Ideal methods for human gene therapy will eventually include direct gene transfer to defective tissues in a patient in vivo. Toward that goal, we have used high titer, pseudotyped retroviral vectors expressing genes for the Escherichia coli beta-galactosidase (lacZ) or hepatitis B virus surface antigen (HBsAG) to infect mouse liver by in vivo direct injection into the liver parenchyma. We have found that a single percutaneous injection of small volumes of vectors into the newborn mouse liver leads to transduction of at least 25-30% of the hepatocytes throughout the liver, as judged by in situ staining of liver sections for beta-gal activity at 4 weeks after injection. We have demonstrated that stable levels of HBsAg were also detected in the circulation of injected mice up to 4 months after HBsAg-vector injection. We suggest that the high efficiency of in vivo transduction in the neonatal liver and subsequent stable transgene expression by high-titer pseudotyped retroviral vectors in the absence of an invasive partial

Retroviral vectors have been central components in many studies leading to human gene therapy. However, the generally low titers and inefficient infectivity of retroviral vectors in human cells have limited their use. We previously reported that the G protein of vesicular stomatitis virus can serve as the exclusive envelope protein component for one specific retroviral vector, LGRNL, that expresses vesicular stomatitis virus G. We now report a more useful general transient transfection scheme for producing very high-titer vesicular stomatitis virus G-enveloped pseudotypes from any Moloney murine leukemia-based retroviral vector without having to rely on the expression of the cytotoxic G protein from the retroviral vector itself. We also demonstrate very high efficiency of infection with a pseudotyped lacZ vector in primary mouse hepatocytes. We suggest that pseudotyped retroviral vectors carrying reporter genes will permit genetic studies in many previously inaccessible vertebrate and invertebrate systems. Furthermore, because these vectors represent retroviral vectors of sufficiently high titer to allow efficient direct retroviral-mediated in vivo gene transfer, we also suggest that pseudotyped vectors carrying potentially therapeutic genes will become useful to test the potential for in vivo gene therapy.

Hepatitis B virus (HBV) infection is associated with the development of hepatocellular carcinoma in humans. HBV infection is primarily restricted to hepatocytes, and replication and gene expression of HBV require liver-specific transcription factors. Regulation of HBV gene expression has been shown to be controlled by two enhancers, and liver-specific gene expression of HBV can be attributed largely to the activity of enhancer 2. In this study, we have used mutational analysis to identify a 20-base-pair sequence motif essential for the liver-specific enhancer 2 activity. Analysis of the sequence reveals that this motif is similar to the regulatory region of several other liver-specific cellular genes, suggesting that common transcription factors may be involved in the activation of cellular as well as HBV gene expression in hepatocytes.

The mutations in three polyoma ts-a mutants have been determined. Two mutants, ts-25 and ts-52, have different single-base changes at the same position (2883) in the early region corresponding to a conserved glycine residue very near the C-terminus of the polyoma large T antigen. Mutant ts-48 has a single-base change at position 2341, as well as a second change at position 1228, in the region of large T antigen shared with medium T antigen.

A series of clones that contain human Alu family elements are actively transcribed in soluble in vitro RNA polymerase III systems. The 5' ends of the in vitro transcripts are located about 170 nucleotides upstream of the eponymous Alu I site of the repeat, while a region associated with specifying of the initiation site for in vitro transcription lies in the region between 79 and 106 nucleotides upstream of the central Alu site. Thus, the RNA polymerase III transcription unit defined by the human Alu family is similar to other RNA polymerase III transcription units in possessing an internal region that is required for active transcription in vitro.

We present the 5295 nucleotide-long sequence of the polyoma genome and the restriction enzyme digestion sites predicted from this sequence.

The early region of the polyoma virus genome encodes proteins required for cell transformation. The proteins encoded in this region can be identified by immunoprecipitation of infected-cell extracts, using serum from tumor-bearing animals. The proteins identified in this way are called T antigens. At least three T antigens are encoded in the polyoma virus genome (Ito et al. 1977a; Hutchinson et al. 1978; Schaffhausen et al. 1978). These three T antigens, referred to as large, middle, and small T antigens, have apparent molecular weights of 90,000–100,000, 50,000–90,000, and 22,000, respectively, by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis.

The nucleotide sequence of the late region of the polyoma genome has been determined. It consists of 2366 bp and encodes the virion capsid proteins VP1, VP2 and VP3. Extensive open reading frames identify the possible coding sequences of VP2 and VP3 toward the 5′ end of the late region, and of the major capsid protein VP1 toward the 3′ end of the late region. The 5′ end of the sequence encoding VP1 overlaps the 3′ VP2/VP3 region by 29 nucleotides and is in a different reading frame. The predicted amino acid sequences for all three known capsid proteins show extensive homology with the analogous capsid proteins of SV40 throughout most of their length. The VP2/VP3 amino acid homology between the two viruses is 34%, while the major capsid protein VP1 is much more highly conserved, showing 54% homology. These homologies together with the extent of open reading frames help to define the extent of the coding sequences. The VP2 initiator begins at position 269 and the coding region extends to the first termination codon beginning at 1226. The predicted size of VP2 is 35,007 daltons. A probable VP3 initiator is within the VP2 coding sequence at position 614 and is in the same frame as VP2. This coding sequence can also utilize the terminator at position 1226, and the predicted size of the VP3 translation product is 22,979 daltons. The VP1 coding region begins at position 1197 and continues in a frame different from that of VP2/ VP3 to a termination point at 2349. The molecular weight of VP1 is predicted to be 42,834 daltons. The 5′ untranslated region contains sequences that resemble a potential ribosomal binding site and a possible mRNA capping sequence similar to those found in other eucaryotic systems. There is also a sequence (5′-TCAAGTAAGTGA-3′) almost identical to one found in two regions containing potential splice sites in the early region of polyoma. The 5′ untranslated region does not show the extensive repeated sequences found in the similar region of SV40. The 3′ untranslated region contains the sequence 5′-AATAAA-3′, thought to represent a polyadenylation signal. As in the early region of polyoma, the extensive nucleotide and deduced amino acid homology with SV40 indicate a close evolutionary relationship between the two viruses, and help to identify regions of common and important structure-function relationships.

The nucleotide sequence of the early region of the polyoma genome has been determined by the chemical method of Maxam and Gilbert (1977) and the primed synthesis method of Sanger, Nicklen and Coulson (1978). The sequence consists of 3013 nucleotide pairs and contains the regions encoding the three related forms of the tumor antigens, as well as the regions encoding the origin of DNA replication and sequences regulating messenger RNA transcription and splicing. The extent of the open reading frames, together with estimated mRNA splice positions, defines potential coding regions and approximate sizes for the three forms of the tumor (T) antigens. There are uninterrupted open frames corresponding to the probable small t coding region, the 5′ ends of the large T and medium T coding regions, the maximum length for the 3′ end of the large T coding region, and a region potentially available for the 3′ end of the medium T antigen. The 5′ untranslated region contains sequences that are capable of interacting in several different ways with the 3′ end of the 18S ribosomal RNA, thereby representing a possible ribosomal binding site. There are two other 5′ untranslated sequences similar to sequences found at the 5′ ends of other eucaryotic genes that might represent capping signals and/or promoter signals. The 3′ untranslated region contains the sequence 5′-AA-TAAA-3′ just 3′ to the TGA termination triplet, a sequence that may constitute an eucaryotic polyadenylation signal. Codon utilization in the polyoma early region shows deviation from random, especially in its deficiency of triplets ending in TC and in its paucity of CG. There is extensive nucleotide, and deduced amino acid, homology with the early region of SV40 (Fiers et al., 1978; Reddy et al., 1978) throughout regions thought to encode the respective small t and large T antigens. Furthermore, a portion of the possible medium T antigen coding region shows homology with the 3′ end of the SV40 large T antigen coding region, suggesting that the function of the polyoma medium T antigen is performed by the SV40 large T antigen.

The early region of the polyoma genome encodes three T antigens. We have analyzed the organization of the coding regions for the T antigens, using the nucleotide sequence of polyoma DNA and peptides derived from purified, radio-labeled T antigens, separated by two-dimensional electrophoresis and chromatography. We compared the peptides, predicted from the nucleotide sequence of the DNA, with those derived from the purified T antigens. We also compared chemically synthesized peptides, predicted from the DNA sequence, with observed peptides. The results show that the three polyoma T antigens are encoded in overlapping regions of the viral DNA, translated, in part, in two different reading frames.

The nucleotide sequence of polyoma DNA, from near the Hpa II 3/5 unction to the Hpa II 4/ae III 18 junction has been determined by the chemical method of Maxam and Gilbert (Maxam, A., and Gilbert, W. (1977) Proc. Natl. Acad. Sci. U. S. A. 74, 560--564). The sequence contains 878 base paris, including the origin of DNA replication and the region known to encode the hr-t function. The region corresponding to the origin of DNA replication contains several short-repeated sequences and palindromes. There is a 30-base-pair region with striking similarity to the corresponding region of SV40, and, as in SV40, a portion of that sequence is capable of forming a stable hairpin loop. In the region encoding the hr-t function, there is apparently a single open reading frame extending from position 188 to theHpa III 4/Hae III 18 junction. The potential translation product of this open frame begins with an initiation codon starting at position 188, and the first five amino acids of this product are Met-Asp-Arg-Val-Leu. This sequence is similar to the NH2-terminal five amino acids of SV40 small t-antigen known from nucleotide and amino acid sequencing to be Met-Asp-Lys-Val-Leu.