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Biomedical subjects

A Rein

Publications and source records attributed to A Rein.

At least 37 records · Page 2Linked to original sources
PubMed1996

HIV-1 nucleocapsid protein induces "maturation" of dimeric retroviral RNA in vitro.

After a retrovirus particle is released from the cell, the dimeric genomic RNA undergoes a change in conformation. We have previously proposed that this change, termed maturation of the dimer, is due to the action of nucleocapsid (NC) protein on the RNA within the virus particle. We now report that treatment of a 345-base synthetic fragment of Harvey sarcoma virus RNA with recombinant or synthetic HIV-1 NC protein converts a less stable form of dimeric RNA to a more stable form. This phenomenon thus appears to reproduce the maturation of dimeric retroviral RNA in a completely defined system in vitro. To our knowledge, maturation of dimeric RNA within a retrovirus particle is the first example of action of an "RNA chaperone" protein in vivo. Studies with mutant NC proteins suggest that the activity depends upon basic amino acid residues flanking the N-terminal zinc finger and upon residues within the N-terminal finger, including an aromatic amino acid, but do not require the zinc finger structures themselves.

Amino Acid Sequence↗
PubMed1996

Human immunodeficiency virus type 1 nucleocapsid protein reduces reverse transcriptase pausing at a secondary structure near the murine leukemia virus polypurine tract.

In an earlier study on minus-strand DNA synthesis catalyzed by murine leukemia virus reverse transcriptase, we described a prominent pause site near the polypurine tract (J. Guo, W. Wu, Z. Y. Yuan, K. Post, R. J. Crouch, and J. G . Levin, Biochemistry 34:5018-5029, 1995). We now report that pausing at this site is due to a stem-loop structure in the RNA template, formed by interaction of a number of bases in the polypurine tract, including the six G's, and a 3' sequence which includes four C's. Addition of human immunodeficiency virus type 1 (HIV-1) nucleocapsid (NC) protein to reverse transcriptase reactions reduces pausing by approximately 8- to 10-fold and stimulates synthesis of full-length DNA. Thus, NC functions as an accessory protein during elongation of minus-strand DNA and increases the efficiency of DNA synthesis, in this case, by apparently destabilizing a region of secondary structure in the template. Since NC is associated with genomic RNA in the viral core and is likely to be part of a viral replication complex, these results suggest that NC may also promote efficient DNA synthesis during virus replication. Mutational analysis indicates that the features of HIV-1 NC which are important for reduction of pausing include the basic amino acids flanking the first zinc finger, the zinc fingers, and the cysteine and aromatic amino acids within the fingers. These findings suggest that reverse transcription might be targeted by drugs which inactivate the zinc fingers of HIV-1 NC.

Amino Acid Sequence↗
PubMed1996

Genetic analysis of the zinc finger in the Moloney murine leukemia virus nucleocapsid domain: replacement of zinc-coordinating residues with other zinc-coordinating residues yields noninfectious particles containing genomic RNA.

The effect of changing zinc (Zn2+)-coordinating residues in the nucleocapsid protein of Moloney murine leukemia virus was investigated by introducing a His-34-to-Cys or Cys-39-to-His mutation into the putative Zn2+ finger. Mutant virions contained normal levels of properly processed Gag and Env proteins and wild-type levels of full-length viral RNA. However, the specific infectivity of the mutants was approximately 4 x 10(-4) that of wild-type particles. They were probably noninfectious because of the inability of the particles to synthesize cDNA transcripts, since full-length viral DNA could not be detected in Hirt supernatants of NIH 3T3 cells infected with the CCCC or CCHH virus. These mutants will provide an extremely valuable tool for analysis of the role of retroviral Zn2+ fingers in infection processes, independent of viral RNA recognition and packaging.

3T3 Cells↗
PubMed1996

Therapeutic effect of Gag-nuclease fusion protein on retrovirus-infected cell cultures.

Capsid-targeted viral inactivation is a novel protein-based strategy for the treatment of viral infections. Virus particles are inactivated by targeting toxic fusion proteins to virions, where they destroy viral components from within. We have fused Staphylococcus nuclease (SN) to the C-terminal end of Moloney murine leukemia virus Gag and demonstrated that expression of this fusion protein in chronically infected chicken embryo fibroblasts resulted in its incorporation into virions and subsequent inactivation of the virus particles by degradation of viral RNA. Release of particles incorporating Gag-SN fusion proteins into the extracellular milieu activates the nuclease and results in destruction of the virion from within. By comparing the effects of incorporated SN and SN*, an enzymatically inactive missense mutant form of SN, on the infectivity of virus particles, we have clearly demonstrated that nucleolytic activity is the antiviral mechanism. Expression of Gag-SN fusion proteins as a therapeutic agent causes a stable reduction of infectious titers by 20- to 60-fold. The antiviral effect of capsid-targeted viral inactivation in our model system, using both prophylactic and therapeutic approaches, suggests that a similar anti-human immunodeficiency virus strategy might be successful.

Animals↗
PubMed1996

Inactivation of murine leukemia virus by compounds that react with the zinc finger in the viral nucleocapsid protein.

All retroviral nucleocapsid (NC) proteins, except those of spumaretroviruses, contain one or two copies of the conserved sequence motif C-X2-C-X4-H-X4-C. The conserved cysteine and histidine residues coordinate a zinc ion in each such motif. Rice et al. (W. G. Rice, J. G. Supko, L. Malspeis, R. W. Buckheit, Jr., D. Clanton, M. Bu, L. Graham, C. A. Schaeffer, J. A. Turpin, J. Domagala, R. Gogliotti, J. P. Bader, S. M. Halliday, L. Coren, R. C. Sowder II, L. 0. Arthur, and L. E. Henderson, Science 270:1194-1197, 1995) have described a series of compounds which inactivate human immunodeficiency virus type 1 (HIV-1) particles and oxidize the cysteine thiolates in the NC zinc finger. We have characterized the effects of three such compounds on Moloney murine leukemia virus (MuLV). We find that, as with HIV-1, the compounds inactivate cell-free MuLV particles and induce disulfide cross-linking of NC in these particles. The killed MuLV particles were found to be incapable of synthesizing full-length viral DNA upon infection of a new host cell. When MuLV particles are synthesized in the presence of one of these compounds, the normal maturational cleavage of the Gag polyprotein does not occur. The compounds have no effect on the infectivity of human foamy virus, a spumaretrovirus lacking zinc fingers in its NC protein. The resistance of foamy virus supports the hypothesis that the zinc fingers are the targets for inactivation of MuLV and HIV- I by the compounds. The absolute conservation of the zinc finger motif among oncoretroviruses and lentiviruses and the lethality of all known mutations altering the zinc-binding residues suggest that only the normal, wild-type structure can efficiently perform all of its functions. This possibility would make the zinc finger an ideal target for antiretroviral agents.

3T3 Cells↗
PubMed1995

Targeting of a nuclease to murine leukemia virus capsids inhibits viral multiplication.

Capsid-targeted viral inactivation is an antiviral strategy in which toxic fusion proteins are targeted to virions, where they inhibit viral multiplication by destroying viral components. These fusion proteins consist of a virion structural protein moiety and an enzymatic moiety such as a nuclease. Such fusion proteins can severely inhibit transposition of yeast retrotransposon Ty1, an element whose transposition mechanistically resembles retroviral multiplication. We demonstrate that expression of a murine retrovirus capsid-staphylococcal nuclease fusion protein inhibits multiplication of the corresponding murine leukemia virus by 30- to 100-fold. Staphylococcal nuclease is apparently inactive intracellularly and hence nontoxic to the host cell, but it is active extracellularly because of its requirement for high concentrations of Ca2+ ions. Virions assembled in and shed from cells expressing the fusion protein contain very small amounts of intact viral RNA, as would be predicted for nuclease-mediated inhibition of viral multiplication.

3T3 Cells↗
PubMed1995

Multiple regions of Harvey sarcoma virus RNA can dimerize in vitro.

Retroviruses contain a dimeric RNA consisting of two identical molecules of plus-strand genomic RNA. The structure of the linkage between the two monomers is not known, but they are believed to be joined near their 5' ends. Darlix and coworkers have reported that transcripts of retroviral RNA sequences can dimerize spontaneously in vitro (see, for example, E. Bieth, C. Gabus, and J. L. Darlix, Nucleic Acids Res. 18:119-127, 1990). As one approach to identification of sequences which might participate in the linkage, we have mapped sequences derived from the 5' 378 bases of Harvey sarcoma virus (HaSV) RNA which can dimerize in vitro. We found that at least three distinct regions, consisting of nucleotides 37 to 229, 205 to 272, and 271 to 378, can form these dimers. Two of these regions contain nucleotides 205 to 226; computer analysis suggests that this region can form a stem-loop with an inverted repeat in the loop. We propose that this hypothetical structure is involved in dimer formation by these two transcripts. We also compared the thermal stabilities of each of these dimers with that of HaSV viral RNA. Dimers of nucleotides 37 to 229 and 205 to 272 both exhibited melting temperatures near that of viral RNA, while dimers of nucleotides 271 to 378 are quite unstable. We also found that dimers of nucleotides 37 to 378 formed at 37 degrees C are less thermostable than dimers of the same RNA formed at 55 degrees C. It seems possible that bases from all of these regions participate in the dimer linkage present in viral RNA.

Base Sequence↗
PubMed1995

Positioning post-outpatient cardiac catheterization.

UNLABELLED: The optimal positioning post-outpatient cardiac catheterization is a largely unexplored area of research. Traditionally, patients have been placed supine, with the head of the bed (HOB) flat. This study sought to explore the influence of three different post-procedure positions on the incidence of complications and patient perceptions of satisfaction. Sixty-nine patients were randomized into three groups immediately after hemostasis of the arterial puncture site was achieved. Group 1 patients were placed supine with the HOB flat. Group 2 patients were placed on their sides, with the affected extremity straight. Group 3 patients were placed on their backs, with the HOB at 15-30 degrees. Endpoints included the presence of any complication (bleeding, hematoma formation or expansion, back pain and urinary retention) and patient comfort and satisfaction with the randomized position. Patients were asked prior to discharge to select the position they would have preferred post-procedure. Selection of an alternative position was felt to indicate dissatisfaction with the randomized position. Ten patients in Group 1, and nine patients in Group 2 and Group 3 experienced a complication (X2 = 3.682, df = 1, p = 0.05). Eighty-five percent of Group 1 patients selected an alternative position vs. 24% of those patients in Group 2 and Group 3 (X2 = 27.6, df = 1, p < .001). CONCLUSION: varying patient position post-outpatient cardiac catheterization is at least as safe as the traditional supine position and is more comfortable for patients.

Adult↗
PubMed1994

10A1 MuLV induces a murine leukemia that expresses hematopoietic stem cell markers by a mechanism that includes fli-1 integration.

The 10A1 murine leukemia virus induces tumors that lack lineage-specific markers found on myeloid, T-cell, and B-cell lineages. Either erythroid or multipotent stem cells can have this phenotype; therefore we have used fluorescence-activated cell sorter analysis with either multipotent stem cell markers or markers found on lineage-restricted precursors to differentiate between these two possibilities. The results showed that tumors induced by 10A1 expressed multipotent stem cell markers as well as some lineage-restricted precursor markers. To further study the tumor phenotype, we analyzed total RNAs from 10A1-induced tumors by Northern blotting for c-kit, erythropoietin receptor, and T-cell gamma receptor mRNAs. Most of the tumors contained these mRNAs, which are characteristic of early hematopoietic cells. These results are consistent with the hypothesis that 10A1-induced tumor cells are early multipotent hematopoietic stem cells. Southern blot analysis revealed that 14 of 14 10A1-induced tumor cell DNAs examined contained MuLV integrations into the fli-1 gene. The results strongly suggested that promoter insertion into fli-1 is required for tumor formation.

Animals↗
PubMed1994

Retroviral RNA packaging: a review.

In retroviruses, the "Gag" or core polyprotein is capable of assembling into virus particles and packaging the genomic RNA of the virus. How this protein recognizes viral RNA is not understood. Gag polyproteins contain a zinc-finger domain; mutants with changes in this domain assemble into virions, but a large fraction of these particles lack viral RNA. Thus, one crucial element in the RNA packaging mechanism is the zinc-finger domain. RNA sequences required for packaging ("packing signals") have been studied both by deletion analysis and by measuring encapsidation of nonviral mRNAs containing limited insertions of viral sequence. These experiments show that all or part of the packaging signal in viral RNA is located near the 5 end of the genome. These signals appear to be quite large, i.e., hundreds of nucleotides. Each virus particle actually contains a dimer of two identical, + strand genomic RNA molecules. The nature of the dimeric linkage is not understood. In some experimental situations (including zinc-finger mutants), only a small fraction of the particles in a virus preparation contain genomic RNA. It is striking that the genomic RNA packaged in these situations is dimeric. Because of this important observation, it is speculated that only dimers are packaged, and that the dimeric structure is an element of the packaging signal. It is also suggested that the dimers undergo a conformational change ("RNA maturation") after the virus is released from the cell, and that this change may depend upon the cleavage of the Gag polyprotein, a post-assembly event catalyzed by the virus-coded protease.

Base Sequence↗
PubMed1994

Function of the cytoplasmic domain of a retroviral transmembrane protein: p15E-p2E cleavage activates the membrane fusion capability of the murine leukemia virus Env protein.

In the murine leukemia viruses (MuLVs), the Env complex is initially cleaved by a cellular protease into gp70SU and pre15ETM. After the virus particle is released from the cell, the C-terminal 16 residues are removed from the cytoplasmic domain of pre15E by the viral protease, yielding the mature p15ETM and p2E. We have investigated the function of this cleavage by generating a Moloney MuLV mutant, termed p2E-, in which the Env coding region terminates at the cleavage site. This mutant synthesizes only the truncated, mature form of TM rather than its extended precursor. When cells expressing this truncated Env protein are cocultivated with NIH 3T3 cells, they induce rapid cell-cell fusion. Thus, the truncated form, which is normally found in virions but not in virus-producing cells, is capable of causing membrane fusion. We conclude that the 16-residue p2E tail inhibits this activity of Env until the virus has left the cell. p2E- virions were found to be infectious, though with a lower specific infectivity than that of the wild type, showing that p2E does not play an essential role in the process of infection. Fusion was also observed with a chimeric p2E- virus in which gp70SU and nearly all of p15ETM are derived from amphotropic, rather than Moloney, MuLV. In a second mutant, an amino acid at the cleavage site was changed. The pre15E protein in this mutant is not cleaved. While the mutant Env complex is incorporated into virions, these particles have a very low specific infectivity. This result suggests that the cleavage event is essential for infectivity, in agreement with the idea that removal of p2E activates the membrane fusion capability of the Env complex.

Amino Acid Sequence↗
PubMed1994

Characterization of human immunodeficiency virus type 1 dimeric RNA from wild-type and protease-defective virions.

We have characterized the dimeric genomic RNA in particles of both wild-type and protease (PR)-deficient human immunodeficiency virus type 1 (HIV-1). We found that the dimeric RNA isolated from PR- mutant virions has a lower mobility in nondenaturing gel electrophoresis than that from wild-type virions. It also dissociates into monomers at a lower temperature than the wild-type dimer. Thus, the dimer in PR- particles is in a conformation different from that in wild-type particles. These results are quite similar to recent findings on Moloney murine leukemia virus and suggest that a postassembly, PR-dependent maturation event is a common feature in genomic RNAs of retroviruses. We also measured the thermal stability of the wild-type and PR- dimeric RNAs under different ionic conditions. Both forms of the dimer were stabilized by increasing Na+ concentrations. However, the melting temperatures of the two forms were not significantly affected by the identity of the monovalent cation present in the incubation buffer. This observation is in contrast with recent reports on dimers formed in vitro from short segments of HIV-1 sequence: the latter dimers are specifically stabilized by K+ ions. K+ stabilization of dimers formed in vitro has been taken as evidence for the presence of guanine quartet structures. The results suggest that guanine quartets are not involved in the structure linking full-length, authentic genomic RNA of HIV-1 into a dimeric structure.

Biopolymers↗
PubMed1994

Evidence that a central domain of nucleocapsid protein is required for RNA packaging in murine leukemia virus.

We have analyzed RNA packaging by a series of mutants altered in the nucleocapsid (NC) protein of Moloney murine leukemia virus (Mo-MuLV). We found that mutants lacking residues 8 through 11 or 44 through 60 of NC package Mo-MuLV RNA with virtually the same efficiency as wild-type Mo-MuLV. In contrast, point mutants altered at the conserved cysteines in the cysteine array (residues 26 and 29) and a mutant lacking residues 16 through 23 packaged Mo-MuLV RNA with approximately 1% of the efficiency of wild-type Mo-MuLV. The deficiency in packaged RNA was observed not only in Northern (RNA) analysis but also in an RNA-PCR assay, which would detect degraded as well as intact RNA. One of the cysteine array mutants was also shown to be defective with respect to encapsidation of hygromycin phosphotransferase mRNA containing a Mo-MuLV packaging signal. We suggest that a central region of NC, consisting of the cysteine array and flanking basic residues, is required for RNA packaging in Mo-MuLV.

Amino Acid Sequence↗
PubMed1993

The two zinc fingers in the human immunodeficiency virus type 1 nucleocapsid protein are not functionally equivalent.

The highly conserved zinc fingers in retroviral nucleocapsid (NC) proteins have the general structure Cys-(X)2-Cys-(X)4-His-(X)4-Cys. Human immunodeficiency virus type 1 (HIV-1) contains two Zn2+ fingers, and mutants were constructed in which the native sequence of each Zn2+ finger was maintained but their positions in the NC protein were changed. Mutants had either two first-finger sequences (pNC1/1), two second-finger sequences (pNC2/2), or reversed first- and second-finger sequences (pNC2/1). Cells transfected with mutant or wild-type clones produced similar levels of Tat, Gag, Pol, and Env proteins, formed syncytia, and shed viruslike particles that were indistinguishable by electron microscopy. However, the pNC2/1 and pNC2/2 mutants were inefficient in packaging genomic RNA (less than 15% of wild-type levels), whereas the pNC1/1 mutant packaged approximately 70% of wild-type levels of RNA. No infectious virus could be detected with either the pNC2/1 or pNC2/2 mutants, whereas the pNC1/1 mutant appeared to sustain a low level of replication and reverted to a competent wild-type-like viral species after a 2- to 4-week lag period. The data strongly suggest that the two Zn2+ fingers of HIV-1 are not functionally equivalent and that the first Zn2+ finger in the Gag precursor plays a more prominent role in RNA selection and packaging. The data also indicate that both Zn2+ fingers in the mature NC protein play as yet unknown roles in viral assembly or the early stages of the viral infection process.

Amino Acid Sequence↗
PubMed1993

Maturation of dimeric viral RNA of Moloney murine leukemia virus.

We have analyzed the dimeric RNA present in Moloney murine leukemia virus (MoMuLV) particles. We found that the RNA in newly released virions is in a conformation different from that in mature virions, since it has a different electrophoretic mobility in nondenaturing agarose gels and dissociates into monomers at a lower temperature. On the basis of these results, we suggest that the RNA initially packaged into nascent virions is already dimeric but that the dimer undergoes a maturation process after the virus is released from the cell. In further experiments, we tested the possibility that this maturation event is linked to the maturation cleavage of the virion proteins, which is catalyzed by the viral protease (PR). We found that the dimeric RNA isolated from PR- mutant virions resembles that from immature virions: it has a lower electrophoretic mobility and a lower sedimentation rate, and it also dissociates at a lower temperature than does RNA from mature wild-type virions. When Kirsten sarcoma virus is rescued by a PR- mutant or by a somewhat leaky cysteine array mutant of MoMuLV, its RNA also exhibits a electrophoretic mobility lower than that in the wild-type pseudotype. These results suggest that the maturation of dimeric RNA in released virus particles requires the cleavage of the Gag precursor and the presence of an intact cysteine array in the released nucleocapsid protein.

3T3 Cells↗
PubMed1993

Benefits of an automated narcotic retrieval system.

A comparison was made in effectiveness of lock and key and automated narcotics retrieval systems. Time required per narcotic averaged 137 seconds for the traditional mode and 48 seconds for the automated system. This resulted in a monthly savings of $594, along with general nurse satisfaction and a perception of increased security.

Automation↗