The neurosurgeon as innovator and entrepreneur.

INNOVATION IS THE driving force behind progress in neurosurgery. Most significant innovations require commercialization to ensure appropriate development and ultimate distribution to patients. There are several key factors that determine whether a particular innovation is likely to be commercially successful. Relationships between academic neurosurgeons and industry are likely to increase in the future. Stronger and more productive relationships between academic neurosurgeons and commercial ventures will provide new opportunities for neurosurgeons to bring innovations to patients more effectively and efficiently. The transfer of innovation from the academic environment to the commercial setting is consistent with the academic mission and can increase funding for basic and clinical neuroscience research.

Sequences in the preC region of duck hepatitis B virus affect pregenomic RNA accumulation.

The pregenomic RNA of hepadnaviruses serves as both the mRNA for the core and polymerase proteins and the RNA template for reverse transcription. We have identified a region in the duck hepatitis B virus pregenomic RNA transcription unit that is critical for the accumulation of this transcript. This 85-nt region, termed alpha, is located within the preC region; deletion of alpha results in drastically reduced steady-state levels of pregenomic RNA. This effect is not due to reduction in transcription initiation or to enhancement of premature polyadenylation at the 5' copy of the viral poly(A) signal. However, this phenotype is suppressed by deletion of a second, larger region (beta) located ca. 1 kb downstream. The activity of the alpha element is tissue- and species-nonspecific; however, it displays absolute orientation-dependence and its activity is influenced by its position within the transcript. Models for its action are discussed.

Sequence-independent RNA cleavages generate the primers for plus strand DNA synthesis in hepatitis B viruses: implications for other reverse transcribing elements.

Reverse transcription of RNA into duplex DNA requires accurate initiation of both minus and plus strand DNA synthesis; this in turn requires the generation of specific primer molecules. We have examined plus strand primer generation in the hepatitis B viruses, small DNA viruses that replicate via reverse transcription. The plus strand primer in these viruses is a short capped RNA derived from the 5' end of the RNA template by cleavage at a specific set of sites. To elucidate the cleavage mechanism we constructed a series of viral mutants bearing alterations in and around the cleavage sites. Our results reveal that the cleavage reaction is sequence-independent and indicate that the cleavage sites are positioned by measurement of the distance from the 5' end of the RNA. Comparison of these findings with what is known about RNase H-mediated primer generation in retroviruses and other retroid elements suggests that, despite many divergent features, some common molecular features are preserved.

cis-acting sequences required for encapsidation of duck hepatitis B virus pregenomic RNA.

Hepadnavirus reverse transcription requires that pregenomic RNA first be selectively packaged into a cytoplasmic core particle. This process presumably requires the presence of specific recognition sequences on the pregenomic RNA. To define the cis-acting sequences required for pregenome encapsidation in the duck hepatitis B virus (DHBV), we assayed the packaging efficiency of a series of pregenomic RNA deletion mutants and hybrid DHBV/lacZ fusion transcripts. The 5' boundary of the packaging signal lies within the precore region, starting approximately 35 nucleotides from the cap site of pregenomic RNA; thus, the DR1 sequence required for proper viral DNA synthesis is not included in this signal. To define the 3' boundary of the encapsidation signal, fusion transcripts bearing foreign (lacZ) sequences fused to DHBV at different sites 3' to the pregenomic RNA start site were examined. A surprisingly large region of the DHBV genome proved to be required for packaging of such chimeras, which are efficiently encapsidated only when they contain the first 1,200 to 1,400 nucleotides of DHBV pregenomic RNA. However, mutant genomes bearing insertions within this region are packaged efficiently, making it likely that the actual recognition elements for encapsidation are smaller discontinuous sequences located within this region.

Effects of insertional and point mutations on the functions of the duck hepatitis B virus polymerase.

The polymerase (P) gene of hepadnaviruses encodes a large polypeptide that appears to participate in several steps in the viral life cycle: packaging of viral RNA, providing the primer for synthesis of minus-strand DNA, synthesizing minus-strand DNA from an RNA template and plus-strand DNA from a DNA template, and degrading viral RNA in RNA-DNA hybrids. To assist in the assignment of these functions to domains of the duck hepatitis B virus polymerase protein, we have constructed a series of substitution mutations and a large insertion mutation, based in part on amino acid sequence comparisons with other proteins known to exhibit reverse transcriptase (RT) and RNase H activities. We found that changes in highly conserved sequences in putative RT and RNase H domains in the carboxy-terminal half of the protein dramatically reduced synthesis of both strands of viral DNA without major effects on RNA packaging into subviral cores. Thus we can uncouple RNA packaging and DNA synthesis but cannot separate RT and RNase H activities as has been done with human hepatitis B virus. The viability of a mutant with a large insertion (123 amino acids) upstream of the RT and RNase H domain indicates that a hinge region may separate parts of the polymerase protein implicated in priming and polymerization.

Polymerase gene products of hepatitis B viruses are required for genomic RNA packaging as wel as for reverse transcription.

All reactions involving reverse transcription of RNA are segregated from the cytosol within a subviral particle or capsid composed of the major capsid protein, the polymerase and the RNA template. A key step in the formation of these particles is the selective encapsidation of the RNA template. Although an important general feature of the reverse transcription pathway, encapsidation has been carefully studied only for retroviruses. We have now examined the encapsidation reaction in a family of enveloped DNA viruses that replicate by reverse transcription--the hepatitis B viruses (hepadnaviruses). Our results indicate that the hepadnaviral polymerase (P) gene product is required for RNA packaging, and that the encapsidation function of the enzyme can be separated from its DNA polymerase activity. To our knowledge, this is the first description of a role for polymerase gene products in this step of the reverse transcription pathway.