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Biomedical subjects

A Rein

Publications and source records attributed to A Rein.

At least 73 records · Page 4Linked to original sources
PubMed1986

Myristylation site in Pr65gag is essential for virus particle formation by Moloney murine leukemia virus.

It was previously reported that the gag proteins of mammalian type C retroviruses are modified by the addition of myristate to the N-terminal glycine residue. We have performed oligonucleotide-directed mutagenesis to change this glycine codon in the Moloney murine leukemia virus genome to an alanine codon and also to specifically delete the glycine codon. Upon transfection into mammalian cells, these mutant genomes direct the synthesis of gag proteins, but these proteins are not myristylated. The mutants do not form virus particles or any recognizable virus-specific structures visible in thin sections with the electron microscope. Further, the mutant gag proteins appear to remain in the cytosol, whereas the wild type is found principally in particulate fractions of the cell. The results are consistent with the theory that myristate is required for the association of the gag protein with the plasma membrane and that this association is necessary for virus assembly.

Acylation↗
PubMed1985

Murine leukemia virus maturation: protease region required for conversion from "immature" to "mature" core form and for virus infectivity.

Murine leukemia virus (MuLV) genome encodes a protease (Y. Yoshinaka, I. Katoh, T.D. Copeland, and S. Oroszlan (1985), Proc. Natl. Acad. Sci. USA 82, 1618-1622), which has been shown to cause maturation, specified as morphological conversion from "immature" to "mature" form of virus cores. To examine whether "immature" particles have infectivity or not, we constructed mutant DNAs with deletions in the protease region. The NIH/3T3 cells transfected with mutant DNAs produced "immature" particles, having immature morphology and containing Pr65gag, a polyprotein precursor of core proteins. The specific infectivity of the extracellularly released and purified particles was shown to be greatly reduced based on reverse transcriptase activity and protein content as compared with the "mature" particles obtained from wild-type DNA-transfected cells. The mutant genomes encoded functionally normal surface glycoprotein, gp70. These results strongly suggest that maturation of MuLV from "immature" to "mature" form of virus particles is indispensable to virus infectivity. The importance of processing of gag and pol, as well as transmembrane protein precursors by the viral protease is discussed.

Amino Acid Sequence↗
PubMed1985

Maturation of murine leukemia virus env proteins in the absence of other viral proteins.

A mutant of Akv which produces env but no detectable gag or pol products (A. Rein, D. R. Lowy, B. I. Gerwin, S. K. Ruscetti, and R. H. Bassin, J. Virol. 41, 626-634 (1982] was examined for maturation of env gene protein products. In comparison with wild-type Akv, the mutant AK 71 synthesizes gPr85env and produces gp70 and Prp15E in normal amounts and with normal kinetics. Cell surface gp70 was found alike in the mutant and wild type. However, cleavage of Pr15E to p15E did not occur in the mutant. This final cleavage step of AK 71 Prp15E could be made to occur by superinfecting cells containing the mutant with baboon endogenous virus. Thus, unlike earlier steps, this final step in maturation of the env gene product appears to require gag or pol gene products. It is proposed that the virus-encoded protease is required for this last step.

Animals↗
PubMed1985

Infection of immune mast cells by Harvey sarcoma virus: immortalization without loss of requirement for interleukin-3.

Cells from adult mouse spleens were cultured in WEHI-3 cell-conditioned medium, which contains the lymphokine interleukin-3 (IL-3). Under these conditions, cells grow well for 4 to 8 weeks; the cultures contain a variety of cell types for the first 1 to 2 weeks but are subsequently composed largely of immune mast cells. We found that infection of these cultures with Harvey sarcoma virus (HaSV) profoundly enhanced the growth potential of the cells, resulting in the reproducible isolation of long-term cell lines. These HaSV-infected cells appeared to be phenotypically identical to the immune mast cells found in uninfected cultures as determined by biochemical, immunological, and cytological tests. Although the cells expressed protein p21Ha-ras at levels similar to those in HaSV-transformed fibroblasts, they continued to require IL-3 for growth in vitro. Similar IL-3-dependent, long-term mast cell lines were also cultured from the enlarged spleens present in HaSV-infected mice. These results suggest that high-level expression of an activated Ha-ras oncogene enhances growth in these cells, perhaps by stimulating the progression of the cells into S, without affecting differentiation or altering the requirements for normal growth factor.

Animals↗
PubMed1985

[2 trials of measuring the flow of the intraorbital ophthalmic artery using pulsed Doppler].

We studied the flow pattern of the ophthalmic artery by pulsed doppler. The technique of visualization of the artery using bidimensional echography and the method of flow calculation by pulsed doppler are described in this preliminary report. This direct, non-invasive and readily feasible technique may play a prominent role in the clinical diagnosis of ophthalmic artery diseases.

Blood Flow Velocity↗
PubMed1984

Different recombinant murine leukemia viruses use different cell surface receptors.

Retroviruses can be grouped by viral interference measurements into classes which use common cell surface receptors. We previously tested a large number of isolates of mink cell focus-inducing (MCF) murine leukemia viruses (MuLVs), and reported that all of them share a distinct receptor on NIH/3T3 cells (A. Rein, Virology 120, 251, 1982). We now extend this generalization to several additional recombinant isolates, including two (SL3-2 and GPA-V2) which would not be considered MCFs on the basis of host-range data. We note the superiority of interference tests, based on positive, unambiguous data, over host-range tests for virus classification. We also show that in contrast to the MCFs, which are all derived from ecotropic MuLVs, a recombinant derived from wild mouse amphotropic MuLV (S. Rasheed et al., Int. J. Cancer 29, 345, 1982) uses a unique receptor on NIH/3T3 cells. This suggests that (a) mouse cells contain more than one type of endogenous env sequence; and (b) there is some specificity in the generation of recombinants, since ecotropic MuLVs appear to give rise only to MCFs, while amphotropic MuLV has generated a distinct type of recombinant. It also represents a second case (in addition to the MCFs) in which an env gene recombinant is more pathogenic than its exogenous parent. We also show that xenotropic MuLV does not interfere with MCFs in NZB mouse cells; thus, despite the close homology between MCF and xenotropic env sequences, the gp70 of xenotropic MuLV appears to have no detectable affinity for the MCF receptor.

Animals↗
PubMed1984

Murine leukemia virus mutant with a frameshift in the reverse transcriptase coding region: implications for pol gene structure.

The molecular defect in the nonconditional B-tropic MuLV pol mutant, clone 23 (Gerwin et al., J. Virol. 31:741-751, 1979), has been characterized by recombinant DNA technology. The entire mutant genome was cloned from an EcoRI digest of integrated cellular DNA into bacteriophage lambda Charon 4A and then subcloned at the EcoRI site of pBR322. NIH-3T3 cells transfected with the plasmid clone, termed pRTM (RTM, reverse transcriptase mutant), reproduced the properties of clone 23 virus-infected cells. In vivo ligation experiments involving cotransfection of subclones of pRTM and wild-type murine leukemia virus localized the defect in the clone 23 genome to an approximately 400-base-pair region in the pol gene between the SalI and XhoI sites. Sequence analysis of this region in the wild-type and mutant genomes revealed that the mutant has one additional C residue located 231 bases downstream of the last base of the SalI recognition site. This 1-base insertion brings three TGA termination codons into phase. Thus, the mutation in clone 23 leads to premature termination of translation, explaining the presence in clone 23 virions of a truncated polymerase with low levels of enzymatic activity. It was previously shown that the gag precursor is cleaved normally in clone 23-infected cells; therefore, if a virus-coded protease is involved in this cleavage, it must be encoded by sequences upstream of the reverse transcriptase region of the pol gene. This consideration, coupled with the observed molecular weight of the mutant polymerase and our precise determination of its C terminus, have led to a proposal for the genetic organization of the murine leukemia virus pol gene.

Animals↗
PubMed1983

Biological, chemical, and immunological studies of Rauscher ecotropic and mink cell focus-forming viruses from JLS-V9 cells.

Two murine leukemia viruses were isolated from JLS-V9 cells which had been infected with Rauscher plasma virus. One virus was XC positive and failed to grow on mink or cat cells and thus was an ecotropic virus. The other virus formed cytopathic foci on mink cells, was XC negative, and fell into the mink cell focus-forming (MCF) viral interference group and was thus an MCF virus. The glycoproteins of the two viruses could be distinguished immunologically, by peptide mapping, and by size in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The MCF virus produced gp69, and the ecotropic virus produced gp71, explaining the origin of the heterogeneous glycoprotein (gp69 and gp71) of Rauscher leukemia virus. Amino-terminal sequences of gp69 and gp71 were determined. The MCF sequence was distinct from the ecotropic sequence, but retained partial homology to it. The data show that the glycoproteins are encoded by related yet distinct genes. The protein structural data support the proposal that MCF virus gp70 molecules have nonecotropic sequences at the amino terminus, with ecotropic sequences occurring at the 3' end of the gene. The Rauscher MCF virus glycoprotein lacks a glycosylation site found at position 12 of the ecotropic sequence.

Animals↗
PubMed1982

Transfer of murine leukaemia and murine sarcoma virus genetic information by transfection with isolated metaphase chromosomes.

Chromosome-mediated transfer of murine leukaemia (MuLV) and murine sarcoma (MuSV) virus genetic information to uninfected recipient cells was investigated. Metaphase chromosomes from AKR MuLV-infected SC-1 mouse cells were incubated with NIH/3T3 cells. After several passages (1 to 3 weeks), infectious virions exhibiting reverse transcriptase activity and the characteristic host range of ecotropic, N-tropic AKR virus appeared in the supernatant fluids of the treated cells. Restriction endonuclease analysis of genomic DNA from transfected cells indicated that AKR proviral DNA was associated with the high molecular weight DNA of the host. These results demonstrate that the AKR MuLV genome can be stably transferred to uninfected recipient cells via isolated metaphase chromosomes. Although AKR virions are not able to infect heterologous cells, chromosome-mediated transfection resulted in the establishment of productive AKR MuLV infection in mink cells. Thus, the use of chromosomes to transfer virus genes can circumvent the natural host restriction barrier. In other experiments, it was shown that normal NIH/3T3 cells were transformed after exposure to metaphase chromosomes isolated from an MuSV-infected, non-producer line. Foci were detected 14 to 21 days after chromosome treatment and were shown to contain true viral transformants since transforming virus was produced after superinfection with MuLV.

Animals↗
PubMed1982

Molecular properties of a gag- pol- env+ murine leukemia virus from cultured AKR lymphoma cells.

We have described the isolation of a replication-defective murine leukemia virus from a culture of AKR lymphoma cells [Rein et al., Nature (London) 282:753-754, 1979]. To facilitate the characterization of this murine leukemia virus, we transmitted it to mink cells and analyzed its genome by restriction mapping of the mink cellular DNA. This genome resembled the Akv genome quite closely, but it had an additional KpnI cleavage site at 1.3 kilobase pairs from the 5' end of the provirus and a small (approximately 50-base-pair) deletion between 1.8 and 3.0 kilobase pairs from the 5' end. When we tested these mink cells by immune precipitation or by competition radioimmunoassay, we found that they synthesized gPr82env, but contained no detectable gag or pol proteins. It seems likely that the KpnI cleavage site at 1.3 kilobase pairs reflects an abnormal sequence at or near the beginning of the gag gene, which prevents gag or pol translation by introducing a frameshift or termination codon into this region.

AKR murine leukemia virus↗
PubMed1982

In situ hybridization: general infectivity assay for retroviruses.

We have devised a general infectivity assay for retroviruses. A virus-specific [32P]DNA probe is hybridized in situ to a monolayer culture, and foci of infected cells in the monolayer are detected by exposure of the hybridized culture to X-ray films. The method is quantitative, in that it gives the same titer for Moloney murine leukemia virus as does the standard UV-XC test. The specificity of the assay is indicated by the fact that murine leukemia virus and baboon endogenous virus do not cross hybridize under the conditions used. The assay is completed within 1 to 3 weeks and should be broadly applicable for retroviruses which replicate without altering cellular morphology: its use is demonstrated with mouse mammary tumor virus and the helper virus of the reticuloendotheliosis complex.

Animals↗
PubMed1982

Mitral valve prolapse and panic attacks.

Patients complaining of recurrent spontaneous panic attacks (RSPA) are frequently seen in emergency rooms and psychiatric outpatient services. The symptomatology of RSPA suggests some underlying cardiac pathology. Recent publications in the psychiatric literature have reported a high incidence of mitral valve prolapse (MVP) in patients complaining of RSPA. In our study we report a 39% incidence of MVP in a psychiatric population with RSPA. The importance of this finding is yet to be determined.

Adult↗
PubMed1981

Baboon endogenous virus genome: molecular cloning and structural characterization of nondefective viral genomes from DNA of a baboon cell strain.

Several heterogeneities in the baboon endogenous virus (BaEV) genomes that are present in the DNA of normal baboon tissues and the baboon cell strain BEF-3 have been described previously. To study these genomes, we cloned BaEV proviruses from BEF-3 cellular DNA into the lambda vector Charon 4A. Of the four full-length clones isolated, one was nondefective as determined by transfection. The sequence of a portion of this clone was found to code for amino acids 61-91 in the p30 region of the gag gene. This identification allowed us to align the restriction map with the BaEV genetic map. One heterogeneity, a BamHI site 2.4 kilobases (kb) from the proviral 5' end, was located close to the gag-pol junction; another, a BamHI site 1.4 kb from the 5' end of the genome, corresponded to the gag p30 coding sequence for amino acids 32-34; and a third, a Xho I site, was near the 3' end of the pol gene. To select the nondefective BaEV genomes from BEF-3 cells, we infected permissive cells with virus produced by BEF-3 cells and also transfected BEF-3 cellular DNA into permissive cells. The BaEV genomes in the permissive recipient cultures were then analyzed by restriction enzyme analysis. These nondefective genomes were found to be heterogeneous with respect to the gag-pol BamHI site and the Xho I site, but all were found to contain the BamHI site 1.4 kb from the 5' end of the genome.

Animals↗