphenicol acetyltransferase (CAT) gene together with a plasmid containing almost the whole B19 genome, we showed that BP06 is transactivated by a B19 gene product. The transactivating viral protein was identified as the nonstructural protein NS-1. NS-1 synthesized in a wheat germ extract specifically stimulates transcription from BP06 in vitro. NS-1 of the minute virus of mice (MVM) activates the analogous MVM promoter, MP04. NS-1, therefore, has a positive feedback effect on the activity of its own promoter. Moreover, NS-1 of MVM activates the human BP06. We have identified, in the genome of B19, a second transcriptional promoter activity at map position 44, before the capsid protein genes. This promoter, BP44, was identified by cloning fragments of B19 DNA upstream of the CAT gene, transfecting the DNA into HeLa cells, and measuring CAT expression. The strength of the BP44 promoter is similar to that of the capsid gene promoter, MP39, of MVM. In (nonpermissive) HeLa cells, the BP44 promoter is not activated by NS-1. Thus, the BP06 promoter apparently does not determine the tissue specificity of B19 virus but BP44 could do so.

We have characterized an immunosuppressive parvovirus related to the minute virus of mice (MVM). The parvovirus, MVM(i), grew efficiently on the murine lymphoma cell line EL-4 and not on the A-9 strain of L-cells which is a host for the prototype MVM. MVM(i) was immunosuppressive for allogeneic mixed leukocyte cultures, inhibiting the generation of cytolytic T lymphocytes. MVM had no effect on mixed leukocyte cultures. MVM and MVM(i) particles were similar in buoyant density, sedimentation rate, appearance in the electron microscope, and polypeptide composition. We present restriction enzyme maps of the DNAs of MVM and MVM(i) which show that they are closely related. Out of 109 restriction endonuclease cleavage sites (representing together about 10% of the nucleotide sequence), 86 sites were shared by MVM and MVM(i), whereas 22 sites were absent from one of the two viruses. MVM(i) DNA had an apparent deletion of about 60 nucleotides relative to MVM, located near the 5' terminus of viral DNA.

The genome of the autonomous parvovirus minute virus of mice (MVM) is organized in two overlapping transcription units: the genes coding for the two non-structural proteins (NS-1 ad NS-2) are transcribed from a promoter (P04) located at map unit 4, whereas the promoter controlling the capsid protein genes (P39) lies at map unit 39. We studied the effect of viral proteins on the activity of the P39 promoter in vivo. By site-directed mutagenesis we constructed clones encoding only one of the two NS proteins. The activity of the P39 promoter was measured in HeLa or EL-4 cells transfected with these clones, either by an RNase protection assay or by following the expression of a reporter gene, CAT (which codes for chloramphenicol acetyltransferase), placed under the control of this promoter. We found that the P39 promoter of strain MVMi is activated in trans by a viral gene product, and evidence to suggest that NS-1 is the only viral gene product responsible for this trans-activation. We also determined that the mechanism of trans-activation is very rapid, since all species of viral mRNAs appear together in non-synchronized infected EL-4 cells within a 2 h interval.

In herpes simplex virus (HSV)-infected cells, there is a sequential expression of viral genes. In vivo experiments have implicated the Mr 175,000 immediate early protein ICP4 (infected-cell polypeptide 4) in the regulation of viral RNA synthesis, but the mechanism whereby ICP4 regulates transcription of viral genes is at present unknown. In this report we describe experiments with an in vitro transcription system and a purified preparation of ICP4 (estimated 5% of total protein). Using DNA from the HSV glycoprotein D gene (gD) as the template, we have observed that specific binding occurs between ICP4 and DNA sequences adjacent to the gD gene promoter and ICP4 stimulates initiation of transcription from the gD gene. The degree of stimulation depends on the amount of ICP4 present in the incubation. The kinetics of RNA synthesis demonstrate that the protein acts at the initiation step of transcription. These results identify ICP4 as a viral transcription factor whose presence on DNA facilitates the formation of transcription complexes.

Papillomaviruses are tissue specific and replicate in differentiating keratinocytes. We are interested in the question of tissue specificity at the level of transcription. We used extracts from human keratinocytes and human fibroblasts at low passage number and from HeLa cells to look for factors binding to the E6 promoter of human papillomavirus type 18 (HPV-18) DNA by footprint and gel mobility shift experiments. We found a factor present in HeLa and keratinocyte extracts but not in fibroblast extracts which bound about 160 base pairs upstream from the start of E6. The binding site included the sequence TGACTAAG, which resembles the consensus binding site for the AP-1 family of proteins. Synthetic oligonucleotides containing this binding site specifically competed with factor binding to HPV-18 DNA, as did the AP-1 sequence of simian virus 40. They also inhibited transcription from the E6 promoter in vitro in extracts from HeLa cells. Thus, the presence of this keratinocyte-specific factor seems to be important for HPV-18 transcription.