Proviral genome of radiation leukemia virus: molecular cloning of biologically active proviral DNA and nucleotide sequence of its long terminal repeat.

The proviral genome of a leukemogenic and thymotropic C57BL/Ka mouse retrovirus, RadLV/VL3(T+L+), was cloned as a biologically active PstI insert in the bacterial plasmid pBR322. Its restriction map was compared with those, already known, of two nonthymotropic and nonleukemogenic viruses of the same mouse strain: the ecotropic BL/Ka(B) virus and the xenotropic constituent of the radiation leukemia virus complex. Differences were observed around the gag-pol gene junction, in the pol gene, and in the env gene. Moreover, the nucleotide sequence of the RadLV/VL3(T+L+) long terminal repeat revealed the existence of two copies of a 43-base-pair sequence, of which BL/Ka(B) possesses only one copy.     

Inability of Kaplan radiation leukemia virus to replicate on mouse fibroblasts is conferred by its long terminal repeat.

The molecularly cloned infectious Kaplan radiation leukemia virus has previously been shown to be unable to replicate on mouse fibroblasts (E. Rassart, M. Shang, Y. Boie, and P. Jolicoeur, J. Virol. 58:96-106, 1986). To map the viral sequences responsible for this, we constructed chimeric viral DNA genomes in vitro with parental cloned infectious viral DNAs from the nonfibrotropic (F-) BL/VL3 V-13 radiation leukemia virus and the fibrotropic (F+) endogenous BALB/c or Moloney murine leukemia viruses (MuLV). Infectious chimeric MuLVs, recovered after transfection of Ti-6 lymphocytes with these recombinant DNAs, were tested for capacity to replicate on mouse fibroblasts in vitro. We found that chimeric MuLVs harboring the long terminal repeat (LTR) of a fibrotropic MuLV replicated well on mouse fibroblasts. Conversely, chimeric MuLVs harboring the LTR of a nonfibrotropic MuLV were restricted on mouse fibroblasts. These results indicate that the LTR of BL/VL3 radiation leukemia virus harbors the primary determinant responsible for its inability to replicate on mouse fibroblasts in vitro. Our results also show that the primary determinant allowing F+ MuLVs (endogenous BALB/c and Moloney MuLVs) to replicate on mouse fibroblasts in vitro resides within the LTR.     

Stoichiometry of murine leukemia virus envelope protein-mediated fusion and its neutralization

Envelope glycoproteins (Envs) of retroviruses form trimers that mediate fusion between viral and cellular membranes and are the targets for neutralizing antibodies. Understanding in detail how Env trimers mediate membrane fusion, and how antibodies interfere with this process, is a fundamental problem in biology with practical implications for the development of antiviral drugs and vaccines. We investigated the stoichiometry of Env-mediated fusion and its inhibition by antibody by inserting an epitope from human immunodeficiency virus for a neutralizing antibody (2F5) into the surface (SU) or transmembrane (TM) protein of murine leukemia virus Env, along with point mutations that abrogate SU and TM function but complement one another. We transfected various combinations of these Env genes and investigated Env-mediated cell fusion and its inhibition by 2F5 antibody. Our results showed that heterotrimers with one functional SU molecule were fusion competent in complementation experiments and that one antibody molecule was sufficient to inactivate the fusion function of a trimer when its epitope was in functional SU or TM. 2F5 antibody could also neutralize trimers with the 2F5 epitope in nonfunctional SU or TM, but less efficiently.     

Receptor-binding domain of murine leukemia virus envelope glycoproteins.

The surface glycoprotein (SU) of murine leukemia viruses (MuLVs) comprises two domains connected by a proline-rich hinge. The interaction of MuLV particles with subgroup-specific cell surface receptors depends primarily on two variable regions (VRA and VRB) located in the amino-terminal domain. To delineate the minimal receptor-binding domains, we examined the capacity of soluble envelope fragments to compete with the entry of virus particles. Amphotropic, ecotropic, polytropic, and xenotropic truncated SUs were produced by inserting stop codons in the env gene of the 4070A, Friend, MCF247 and NZB MuLVs, respectively. These fragments, as well as full-length envelope glycoproteins, were stably expressed in cells bearing the corresponding receptor. Synthesis, posttranslational modifications, transport, and secretion of the env gene products were monitored by immunoprecipitation. Cells expressing the modified SUs or naive cells preincubated with SU-containing conditioned media were infected with different pseudotypes of a retroviral vector carrying a beta-galactosidase marker gene. Reduction of cell susceptibility to infection in the presence of SU was used as a measure of receptor occupancy. The results indicated that the amphotropic and ecotropic envelope amino-terminal domains contain all of the determinants required for receptor binding. In contrast, additional sequences in the proline-rich region were needed for efficient interaction of the polytropic and xenotropic amino-terminal domains with the receptors.     

Molecular cloning of viral DNA from leukemogenic Gross passage A murine leukemia virus and nucleotide sequence of its long terminal repeat.

The viral DNA genome of the leukemogenic Gross passage A virus was cloned in phage Charon 21A as an infectious molecule. The virus recovered by transfection with this infectious DNA was ecotropic, N-tropic, fibrotropic, and XC+. It was leukemogenic when reinjected into newborn SIM mice, indicating that ecotropic murine leukemia virus (MuLV) from an AKR mouse thymoma can harbor leukemogenic sequences. Its restriction map was similar to that of nonleukemogenic AKR MuLV, its putative parent, but differed at the 3' end and in the long terminal repeat (LTR). The nucleotide sequence of the Gross A virus LTR was identical to the AKR MuLV LTR sequence (Van Beveren et al., J. Virol. 41:542-556, 1982) in U5, R, and part of U3. All differences between both LTRs were found in U3. Only one copy of the U3 tandem direct repeat was conserved in the Gross A virus LTR, and it was rearranged by the insertion of a 36-base-pair sequence and by five point mutations. Only one additional point mutation common to several oncogenic MuLVs was present in U3. These structural changes in the U3 LTR and at the 3' end of the genome may be related to the leukemogenicity of this virus.     

A novel intermediate in processing of murine leukemia virus envelope glycoproteins. Proteolytic cleavage in the late Golgi region.

The intracellular processing of the murine leukemia virus envelope glycoprotein precursor Pr85 to the mature products gp70 and p15e was analyzed in the mouse T-lymphoma cell line W7MG1. Kinetic (pulse-chase) analysis of synthesis and processing, coupled with endoglycosidase (endo H) and neuraminidase digestions revealed the existence of a novel high molecular weight processing intermediate, gp95, containing endo H-resistant terminally glycosylated oligosaccharide chains. In contrast to previously published conclusions, our data indicate that proteolytic cleavage of the envelope precursor occurs after the acquisition of endo H-resistant chains and terminal glycosylation and thus after the mannosidase II step. In the same W7MG1 cell line, the type and order of murine leukemia virus envelope protein processing events was identical to that for the mouse mammary tumor virus envelope protein. Interestingly, complete mouse mammary tumor virus envelope protein processing requires the addition of glucocorticoid hormone, whereas murine leukemia virus envelope protein processing occurs constitutively in these W7MG1 cells. We propose that all retroviral envelope proteins share a common processing pathway in which proteolytic processing is a late event that follows acquisition of endo H resistance and terminal glycosylation.     

大肠杆菌青霉素G酰化酶基因及其邻近区域的核苷酸全序列

Some of microorganisms have been known to possess penicillin G acylase activity. The E. coli derived penicillin G acylase (PGA) can catalyze the conversion of penicillin G into phenylacetic acid and 6-amino-penicillanic acid, the latter is used as the starting compound for the industrial formation of semi-synthetic penicillins. Apart from its industrial importance, the enzyme PGA displays a number of interesting properties. Catalytically active enzyme is localized in the periplasmic space of E. coli cells and composed of two dissimilar subunits. The two subunits are apparently produced from a precursor protein, via a processing pathway hitherto unique in its features for a prokaryotic enzyme. The studies on processing of the precursor and on the relationship between structure and function of the mature enzyme are important theoretically. Previously we cloned a 3.5 kb DNA fragment from a strain (E. coli AS 1.76), which displays PGA activity. In this paper, we report a nucleotide sequence of the 3.5 kb DNA fragment containing PGA gene. After insertion of the DNA fragment into EcoR I and Hind III sites in pWR 13, pPGA 20 had been obtained. We subcloned the Hind III and Bg1 II treated fragment of 1.6 kb in length from pPGA 20 into Hind III and BamH I sites of pWR 13 to get a pPGA 1.6, and Bg1 II and EcoR I treated fragment of 1.9 kb in length into BamH I and EcoR I sites of pWR 13 to get a pPGA 1.9. The linearized pPGA 1.9 which were digested with appropriate restriction enzymes were progressively shortened from both ends respectively by digestion with Bal 31 nuclease, followed by cleavage of shortened target DNA off vector DNA molecules with appropriate restriction enzymes. The series of the DNA fragments shortened from EcoR I end were then cloned into plasmid pWR 13 which had previously digested with Hind III and Sma I enzymes (Fig. 1). The DNA fragment cloned in pWR 13 were directly sequenced on the resulted plasmids by using primer I and primer II. Thus we have obtained the complete nucleotide sequence of the 3.5 kb DNA fragment. The 3.5 kb fragment contains an intact PGA gene which is 2.6 kb.(ABSTRACT TRUNCATED AT 400 WORDS)     

Structure of the murine leukemia virus envelope glycoprotein precursor.

The glycosylated env gene precurosr (Pr80env) of Moloney murine leukemia virus has been isolated by selective immunoprecipitation. Use of the drug tunicamycin to inhibit nascent glycosylation or specific cleavage with endoglycosidase H demonstrated that the precursor contained an apoprotein with a molecular weight of 60,000. The finished virion glycoprotein (gp70) was largely resistant to the action of endoglycosidase H. Chromatography of the glycopeptides of Pr80env in conjunction with endoglycosidase H digestion studies suggested that the precursor contained two distinct major glycosylation sites. Analysis of partial proteolytic cleavage fragments of Pr80env before and after endoglycosidase H treatment placed the two glycosylation sites within a 30,000-dalton region of the apoprotein sequence. Kinetic experiments showed that carbohydrate processing as well as proteolytic cleavage are late steps in the maturation of Pr80env.     

Determinants of human immunodeficiency virus type 1 envelope glycoprotein oligomeric structure.

Oligomerization of the human immunodeficiency virus type 1 envelope (env) glycoproteins is mediated by the ectodomain of the transmembrane glycoprotein gp41. We report that deletion of gp41 residues 550 to 561 resulted in gp41 sedimenting as a monomer in sucrose gradients, while the gp160 precursor sedimented as a mixture of monomers and oligomers. Deletion of the nearby residues 571 to 582 did not affect the oligomeric structure of gp41 or gp160, but deletion of both sequences resulted in monomeric gp41 and predominantly monomeric gp160. Deletion of residues 655 to 665, adjacent to the membrane-spanning sequence, partially dissociated the gp41 oligomer while not affecting the gp160 oligomeric structure. In contrast, deletion of residues 510 to 518 from the fusogenic hydrophobic N terminus of gp41 did not affect the env glycoprotein oligomeric structure. Even though the mutant gp160 and gp120 molecules were competent to bind CD4, the mutations impaired fusion function, gp41-gp120 association, and gp160 processing. Furthermore, deletion of residues 550 to 561 or 550 to 561 plus 571 to 582 modified the antigenic properties of the proximal residues 586 to 588 and the distal residues 634 to 664. Our results indicate that residues 550 to 561 are essential for maintaining the gp41 oligomeric structure but that this sequence and additional sequences contribute to the maintenance of gp160 oligomers. Residues 550 to 561 map to the N terminus of a putative amphipathic alpha-helix (residues 550 to 582), whereas residues 571 to 582 map to the C terminus of this sequence.     

Nucleotide sequence of AKV murine leukemia virus.

AKV is an endogenous, ecotropic murine leukemia virus that serves as one of the parents of the recombinant; oncogenic mink cell focus-forming viruses that arise in preleukemic AKR mice. I report the 8,374-nucleotide-long sequence of AKV, as determined from the infectious molecular clone AKR-623. The 5'-leader sequence of AKV extends to nucleotide 639, after which lies a long open reading frame encoding the gag and pol gene products. The reading frame is interrupted by a single amber codon separating the gag and pol genes. The pol gene overlaps the env gene within the 3' region of the AKV genome. The nucleotide sequence of the 5' region of AKV reveals the following features. (i) The 5'-leader sequence lacks any AUG codon to initiate translation of gPr80gag, suggesting that gPr80gag is not required for the replication of AKV. (ii) A short portion of the leader region diverges in sequence from the closely related Moloney murine leukemia virus and appears to be related to a sequence highly repeated in eucaryotic genomes. (iii) As in Moloney murine leukemia virus, there is a potential RNA secondary structure flanking the amber codon that separates the gag and pol genes. This structure might function as a regulatory protein binding site that controls the relative levels of synthesis of the gag and pol precursors. The nucleotide sequence of the 3' region of AKV is compared with sequences reported previously from both infectious and noninfectious molecular clones of AKV.     

Provirus of M7 baboon endogenous virus: nucleotide sequence of the gag-pol region

A 3,023-base nucleotide sequence of the M7 baboon endogenous virus genome, spanning the 5' noncoding region as well as the entire gag gene and part of the pol gene, is reported. Within the 562-base 5' noncoding region, a 21-base sequence complementary to the OH terminus of tRNApro is located immediately downstream from the long terminal repeat. Amino acid sequences were deduced from the 1,596 nucleotides comprising the gag gene, and the four structural gag polypeptides, p12, p15, p30, and p10, appeared to be coded contiguously. Only one termination codon interrupted the M7 gag and pol genes. The data suggest that 55 additional amino acids may be attached to the NH2 terminus of the gag precursor protein. However, such a sequence was not detected in virions or in virus-infected cells. With the exception of the p15 region, nucleotide and amino acid sequences of the gag and pol regions of M7 virus exhibited strong homologies to those of Moloney leukemia virus.