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Abstract Background Mutant HIV (HIV-Env-Tr712) lacking the cytoplasmic tail of the viral glycoprotein (Env-CT) exhibits a cell-type specific replication phenotype such that replicative spread occurs in some T-cell lines (referred to as permissive cells) but fails to do so in most T-cell lines or in PBMCs (referred to as non-permissive cells). We aim to gain insight on the underlying requirement for the Env-CT for viral spread in non-permissive cells. Results We established that in comparison to HIV-Wt, both cell-free and cell-to-cell transmission of mutant HIV-Env-Tr712 from non-permissive cells were severely impaired under naturally low infection conditions. This requirement for Env-CT could be largely overcome by using saturating amounts of virus for infection. We further observed that in permissive cells, which supported both routes of mutant virus transmission, viral gene expression levels, Gag processing and particle release were inherently higher than in non-permissive cells, a factor which may be significantly contributing to their permissivity phenotype. Additionally, and correlating with viral transfer efficiencies in these cell types, HIV-Gag accumulation at the virological synapse (VS) was reduced to background levels in the absence of the Env-CT in conjugates of non-permissive cells but not in permissive cells. Conclusions During natural infection conditions, the HIV-Env-CT is critically required for viral transmission in cultures of non-permissive cells by both cell-free and cell-to-cell routes and is instrumental for Gag accumulation to the VS. The requirement of the Env-CT for these related processes is abrogated in permissive cells, which exhibit higher HIV gene expression levels.

Abstract Background The availability of cell lines releasing fluorescent viral particles can significantly support a variety of investigations, including the study of virus-cell interaction and the screening of antiviral compounds. Regarding HIV-1, the recovery of such biologic reagents represents a very hard challenge due to the intrinsic cytotoxicity of many HIV-1 products. We sought to overcome such a limitation by using a cell line releasing HIV-1 particles in an inducible way, and by exploiting the ability of a HIV-1 Nef mutant to be incorporated in virions at quite high levels. Results Here, we report the isolation and characterization of a HIV-1 packaging cell line, termed 18-4s, able to release valuable amounts of fluorescent HIV-1 based Virus-Like Particles (VLPs) in an inducible way. 18-4s cells were recovered by constitutively expressing the HIV-1 NefG3C mutant fused with the enhanced-green fluorescent protein (NefG3C-GFP) in a previously isolated inducible HIV-1 packaging cell line. The G3C mutation creates a palmitoylation site which results in NefG3C-GFP incorporation into virions greatly exceeding that of the wild type counterpart. Upon induction of 18-4s cells with ponasterone A and sodium butyrate, up to 4 μg/ml of VLPs, which had incorporated about 150 molecules of NefG3C-GFP per viral particle, were released into the culture supernatant. Due to their intrinsic strong fluorescence, the 18-4s VLPs were easily detectable by a novel cytofluorometric-based assay developed here. The treatment of target cells with fluorescent 18-4 VLPs pseudotyped with different glycoprotein receptors resulted in these becoming fluorescent as early as two hours post-challenge. Conclusion We created a stable cell line releasing fluorescent HIV-1 based VLPs upon induction useful for several applications including the study of virus-cell interactions and the screening of antiviral compounds.

Abstract H9-T-cells do not support the replication of mutant HIV-1 encoding Env protein lacking its long cytoplasmic C-terminal domain (Env-CT). Here we describe the generation of a H9-T-cell population constitutively expressing the HIV-1 Env-CT protein domain anchored in the cellular membrane by it homologous membrane-spanning domain (TMD). We confirmed that the Env-TMD-CT protein was associated with cellular membranes, that its expression did not have any obvious cytotoxic effects on the cells and that it did not affect wild-type HIV-1 replication. However, as measured in both a single-round assay as well as in spreading infections, replication competence of mutant pNL-Tr712, lacking the Env-CT, was not restored in this H9 T-cell population. This means that the Env-CT per se cannot transcomplement the replication block of HIV-1 virions encoding C-terminally truncated Env proteins and suggests that the Env-CT likely exerts its function only in the context of the complete Env protein.

Furin, a eukaryotic endoprotease enzyme that resembles subtilisin, enables the human immunodeficiency virus (HIV) to infect cells by helping the viral glycoprotein envelope to attach to the cell membrane. The additional finding that peptidyl-chloromethylketones with the amino acid sequence Arg-X-Lys/Arg-Arg can prevent HIV infection by blocking furin's effect may lead to new anti-HIV drugs.

Murein lipoprotein from the outer membrane of Escherichia coli could be fixed to erythrocytes without pretreatment of the erythrocytes. Passive hemagglutination or immune hemolysis could thus he used as sensitive assays to determine antibodies against lipoprotein. In rabbit antisera prepared against whole cells of E. coli, Salmonella, Arizona and Shigella antibodies against lipoprotein were present. The respective titers were lowest in encapsulated smooth strains and highest in rough mutants. Antisera against deep rough mutants showed even higher anti-lipoprotein titers than anti-R-lipopolysaccharide titers. Correspondingly, absorption of lipoprotein antibodies with enterobacterial strains was most pronounced with deep rough mutants and lowest with smooth strains. Lipoprotein becomes increasingly an immunogen as well as an antigen the more sugar residues are missing in the lipopolysaccharide on the cell surface. In wild-type cells lipoprotein is buried in the outer membrane; its exposure in mutant cells is related to defects at the cell surface. Antisera were prepared in rabbits against erythrocytes coated with various lipoprotein samples which differ at the C-terminal end of the polypeptide chain. Antibodies against lipoprotein containing two attached muropeptides (called lipoprotein I) are directed against the muropeptide attachment region. Antibodies against lipoprotein ending with Iysine-55 (lipoprotein II) were directed specifically against this terminal lysine residue. No serological reaction was observable after splitting off this lysine residue by carboxypeptidase B. Remarkably this antiserum against lipoprotein II neither reacted with the free lipoprotein ending with lysine-58 (lipoprotein IV) nor with the muropeptide containing lipoprotein I. Antisera against the lipoprotein lacking lysine-55 (lipoprotein III) reacted with all lipoprotein samples as did the antibodies in antisera prepared against whole heat-killed cells, thus indicating a common binding site located in the polypeptide chain towards the N-terminal end. No indication of an antigenic reactivity of the covalently linked lipoid was obtained. Lipoprotein treated with boiling 4% sodium dodecylsulfate and acetone reacted strongly with antisera against the native lipoprotein. The recovery of the antigenic sites demonstrates the remarkable stability of the native conformation. The lipoprotein is a new common antigen for closely related Enterobacteriaceae. Lipoprotein antisera might be useful for characterization of outer membrane mutants of Enterobacteriaceae. [ABSTRACT FROM AUTHOR]

The primary structure of a covalent lipoglycoprotein (murein lipoprotein) from the cell wall of Escherichia coli is presented. The amino acid sequence consists of 57 residues. The lipid is attached to the N-terminal serine and the murein is bound to the 6-amino group of the C-terminal lysine of the lipoprotein. The repetitive design of the sequence suggests a very conservative evolution in which a structural gene coding for 15 amino acids was duplicated once and then only half of this gene, thus coding for 7 amino acids was fused four times with the first 29 amino acids. Some deletions but only few exchanges of amino acids apparently occured. This structural membrane lipoprotein aggregates strongly when released from the murein by lysozyme digestion. The molecular weight was therefore determined in 2%, 0.5% and 0.1% dodecylsulfate by polyacrylamide gel electrophoresis and gel chromatography. The molecular weight of 13000 thus found deviated from that of 10000 calculated from the sum of the amino acids, the lipid as known so far and the part of the murein remaining bund to the lipoprotein. This was not due to a difference amount of dodecylsulfate binding by the lipoprotein compared with proteins containing no lipid but may be due to a more spherical shape of the small molecule in contrast to the rod-like shape of standard proteins in dodecylsulfate. [ABSTRACT FROM AUTHOR]