Analysis of testes and semen from rabbits treated by intravenous injection with a retroviral vector encoding the human factor VIII gene: no evidence of germ line transduction.

In a phase 1 clinical trial, we are evaluating a murine leukemia virus (MuLV)-based retroviral vector encoding the human factor VIII gene [hFVIII(V)], administered intravenously, as a therapy for hemophilia A. Preclinical biolocalization studies in adult rabbits revealed vector-specific PCR signals in testis tissue at low levels. In follow-up animal studies we used PCR to (1) estimate the frequency with which a given cell in testis tissue is transduced, and (2) determine whether a positive PCR signal could be detected in semen samples from animals treated with hFVIII(V). Using the 99% confidence bound, results indicate that the probability that a given cell within the testis was transduced is less than 1/709,000 (97 days after treatment). This probability decreased with time after hFVIII(V) administration. Moreover, the rate of provector sequence detection in semen samples collected weekly throughout two cycles of spermatogenesis was 3/4281 reactions (0.07%), which is lower than the rate of false positives (1/800, 0.125%) observed for control animals. Using PCR assays with single-copy sensitivity, we have shown that the small number of transduced cells present in testis tissue does not give rise to detectable transduced cells in semen.

Processing of a cellular polypeptide by 3CD proteinase is required for poliovirus ribonucleoprotein complex formation.

Poliovirus interactions with host cells were investigated by studying the formation of ribonucleoprotein complexes at the 3' end of poliovirus negative-strand RNA which are presumed to be involved in viral RNA synthesis. It was previously shown that two host cell proteins with molecular masses of 36 and 38 kDa bind to the 3' end of viral negative-strand RNA at approximately 3 to 4 h after infection. We tested the hypothesis that preexisting cellular proteins are modified during the course of infection and are subsequently recruited to play a role in viral replication. It was demonstrated that the 38-kDa protein, either directly or indirectly, is the product of processing by poliovirus 3CD/3C proteinase. Only the modified 38-kDa protein, not its precursor protein, has a high affinity for binding to the 3' end of viral negative-strand RNA. This modification depends on proteolytically active proteinase, and a direct correlation between the levels of 3CD proteinase and the 38-kDa protein was demonstrated in infected tissue culture cells. The nucleotide (nt) 5-10 region (positive-strand numbers) of poliovirus negative-strand RNA is important for binding of the 38-kDa protein. Deletion of the nt 5-10 region in full-length, positive-strand RNA renders the RNA noninfectious in transfection experiments. These results suggest that poliovirus 3CD/3C proteinase processes a cellular protein which then plays an essential role during the viral life cycle.

Poliovirus infection enhances the formation of two ribonucleoprotein complexes at the 3' end of viral negative-strand RNA.

To identify proteins involved in the formation of replication complexes at the 3' end of poliovirus negative-strand RNA, a combined in vitro biochemical and in vivo genetic approach was used. Five subgenomic cDNA constructs were generated to transcribe different negative-strand RNA fragments. In UV cross-linking assays, distinct differences in binding of proteins in extracts from poliovirus-infected and uninfected cells to virus-specific, radiolabeled transcripts were observed. Two proteins present in extracts from poliovirus-infected cells with approximate molecular masses of 36 and 38 kDa were shown to cross-link to the 3' end of poliovirus negative-strand RNA. Appearance of the 36- and 38-kDa proteins in UV cross-linking assays can be detected 3 to 3.5 h after infection, and cross-linking reaches maximum levels by 5 h after infection. The binding site for the 36-kDa protein overlaps with the computer-predicted loop b region of stem-loop I, the so-called cloverleaf structure, and the RNA sequence of this region is required for efficient binding. Transfection of full-length, positive-sense RNA containing a five-nucleotide substitution (positions 20 to 25) in the loop b region of stem-loop I into tissue culture cells yielded only viral isolates with a reversion at position 24 (U-->C). This finding demonstrates that the wild-type cytidine residue at position 24 is essential for virus replication. RNA binding studies with transcripts corresponding to the 3' end of negative-strand RNA suggest that complex formation with the 36-kDa protein plays an essential role during the viral life cycle.

Regulation of the cellular thymidine kinase gene promoter in simian virus 40-infected cells.

We examined the regulation of the cellular thymidine kinase (TK) gene promoter in simian virus 40 (SV40)-infected simian CV1 cells. Nuclear run-on transcription assays demonstrated a three- to fourfold increase in the rate of transcription of the endogenous gene at 14 to 16 h following viral infection. In addition, hybrid genes containing the human TK promoter linked to the bacterial neomycin resistance gene were induced by SV40 in stably transfected cells, indicating that promoter sequences are sufficient to confer viral regulation. Analysis of human TK promoter deletion mutants indicated that sequences localized between -67 and +30 bp relative to the transcriptional initiation site are sufficient to confer regulation on SV40-infected cells. These sequence elements are distinct from those required for serum induction, which were previously localized to the region between -135 and -67. These results suggest that SV40 activates novel cellular pathways that are not activated by serum stimulation of quiescent cells.

Identification of a G1-S-phase-regulated region in the human thymidine kinase gene promoter.

We have identified a regulatory region in the human thymidine kinase gene promoter. A set of promoter deletion mutants was constructed, linked to the bacterial neomycin resistance gene, and stably transfected into Rat3 cells. It was shown that the region between 135 and 67 base pairs upstream of the cap site is required for conveying G1-S-phase regulation to the linked neo gene. In addition, primer extension assays demonstrated that the same transcriptional start sites were used in G1- and S-phase cells and in the various deletion mutants tested.