Mahfuz Khan

HIV Type 1 Nef is released from infected cells in CD45(+) microvesicles and is present in the plasma of HIV-infected individuals.

HIV-1 Nef has been demonstrated to be integral for viral persistence, infectivity, and the acceleration of disease pathogenesis (AIDS) in humans. Nef has also been detected in the plasma of HIV-infected individuals and is released from infected cells. The form in which Nef is released from infected cells is unknown. However, Nef is a myristoylated protein and has been shown to interact with the intracellular vesicular trafficking network. Here we show that Nef is released in CD45-containing microvesicles. This microvesicular Nef (mvNef) is detected in the plasma of HIV-infected individuals at relatively high concentrations (10 ng/ml). It is also present in tissue culture supernatants of Jurkat cells infected with HIV(MN). Interestingly, plasma mvNef levels in HIV(+) patients did not significantly correlate with viral load or CD4 count. Microvesicular Nef levels persisted in the plasma of HIV-infected individuals despite the use of antiretroviral therapy, even in individuals with undetectable viral loads. Using cell lines, we found Nef microvesicles induce apoptosis in Jurkat T-lymphocytes but had no observed effect on the U937 monocytic cell line. Given the large amount of mvNef present in the plasma of HIV-infected individuals, the apoptotic effect of mvNef on T cells, and the observed functions of extracellular soluble Nef in vitro, it seems likely that in vivo mvNef may play a significant role in the pathogenesis of AIDS.

Invasion strategies of the oral pathogen porphyromonas gingivalis: implications for cardiovascular disease.

Microorganisms have evolved a variety of mechanisms designed to evade detection and/or destruction by the host. Many pathogens evade host defenses by invading cells, thus providing the bacterium with an environment free of competing microorganisms. Adherence and invasion are active processes in which microorganisms often use host proteins and enzymes to gain entry into the cell, thus stimulating their own uptake. The investigation of invasion by the periopathogen Porphyromonas gingivalis is in its infancy in comparison with that of the enteric pathogens. However, recent studies with P. gingivalis have revealed that these organisms have developed invasion strategies and mechanisms similar to those of the enteric pathogens for both epithelial and endothelial cells. The study and elucidation of the mechanisms by which microorganisms such as P. gingivalis persist in chronic infection will provide valuable insight into the pathogenesis of P. gingivalis-mediated periodontal disease. The ability to multiply in and to activate endothelial cells may be one of the pathogenic mechanisms exerted by P. gingivalis that may explain the recently described association between this organism and cardiovascular disease.

Genetic characterization of HIV type 1 Nef-induced vesicle secretion.

The HIV-1 Nef protein is known to be secreted, and our group has shown that Nef is secreted from nef-transfected and HIV-1-infected cells in small exosome-like vesicles (d. 40-100 nm). The role of secreted Nef remains to be fully characterized. Thus, it is important to characterize the nature of and the mechanisms regulating Nef secretion. We hypothesized that specific structural domains on the Nef protein interact with components of the endosomal trafficking machinery, sorting Nef into multivesicular bodies (MVB) and packaging it in exosome-like vesicles. To identify those domains, a series of mutants spanning the entire nef sequence were made and cloned into the expression vector pQB1, which expresses the mutants as Nef-GFP fusion proteins. These constructs were used in transient transfection assays to identify sequences necessary for secretion of the Nef-GFP fusion protein. N-terminal domains were identified as critical for Nef-induced vesicle secretion: (1) a basic cluster of four arginine residues (aa 17, 19, 21, 22), (2) the phosphofurin acidic cluster sequence (PACS; Glu62-65), and (3) a previously uncharacterized domain spanning amino acid residues 66-70 (VGFPV), which we named the secretion modification region (SMR). Additional amino acids P25, 29GVG31, and T44 were identified in HIV-1 Nef as regulating its secretion. These residues have not been associated with other reported Nef functions. The myristoylation domain, ubiquitination lysine residues, and the C-terminal portion of Nef (aa 71-206) had no effect on secretion. A minimal HIV-1 Nef sequence, comprising the identified motifs, was sufficient for Nef-induced vesicle secretion.

Characterization of Nef-CXCR4 Interactions Important for Apoptosis Induction

ABSTRACT The HIV-1 Nef protein was analyzed for apoptotic structural motifs that interact with the CXCR4 receptor and induce apoptosis in CD4+ lymphocytes. Two apoptotic motifs were identified. One centered on Nef amino acids (aa) 50 to 60, with the overlapping 20-mer peptides retaining about 82% of the activity of the full Nef protein. The second centered on aa 170 to 180, with the overlapping 20-mer peptides retaining about 30% of the activity of the full protein. Significant apoptotic abilities were observed for 11-mer motif peptides spanning aa 50 to 60 and aa 170 to 180, with a scrambled version of the 11-mer motif peptide corresponding to aa 50 to 60 showing no apoptotic ability. Hallmarks of apoptosis, such as the formation of DNA ladders and caspase activation, that were observed with the full-length protein were equally evident upon exposure of cells to these motif peptides. A CXCR4 antibody and the endogenous ligand SDF-1α were effective in blocking Nef peptide-induced apoptosis as well as the physical binding of a fluorescently tagged Nef protein, while CCR5 antibodies were ineffective. The CXCR4-negative cell line MDA-MB-468 was resistant to the apoptotic peptides and became sensitive to the apoptotic peptides upon transfection with a CXCR4-expressing vector. A fluorescently tagged motif peptide and Nef protein displayed physical binding to CXCR4-transfected MDA-MB-468 cells, but not to CCR5-transfected cells. The removal of the apoptotic motif sequences from the full-length protein completely eliminated the ability of Nef to induce apoptosis. However, these modified Nef proteins still retained the ability to enhance viral infectivity. Thus, specific sequences in the Nef protein appear to be necessary for Nef protein-induced apoptosis as well as for physical interaction with CXCR4 receptors.

Apoptotic effects in primary human umbilical vein endothelial cell cultures caused by exposure to virion-associated and cell membrane-associated HIV-1 gp120

Summary: During the course of HIV‐1 infection, free virus, infected cells, and free HIV‐1 proteins circulate within the host, exposing the host endothelium to these viral factors. We have previously presented evidence showing that soluble HIV‐1 gp120 protein interacts with chemokine receptors on primary human endothelium and (through those interactions) induces apoptosis as well as other intracellular effects. The current study examines the effect of exposure of vascular endothelium to gp120 IIIb expressed on the surface of Jurkat cells and in the context of viral particles. Apoptosis was observed in human umbilical vein endothelial cell (HUVEC) cultures exposed to gp160‐transfected Jurkat cells as well as to virion particles with gp120 on their surface. Additional experiments show that this apoptotic effect was caused by gp120 protein acting through chemokine receptors on the HUVEC surface, primarily the CXCR4 receptor. At higher concentrations of gp120, this lymphotrophic variant, which has been shown to interact predominantly with CXCR4, seems to interact with and induce apoptosis through the CCR5 receptor. Finally, this apoptotic effect in HUVEC cultures occurs at low levels of the inducing agent, gp120, on cell membranes or on virion particles. These results demonstrate that HIV‐1 gp120 is capable of interacting with and killing vascular endothelial cells in multiple in vivo contexts.

Restoration of wild-type infectivity to human immunodeficiency virus type 1 strains lacking nef by intravirion reverse transcription

ABSTRACT Human immunodeficiency virus type 1 (HIV-1) Nef protein exerts several effects, both on infected cells and as a virion protein, which work together to enhance viral replication. One of these activities is the ability to enhance infectivity and the formation of proviral DNA. The mechanism of this enhancement remains incompletely understood. We show that virions with nef deleted can be restored to wild-type infectivity by stimulating intravirion reverse transcription. Particle composition and measures of reverse transcriptase activity remain the same for Nef+ and Nef− virions both before and after natural endogenous reverse transcription (NERT) treatment. The effect of NERT treatment on virions pseudotyped with murine leukemia virus envelope protein was similar to that on particles pseudotyped with HIV-1 envelope protein. However, virions pseudotyped with vesicular stomatitis virus G envelope protein showed no influence of Nef on NERT enhancement of infectivity. These observations suggest that Nef may function at a level prior to reverse transcription. Since NERT treatment results in partial disassembly of the viral core, we speculate that Nef may function at the level of core particle disassembly.

Isolation of a contact-dependent haemolysin from Mycobacterium tuberculosis.

Contact-dependent haemolytic activity was observed with cells of Mycobacterium tuberculosis H(37)Rv and M. tuberculosis H(37)Ra, but not with those of M. bovis, M. bovis BCG and M. africanum. Culture filtrates of all these strains did not exhibit any haemolytic activity. M. tuberculosis H(37)Rv was subsequently used for the isolation of haemolysin. Haemolytic activity was retained in the cell debris even after sonication of the cells and treatment with Tween 80 and lysozyme. Solubilisation of haemolysin was possible only after the cell debris was washed with ethanol 70% and then treated with Tween 80 0.1%. The haemolysin thus obtained showed a micellar M(r) of >200000 by gel-filtration on Sephadex G-200 and a subunit M(r) of 66000 by SDS-PAGE. It was sensitive to trypsin but stable when heated at 60 degrees C for 10 min. Polyclonal serum raised in rabbits against the haemolysin neutralised the haemolytic activity. The N-terminal amino-acid sequence of the 66-kDa subunit of haemolysin showed identity with TB66, the 66-kDa secretory protein of M. tuberculosis, and 30% homology with the haemolysin A precursor of Vibrio cholerae. Phosphatidylglycerol inhibited lysis of sheep erythrocytes by the haemolysin and is probably the receptor for the haemolysin. Haemolysin not only lysed erythrocytes, but was also cytotoxic to human lung cells. It appears that, among the members of the M. tuberculosis complex, the cell-bound contact-dependent haemolysin/cytolysin is restricted to M. tuberculosis and it may be associated with the pathogenesis of M. tuberculosis.

Effect of polypurine tract (PPT) mutations on human immunodeficiency virus type 1 replication: a virus with a completely randomized PPT retains low infectivity.

ABSTRACT We introduced polypurine tract (PPT) mutations, which we had previously tested in an in vitro assay, into the viral clone NL4-3KFSΔnef. Each mutant was tested for single-round infectivity and virion production. All of the PPT mutations had an effect on replication; however, mutation of the 5′ end appeared to have less of an effect on infectivity than mutation of the 3′ end of the PPT sequence. Curiously, a mutation in which the entire PPT sequence was randomized (PPTSUB) retained 12% of the infectivity of the wild type (WT) in a multinuclear activation of galactosidase indicator assay. Supernatants from these infections contained viral particles, as evidenced by the presence of p24 antigen. Two-long terminal repeat (2-LTR) circle junction analysis following PPTSUB infection revealed that the mutant could form a high percentage of normal junctions. Quantification of the 2-LTR circles using real-time PCR revealed that number of 2-LTR circles from cells infected with the PPTSUB mutant was 3.5 logs greater than 2-LTR circles from cells infected with WT virus. To determine whether the progeny virions from a PPTSUB infection could undergo further rounds of replication, we introduced the PPTSUB mutation into a replication-competent virus. Our results show that the mutant virus is able to replicate and that the infectivity of the progeny virions increases with each passage, quickly reverting to a WT PPT sequence. Together, these experiments confirm that the 3′ end of the PPT is important for plus-strand priming and that a virus that completely lacks a PPT can replicate at a low level.

Activation of the Inositol (1,4,5)-Triphosphate Calcium Gate Receptor Is Required for HIV-1 Gag Release

ABSTRACT The structural precursor polyprotein, Gag, encoded by all retroviruses, including the human immunodeficiency virus type 1 (HIV-1), is necessary and sufficient for the assembly and release of particles that morphologically resemble immature virus particles. Previous studies have shown that the addition of Ca2+ to cells expressing Gag enhances virus particle production. However, no specific cellular factor has been implicated as mediator of Ca2+ provision. The inositol (1,4,5)-triphosphate receptor (IP3R) gates intracellular Ca2+ stores. Following activation by binding of its ligand, IP3, it releases Ca2+ from the stores. We demonstrate here that IP3R function is required for efficient release of HIV-1 virus particles. Depletion of IP3R by small interfering RNA, sequestration of its activating ligand by expression of a mutated fragment of IP3R that binds IP3 with very high affinity, or blocking formation of the ligand by inhibiting phospholipase C-mediated hydrolysis of the precursor, phosphatidylinositol-4,5-biphosphate, inhibited Gag particle release. These disruptions, as well as interference with ligand-receptor interaction using antibody targeted to the ligand-binding site on IP3R, blocked plasma membrane accumulation of Gag. These findings identify IP3R as a new determinant in HIV-1 trafficking during Gag assembly and introduce IP3R-regulated Ca2+ signaling as a potential novel cofactor in viral particle release.

Sprouty 2 Binds ESCRT-II Factor Eap20 and Facilitates HIV-1 Gag Release

ABSTRACT The four ESCRT (endocytic sorting complexes required for transport) complexes (ESCRT-0, -I, -II, and -III) normally operate sequentially in the trafficking of cellular cargo. HIV-1 Gag trafficking and release as virus-like particles (VLPs) require the participation of ESCRTs; however, its use of ESCRTs is selective and nonsequential. Specifically, Gag trafficking to release sites on the plasma membrane does not require ESCRT-0 or -II. It is known that a bypass of ESCRT-0 is achieved by the direct linkage of the ESCRT-I component, Tsg101, to the primary L domain motif (PTAP) in Gag and that bypass of ESCRT-II is achieved by the linkage of Gag to ESCRT-III through the adaptor protein Alix. However, the mechanism by which Gag suppresses the interaction of bound ESCRT-I with ESCRT-II is unknown. Here we show (i) that VLP release requires the steady-state level of Sprouty 2 (Spry2) in COS-1 cells, (ii) that Spry2 binds the ESCRT-II component Eap20, (iii) that binding Eap20 permits Spry2 to disrupt ESCRT-I interaction with ESCRT-II, and (iv) that coexpression of Gag with a Spry2 fragment that binds Eap20 increases VLP release. Spry2 also facilitated release of P7L-Gag (i.e., release in the absence of Tsg101 binding). In this case, rescue required the secondary L domain (YPXnL) in HIV-1 Gag that binds Alix and the region in Spry2 that binds Eap20. The results identify Spry2 as a novel cellular factor that facilitates release driven by the primary and secondary HIV-1 Gag L domains.