Faculty Bibliography

Palmitoylation of the cytoplasmic domain of the human immunodeficiency type virus type 1 (HIV-1) envelope (Env) transmembrane protein, gp41, has been implicated in Env targeting to detergent-resistant lipid rafts, Env incorporation into the virus, and viral infectivity. In contrast, we provide evidence here to show that HIV-1 infectivity, Env targeting to lipid rafts, and Env incorporation into the virus are independent of cytoplasmic tail palmitoylation. The T-cell (T)-tropic HXB2-based virus, which utilizes CXCR4 as the entry coreceptor, carrying a Cys-to-Ser mutation at residue 764 or 837 or at both replicated with wild-type (WT) virus replication kinetics in CD4+ T cells. The properties of Env expression, precursor processing, cell surface expression, and Env incorporation of these three mutant viruses were normal compared to those of the WT virus. These three mutant Env proteins all effectively mediated one-cycle virus infection. When the Cys residues were replaced by Ala residues, all single and double mutants still retained the phenotypes of infectivity, Env incorporation, and lipid raft localization of the WT Env. When Cys-to-Ala substitutions were introduced into the macrophage (M)-tropic ConB virus, which utilizes CCR5 as the coreceptor, these mutations did not affect the replication potential, Env phenotypes, lipid raft targeting, or Env assembly into the virus of the WT Env. These T- and M-tropic mutants also productively replicated in human primary CD4+ T cells. Moreover, mutations at both Cys residues significantly reduced the level of palmitoylation of the Env. Our results together support the notion that palmitoylation of the cytoplasmic tail of the HIV-1 Env is not essential for the HIV-1 virus life cycle.

The objective of the present study was to evaluate human immunodeficiency virus (HIV) levels in the aqueous humor and vitreous fluid in patients with and those without ocular involvement due to AIDS-related cryptococcosis. We also assessed whether cryptococcosis infection in the central nervous system (CNS) was associated with elevated HIV levels in the cerebrospinal fluid (CSF). From 1993 to 2003, we obtained 16 CSF samples from 9 AIDS patients with cryptococcal meningitis and 7 AIDS patients without CNS opportunistic infection. Samples of intraocular fluids were obtained from all 9 patients with cryptococcal meningitis. Five cases presented with ocular involvement in patients with meningitis. By using the method of reverse transcriptase-polymerase chain reaction, we detected higher HIV loads in aqueous humor (27,244+/-4,123 copies/ml) and vitreous fluid (84,930+/-5,071 copies/ml) in patients with concomitant CNS and ocular involvement due to AIDS-related cryptococcosis (p<0.05). HIV levels in the vitreous fluid were correlated with levels in CSF (r=0.77). Mean HIV level in the CSF (209,761+/-18,787 copies/ml) was significantly elevated in AIDS patients with cryptococcoal meningitis (p<0.05). To our knowledge, this is the first report to study the level of HIV loads in the CSF and intraocular fluid simultaneously in AIDS patients with cryptococcosis. Our results revealed the intrathecal and intraocular HIV replication in patients with cryptococcosis.

The biological significance of the presence of a long cytoplasmic domain in the envelope (Env) transmembrane protein gp41 of human immunodeficiency virus type 1 (HIV-1) is still not fully understood. Here we examined the effects of cytoplasmic tail elongation on virus replication and characterized the role of the C-terminal cytoplasmic tail in interactions with the Gag protein. Extensions with six and nine His residues but not with fewer than six His residues were found to severely inhibit virus replication through decreased Env electrophoretic mobility and reduced Env incorporation compared to the wild-type virus. These two mutants also exhibited distinct N glycosylation and reduced cell surface expression. An extension of six other residues had no deleterious effect on infectivity, even though some mutants showed reduced Env incorporation into the virus and/or decreased cell surface expression. We further show that these elongated cytoplasmic tails in a format of the glutathione S-transferase fusion protein still interacted effectively with the Gag protein. In addition, the immediate C terminus of the cytoplasmic tail was not directly involved in interactions with Gag, but the region containing the last 13 to 43 residues from the C terminus was critical for Env-Gag interactions. Taken together, our results demonstrate that HIV-1 Env can tolerate extension at its C terminus to a certain degree without loss of virus infectivity and Env-Gag interactions. However, extended elongation in the cytoplasmic tail may impair virus infectivity, Env cell surface expression, and Env incorporation into the virus.

We have previously demonstrated that a human immunodeficiency virus (HIV) chimeric Gag protein containing a partial replacement of the matrix domain by the viral protease domain (PR) could undergo autoprocessing with no virus particle production [J. Virol. 74 (2000) 3418]. To further analyze the effects of repositioned PR on virus particle production and Gag-Pol incorporation, we introduced the chimeric PR construct into a PR-negative Gag-Pol expression plasmid and coexpressed the resultant construct with a Pr55(gag) expression plasmid (pGAG) in 293T cells. Analysis indicated that the chimeric PR was similar to native PR in that both could prevent virus particle production in cotransfections with an equivalent amount of pGAG plasmid DNA, suggesting an efficient trans processing of Pr55(gag) by the chimeric PR. In cotransfections with the pGAG at a DNA ratio of 1:10 to 1:20, which resembles the normal intracellular expression ratio of Gag-Pol to Gag, Gag-Pol carrying the PR in the Gag coding region could undergo autoprocessing in cells and was incorporated into virus particles at a level about 20-40% of that of wild-type Gag-Pol. However, the incorporated chimeric Gag-Pol was unable to autocleave and unable to process the Gag particles properly, as mature particle-associated reverse transcriptase (RT) and p24(gag) proteins were barely detected. Our data strongly suggest that positioning an active HIV PR in the matrix region significantly affects the PR-mediated virus particle maturation.

The CC chemokine receptor 6 (CCR6) is selectively expressed on memory T cells, B cells, and dendritic cells and appears to be involved in the initiation of a memory immune response. The only chemokine ligand for CCR6 is CCL20/MIP-3alpha. In the present study, we attempted to define the extracellular domains (ECDs) of CCR6 responsible for CCL20/MIP-3alpha binding using a domain-swapping approach in which the ECDs of CCR6 were substituted with the corresponding CCR5 domains to generate various CCR6/CCR5 chimeras. These chimeras were tested for receptor expression, ligand binding, and functional activity as evaluated by calcium flux and chemotaxis. All chimeras showed respectable surface expression; however only one, substituted with extracellular loop 1 from CCR5, showed reduced functional activity. The general failure of functionality of the CCR6/CCR5 chimeras may imply that characteristics of each ECD are critical for coordination among all the ECDs of CCR6. Additionally, of interest, a chimera containing all of the ECDs from CCR5 in the context of CCR6 neither responded to CCR5 ligands nor served as a coreceptor for macrophage-tropic HIV-1. These results suggest that not only ECDs but also transmembrane and intracellular domains of CCR5 are involved in both ligand binding and coreceptor activity.

The aim of the present study was to evaluate the impact of highly active antiretroviral therapy (HAART) on HIV viral load of plasma and intraocular fluids in AIDS patients with ophthalmic opportunistic infections. We further compared the treatment effect of HAART on these patients. From June 1997 to July 2003, we examined and followed up the ophthalmic conditions of 49 patients receiving HAART with ophthalmic diseases during this period. The method of reverse-transcriptase polymerase chain reaction was used to detect and monitor HIV load in plasma and/or aqueous humor of AIDS patients. Before HAART, the HIV levels in the plasma and aqueous humor in 8 AIDS patients with ophthalmic opportunistic infections were significantly higher than those in 6 patients with HIV-related retinopathy (p < 0.05). Compared to the eye findings and clinical improvement, HIV loads of aqueous humor in 10 of 14 AIDS patients (6 with HIV-related retinopathy, 5 with cytomegalovirus retinitis, 2 with toxoplasmic retinitis and 1 with cryptococcal chorioretinitis) declined to undetectable levels (< 400 copies/ml) after 4-8 months of HAART. HIV virus levels in the plasma of AIDS patients were significantly decreased, and the CD4 counts of these patients were significantly increased (Wilcoxon test) after initiation of HAART.

Human cytomegalovirus (HCMV) retinitis is the most common ocular opportunistic infection in AIDS. It often leads to blindness if left untreated. The questions as to how HCMV infection causes retinal immunopathogenesis and visual destruction in AIDS patients have not been completely established. Here we reported that the nitric oxide (NO) levels in aqueous humor samples in 10 AIDS patients with CMV retinitis (104.3 +/- 27.1 microM) were higher than the levels in 7 AIDS patients without CMV retinitis (36.1 +/- 10.4 micro M; p < 0.001). After ganciclovir treatment, the NO level in the vitreous body of 5 patients declined dramatically (53.4 +/- 11.8 micro M). By using immunohistochemistry assay, we found that the aggregates of macrophages infiltrated in the CMV-infected retina of 4 AIDS patients. Moreover, the expression of inducible-form NO synthase was detected in the infected retina of these patients. These results suggest that NO production in the eye may play a fundamental role in the immunopathogenesis of AIDS patients with CMV retinitis.

To understand the role of the lentivirus lytic peptide-1 region of the human immunodeficiency virus type 1 transmembrane glycoprotein (gp) 41 in viral infection, we examined the effects on virus replication of single amino acid deletions spanning this region in an infectious provirus of the HXB2 strain. Among the mutants analyzed, only the deletion of one of the two adjacent valine residues located at positions 832 and 833 (termed the Delta 833 mutant for simplicity) greatly reduced the steady-state, cell-associated levels of the Env precursor and gp120, as opposed to the wild-type virus. The altered Env phenotype resulted in severely impaired virus infectivity and gp120 incorporation into this mutant virion. Analyses of additional mutants with deletions at Ile-830, Ala-836, and Ile-840 demonstrated that the Delta 830 mutant exhibited the most significant inhibitory effect on Env steady-state expression. These results indicate that the N terminus of the lentivirus lytic peptide-1 region is critical for Env steady-state expression. Among the mutant viruses encoding Env proteins in which residues Val-832 and Val-833 were individually substituted by nonconserved amino acids Ala, Ser, or Pro, which were expected to disrupt the alpha-helical structure in the increasingly severe manner of Pro > Ser > Ala, only the 833P mutant exhibited significantly reduced steady-state Env expression. Pulse labeling and pulse-chase studies demonstrated that the Delta 830, Delta 833, and 833P mutants of Env proteins degraded more rapidly in a time-dependent manner after biosynthesis than did the wild-type Env. The results indicate that residue 830 and 833 mutations are likely to induce a conformational change in Env that targets the mutant protein for cellular degradation. Our study has implications about the structural determinants located at the N terminus of the lentivirus lytic peptide-1 sequence of gp41 that affect the fate of Env in virus-infected cells.

Human cytomegalovirus (HCMV) retinitis is the most common ocular opportunistic infection in immunocompromised patients and AIDS. It often leads to blindness if left untreated. The question as to how HCMV infection causes retinal pathogenesis and visual destruction in AIDS patients remains unresolved. To answer the question, by using terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling assay, we detected the significant signals of apoptotic cells at the same sites in the HCMV-infected retina of AIDS patients as compared to AIDS patients without HCMV retinitis. In vitro study also revealed apoptosis induced by HCMV infection in human retinal pigment epithelium cells, mediated by activation of caspase 3 and poly(ADP-ribose) polymerase pathway. These results strongly suggest the fundamental role of HCMV-induced apoptosis in mediating cell death in infected human retina and retinal pigment epithelium cells to make severe visual impairment.