Biomedical subjects

Alan Rein

Publications and source records attributed to Alan Rein.

24 records · Page 2Linked to original sources

PubMed2002

Identification of HIV-1 nucleocapsid protein: nucleic acid antagonists with cellular anti-HIV activity.

The crucial functions of HIV-1 nucleocapsid-p7 protein (NC-p7) at different stages of HIV replication are dependent on its nucleic acid binding properties. In this study, a search has been made to identify antagonists of the interaction between NC-p7 and d(TG)(4). A chemical library of approximately 2000 small molecules (the NCI Diversity Set) was screened, of the 26 active inhibitors that were identified, five contained a xanthenyl ring structure. Further analysis of 63 structurally related compounds led to the identification of 2,3,4,5-tetrachloro-6-(4('),5('),6(')-trihydroxy-3(')-oxo-3H-xanthen-9(')-yl)benzoic acid, which binds to NC-p7 stoichiometrically. This compound exerted a significant anti-HIV activity in vitro with an IC(50) of 16.6+/-4.3 microM (means+/-SD). Synthetic variants lacking the two hydroxyls at positions 4(') and 5(') in the xanthenyl ring system failed to bind NC-p7 and showed significantly less protection against HIV infection. Molecular modeling predicts that these hydroxyl groups would bind to the amide nitrogen of Gly(35) with other contacts at the carbonyl oxygens of Gly(40) and Lys(33).

Anti-HIV Agents↗

PubMed2002

Reversal by dithiothreitol treatment of the block in murine leukemia virus maturation induced by disulfide cross-linking.

We previously reported that if murine leukemia virus particles are produced in the presence of the mild oxidizing agent disulfide-substituted benzamide-2, they fail to undergo the normal process of virus maturation. We now show that treatment of these immature particles with a reducing agent (dithiothreitol) induces their maturation in vitro, as evidenced by proteolytic cleavage of Gag, Gag-Pol, and Env proteins and by their morphology. The identification of partial cleavage products in these particles suggests the sequence with which the cleavages occur under these conditions. This may be a useful experimental system for further analysis of retroviral maturation under controlled conditions in vitro.

3T3 Cells↗

PubMed2002

Murine leukemia virus nucleocapsid mutant particles lacking viral RNA encapsidate ribosomes.

A single retroviral protein, termed Gag, is sufficient for assembly of retrovirus-like particles in mammalian cells. Gag normally selects the genomic RNA of the virus with high specificity; the nucleocapsid (NC) domain of Gag plays a crucial role in this selection process. However, encapsidation of the viral RNA is completely unnecessary for particle assembly. We previously showed that mutant murine leukemia virus (MuLV) particles that lack viral RNA because of a deletion in the cis-acting packaging signal ("Psi") in the genomic RNA compensate for the loss of the viral RNA by incorporating cellular mRNA. The RNA in wild-type and Psi- particles was also found to be necessary for virion core structure. In the present work, we explored the role of RNA in MuLV particles that lack genomic RNA because of mutations in the NC domain of Gag. Using a fluorescent dye assay, we observed that NC mutant particles contain the same amount of RNA that wild-type virions do. Surprisingly enough, these particles contained large amounts of rRNAs. Furthermore, ribosomal proteins were detected by immunoblotting, and ribosomes were observed inside the particles by electron microscopy. The biological significance of the presence of ribosomes in NC mutant particles lacking genomic RNA is discussed.

Animals↗

PubMed2002

Reversible binding of recombinant human immunodeficiency virus type 1 gag protein to nucleic acids in virus-like particle assembly in vitro.

Recombinant human immunodeficiency virus type 1 (HIV-1) Gag protein can assemble into virus-like particles (VLPs) in suitable buffer conditions with nucleic acid. We have explored the role of nucleic acid in this assembly process. HIV-1 nucleocapsid protein, a domain of Gag, can bind to oligodeoxynucleotides with the sequence d(TG)(n) with more salt resistance than to d(A)(n) oligonucleotides. We found that assembly of VLPs on d(TG)(n) oligonucleotides was more salt resistant than assembly on d(A)(n); thus, the oligonucleotides do not simply neutralize basic residues in Gag but provide a binding surface upon which Gag molecules assemble into VLPs. We also found that Gag molecules could be "trapped" on internal d(TG)(n) sequences within 40-base oligonucleotides, rendering them unable to take part in assembly. Thus, assembly on oligonucleotides requires that Gag proteins bind near the ends of the nucleic acid, and binding of Gag to internal d(TG)(n) sequences is apparently cooperative. Finally, we showed that nucleic acids in VLPs can exchange with nucleic acids in solution; there is a hierarchy of preferences in these exchange reactions. The results are consistent with an equilibrium model of in vitro assembly and may help to explain how Gag molecules in vivo select genomic RNA despite the presence in the cell of a vast excess of cellular mRNA molecules.

Gene Products, gag↗

PubMed2002

Retrovirus-specific packaging of aminoacyl-tRNA synthetases with cognate primer tRNAs.

The tRNAs used to prime reverse transcription in human immunodeficiency virus type 1 (HIV-1), Rous sarcoma virus (RSV), and Moloney murine leukemia virus (Mo-MuLV) are, tRNA(Trp), and tRNA(Pro), respectively. Using antibodies to the three cognate human aminoacyl-tRNA synthetases, we found that only lysyl-tRNA synthetase (LysRS) is present in HIV-1, only tryptophanyl-tRNA synthetase (TrpRS) is present in RSV, and neither these two synthetases nor prolyl-tRNA synthetase (ProRS) is present in Mo-MuLV. LysRS and TrpRS are present in HIV-1 and RSV at approximately 25 and 12 molecules/virion, respectively. These results support the hypothesis that, in HIV-1 and RSV, the cognate aminoacyl-tRNA synthetase may be used as the signal for targeting the selective packaging of primer tRNAs into retroviruses. The absence of ProRS in Mo-MuLV is consistent with reports that selective packaging of tRNA(Pro) in this virus is less important for achieving optimum annealing of the primer to Mo-MuLV genomic RNA.

3T3 Cells↗

PubMedDate not supplied

Modulation of entry of enveloped viruses by cholesterol and sphingolipids (Review).

Enveloped animal viruses infect host cells by fusion of viral and target membranes. This crucial fusion event occurs either with the plasma membrane of the host cells at the physiological pH or with the endosomal membranes at low pH and is triggered by specific glycoproteins in the virus envelope. Both lipids and proteins play critical and co-operative roles in the fusion process. Interactions of viral proteins with their receptors direct which membranes fuse and viral fusion proteins then drive the process. These fusion proteins operate on lipid assemblies, whose physical and mechanical properties are equally important to the proper functioning of the process. Lipids contribute to the viral fusion process by virtue of their distinct chemical structure, composition and/or their preferred partitioning into specific microdomains in the plasma membrane called 'rafts'. An involvement of lipid rafts in viral entry and membrane fusion has been examined recently. However, the mechanism(s) by which lipids as dynamic raft components control viral envelope-glycoprotein-triggered fusion is not clear. This paper will review literature findings on the contribution of the two raft-associated lipids, cholesterol and sphingolipids in viral entry.

Animals↗