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.

Mother-to-child discordance in HLA-G exon 2 is associated with a reduced risk of perinatal HIV-1 transmission.

Definitive genetic parameters correlating with mother-to-child transmission (MCT) of HIV have not been fully established. We screened for the potential correlation between HLA-G variants and MCT, in a cohort of mother-child pairs. Discordance in exon 2 of HLA-G was significantly more common among non-transmitting (93%) than transmitting mother-child pairs (40%). Our results suggest that mother-child pairs both carrying the identical mutation in HLA-G exon 2 may be at higher risk of MCT of HIV-1.

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

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.

Identification of non-B human immunodeficiency virus type 1 subtypes in rural Georgia.

As part of an ongoing molecular epidemiological investigation of human immunodeficiency virus type 1 (HIV-1) in rural Georgia, the 5' half of reverse transcriptase (RT) genotypes from 30 patients was sequenced and phylogenetically analyzed. Two patients, GA132 and GA169, were infected with pol sequences of non-B subtype origin that were found to cluster phylogenetically with subtype A-E of Thai origin. Sliding window bootstrap analysis of GA169 showed clear evidence of A/B recombination within the pol gene segment, whereas in the other patient, GA132, no break point within RT could be identified. Interestingly, pairwise comparisons between these 2 patients' C2-V3 env region revealed a 13.5% divergence. However, similar comparisons within the non-B pol segments yielded a 1.23% nucleotide divergence, which suggests a complex phylogenetic and epidemiological history of the subtype A pol genotype in this region. These data demonstrate an increasing diversity of HIV-1 subtypes and the potential emergence of previously unidentified HIV-1 A-E/B recombinants in the rural United States.

Effects of extracellular human immunodeficiency virus type 1 vpr protein in primary rat cortical cell cultures.

Recent evidence suggests that HIV-1 Vpr exists in soluble form in the serum and cerebrospinal fluid (CSF). Further, its abundance in the bloodstream, and the CSF, and its activity on other cell types suggest that it could have an effect on brain activity. Using mixed embryonic rat brain cultures as a model to examine the effects of physiological concentrations of extracellular Vpr protein, Vpr-induced cell death was observed. We also observed similar Vpr-induced effects in enriched primary cortical rat astrocytes, as well as in the C6 glioma cell line. Vpr-induced cell death observed in the astrocytic cells appeared to be caused primarily by a necrotic mechanism, although a few apoptotic nuclei were also present. We did not observe Vpr-induced effects on any primary cortical neurons, although we did observe Vpr-induced cell death in hippocampal neurons and astrocytes. Finally, we observed no cell cycle effects due to extracellular Vpr protein. This data points out that different cell types are affected by the toxic effects of extracellular Vpr protein, and that differential toxic effects of extracellular Vpr protein are observed in similar cell types.

Molecular analysis of HIV-1 infection in Barbados

OBJECTIVE: To determine the sub-type(s) of HIV-1 in patients with advanced disease in Barbados. DESIGN AND METHODS: A molecular analysis was performed in sequences obtained from 38 HIV-1 infected persons. A 375 base pair fragment was amplified from the V3 loop of the env region of the HIV genome, using a nested RT-PCR method. The resulting fragment was sequenced directly and alignment was performed using the BLAST (Basic Local Alignment Search Tool) search. RESULTS: Of 38 patients with advanced HIV-1 infection, all were found to be infected with HIV-1 subtype B, which closely relates to a similar subtype found in North America and Europe. This is the first time that HIV subtypes have been described from Barbadian patients with advanced HIV disease. CONCLUSIONS: Our findings support the hypothesis that HIV infection in Barbados was derived initially from North America and Europe, where type B HIV infection is most common. Moreover, these findings indicate that vaccines suitable for use in North America will probably be effective in our population.(Au)

Involvement of protein kinase C in HIV-1 gp120-induced apoptosis in primary endothelium.

We previously showed that HIV-1 gp120-induced apoptosis in primary human umbilical vein endothelial cell cultures (HUVEC), through CCR5 and CXCR4. Here, we have found that agonists of protein kinase C (PKC), basic fibroblast growth factor (bFGF), and short exposure to low concentrations of phorbol esters were found to block gp120-induced apoptosis in HUVEC cultures. PKC antagonists, sphingosine, H7, and extended exposure of cultures to high concentrations of phorbol esters were also found to block gp120-induced apoptosis in HUVEC cultures. A significant increase in the total amount of cellular PKC enzymatic activity was observed on exposure of HUVEC to gp120. No increase in total PKC activity was observed on exposure of HUVECs to the natural ligands SDF-1alpha, or regulated-on-activation normal T-expressed and secreted (RANTES) cells, and gp120-induced PKC induction was found to be totally blocked by CXCR4 antibodies and partially blocked by the caspase 3 inhibitor, DEVD-CHO. Alternatively, CXCR4 antibodies and DEVD-CHO totally blocked apoptosis. Finally, gp120-induced effects were found to be insensitive to pertussis toxin. Accumulated evidence suggests PKC involvement at multiple points in the gp120-induced apoptotic pathway; also suggests involvement of the CXCR4 receptor internalization pathway, and potentially suggests different downstream effects of gp120-receptor interactions and natural ligand-receptor interactions.

Effect of extracellular human immunodeficiency virus type 1 glycoprotein 120 on primary human vascular endothelial cell cultures.

During the course of an HIV-1 infection, free infectious and noninfectious virus particles, and free HIV-1 proteins, circulate within the host, exposing the host endothelium to these viral factors, even if the endothelium is not infected. This suggests that extracellular HIV-1 proteins could influence endothelial cell function, leading to pathogenesis. In light of this, we have used primary cultured human vascular endothelial cells (HUVECs) to screen for effects of the HIV-1 protein gp120 on endothelial cell function. The results of this study show that short exposure of HUVEC cultures to this protein causes significant levels of cytotoxicity. Further, using several different assays, we have shown that this cytotoxic effect on HUVECs appears to be due to induction of an apoptotic program. The biphasic nature of gp120 titration curves suggests that multiple cellular factors are mediating these gp120-induced effects. Competition studies appear to confirm this by showing that the apoptotic effect is mediated through two cell surface receptors on HUVECs, CCR5 and CXCR4. Alternatively, competition studies examining CD4 receptors suggests that CD4 played no role in gp12O-induced effects on HUVECs.

Transposition of the endogenous insertion sequence element IS1126 modulates gingipain expression in Porphyromonas gingivalis.

We have previously reported on a Tn4351-generated mutant of Porphyromonas gingivalis (MSM-3) which expresses enhanced arginine-specific proteinase activity and does not utilize hemin or hemoglobin for growth (C. A. Genco et al., Infect. Immun. 63:2459-2466, 1995). In the process of characterizing the genetic lesion in P. gingivalis MSM-3, we have determined that the endogenous P. gingivalis insertion sequence element IS1126 is capable of transposition within P. gingivalis. We have also determined that IS1126 transposition modulates the transcription of the genes encoding the lysine-specific proteinase, gingipain K (kgp) and the arginine-specific proteinase, gingipain R2 (rgpB). Sequence analysis of P. gingivalis MSM-3 revealed that Tn4351 had inserted 60 bp upstream of the P. gingivalis endogenous IS element IS1126. Furthermore, P. gingivalis MSM-3 exhibited two additional copies of IS1126 compared to the parental strain A7436. Examination of the first additional IS1126 element, IS1126(1), indicated that it has inserted into the putative promoter region of the P. gingivalis kgp gene. Analysis of total RNA extracted from P. gingivalis MSM-3 demonstrated no detectable kgp transcript; likewise, P. gingivalis MSM-3 was devoid of lysine-specific proteinase activity. The increased arginine-specific proteinase activity exhibited by P. gingivalis MSM-3 was demonstrated to correlate with an increase in the rgpA and rgpB transcripts. The second additional IS1126 element, IS1126(2), was found to have inserted upstream of a newly identified gene, hmuR, which exhibits homology to a number of TonB-dependent genes involved in hemin and iron acquisition. Analysis of total RNA from P. gingivalis MSM-3 demonstrated that hmuR is transcribed, indicating that the insertion of IS1126 had not produced a polar effect on hmuR transcription. The hemin-hemoglobin defect in P. gingivalis MSM-3 is proposed to result from the inactivation of Kgp, which has recently been demonstrated to function in hemoglobin binding. Taken together, the results presented here demonstrate that the introduction of Tn4351 into the P. gingivalis chromosome has resulted in two previously undocumented phenomena in P. gingivalis: (i) the transposition of the endogenous insertion sequence element IS1126 and (ii) the modulation of gingipain transcription and translation as a result of IS1126 transposition.

Application of random amplified polymorphic DNA PCR for genomic analysis of HIV-1-infected individuals.

Random amplified polymorphic DNA polymerase chain reaction (RAPD-PCR) is a DNA fingerprinting technique used to detect genomic polymorphisms. We employed sixteen different RAPD-PCR 10-mer primers to amplify DNA from the peripheral blood mononuclear cells (PBMC) of 80 HIV-1-infected individuals. These individuals were previously identified as either heterozygotes (+ /delta32) and homozygotes (+/+) for the CCR5 locus by PCR with gene specific primers. Four of the sixteen randomly selected RAPD primers produced distinguishable banding profiles between CCR5 (+/delta32) heterozygotes and CCR5 (+/+ ) homozygotes. Direct sequencing of some RAPD-PCR products obtained with one of the four RAPD primers that were tested yielded clearly readable, but limited sequences, which were similar to portions of the previously published sequences for (+/+ ) homozygotes (98% similarity) and (+/delta32) heterozygotes (87% similarity) of the CCR5 alleles. Thus, the RAPD-PCR technique may be useful for the identification of human molecular markers that may correlate with susceptibility to HIV-1-infection, or differences in disease progression among HIV-l-infected individuals.

HIV type 1 envelope sequences from seroconverting patients in Barbados.

PIP: Because of the prevalence of leptospirosis in Barbados, patients who present to the hospital with febrile illnesses are routinely screened for Leptospira infection and their sera are stored for future reference. While the majority of patients are infected with Leptospira, some are not. Since some symptoms of acute HIV-1 illness are similar to those of leptospirosis, patient records were reviewed to identify patients whose clinical symptoms may have been due to HIV-1 infection. 10 HIV-1-positive patients originally hospitalized during 1990-94 were identified whose medical histories suggested the occurrence of acute HIV-1 illness at the time of Leptospira testing. Stored sera from those patients were then tested for the presence of HIV-1 p24 antigen and by Western blotting. Evidence of acute HIV-1 infection was considered to be a positive p24 test or a characteristic Western blot profile occurring at or shortly before the time of seropositivity for HIV-1 antibody. The authors determined the sequence of viral RNA from the 12 remaining sera samples from 8 patients, including paired samples drawn at 3- or 4-day intervals from 4 people. The Barbados patient variants aligned more closely with HIV-1 clade B reference strains than with the other subtypes. 2 variants, however, align separately from the classic B subtype and somewhat closer to variants from clades A and C. The Venezuelan isolate, although different from the patient sequences, is also separate from the other B variants.^ieng

Identification of a second endogenous Porphyromonas gingivalis insertion element.

In this study a second endogenous Porphyromonas gingivalis insertion element (IS element) that is capable of transposition within P. gingivalis was identified. Nucleotide sequence analysis of the Tn4351 insertion site in a P. gingivalis Tn4351-generated transconjugant showed that a complete copy of the previously unidentified IS element, designated PGIS2, had inserted into IS4351R in Tn4351. PGIS2 is 1,207 bp in length with 19-bp imperfect terminal inverted repeats, and insertion resulted in a duplicated 10-bp target sequence. Results of Southern hybridization of chromosomal DNA isolated from several P. gingivalis strains with a PGIS2-specific probe demonstrated that the number of copies of PGIS2 per genome varies among different P. gingivalis strains. Computer analysis of the putative polypeptide encoded by PGIS2 revealed strong homologies to the products encoded by IS1358 from Vibrio cholerae, ISAS1 from Aeromonas salmonicida, and H-rpt in Escherichia coli K-12.

Effects of the human immunodeficiency virus type 1 Rev protein on reporter gene and host T-cell gene expression.

The HIV-1 encoded regulatory Rev protein acts to selectively increase the cytoplasmic concentration of incompletely spliced viral mRNAs through interaction with the Rev responsive element (RRE). In addition, the Rev activation domain, believed to be a nuclear export sequence, has been shown to modulate the export of non-RRE containing RNAs (e.g. 5S rRNA, splicesomal U snRNAs). Recent evidence suggests Rev activity depends on interactions with cellular cofactors, leading to speculation that Rev utilizes a cellular RNA and/or a protein export pathway. Rev interactions with cellular cofactors could lead to sequestration of those cofactors from normal cellular activities, suggesting potential Rev effects on cellular gene products and their resultant activity. We have examined the role of Rev in modulating the expression of cellular gene products. Through transient cotransfection assays, we observed a consistent and significant decrease in the levels of luciferase and B-galactosidase activity in the presence of a Rev expressing construct. Cell fractionation studies demonstrated the nuclear retention of the luciferase gene transcripts. Surprisingly, similar effects were observed on constitutively expressed RNAs such as gamma-actin transcripts, and the 18S and 28S rRNAs. These results suggest Rev can disrupt the nuclear export of multiple classes of RNAs.

Epidemiology of HIV-1 infection in rural Georgia: demographic trends and analysis at the Medical College of Georgia.

We have conducted a retrospective study of 100 HIV-infected patients enrolled in an AZT monotherapy clinical study at the Medical College of Georgia (MCG) in Augusta, Georgia. When compared to the national trends, our results confirm previous studies that describe an overall increase in the burden of HIV infections among blacks, and, in particular, black women in the rural Southeast. In our cohort, infections due to homosexual contact accounted for approximately 40% of all cases while heterosexual contact and intravenous drug use (IDU) comprised 33% and 13%, respectively. Infections attributable to all other risk factors accounted for the remaining 14%. Relative to national surveillance data, we observed an increase in the prevalence of HIV infections among blacks, and heterosexually acquired infections, particularly among black women. Our analysis illustrates the dynamic nature of the current U.S. epidemic which appears to be shifting both in terms of its demographic and epidemiological profile. These data may indicate that national surveillance data may not reflect the dynamic nature of current demographic trends in HIV incidence, particularly as evidenced in the rural Southeast. This suggests that hospital or laboratory based cross-sectional studies, like ours, that analyze demographic variables of HIV-infected clinic attendees may be necessary to more accurately assess the leading edge of the HIV epidemic in rural, non-metropolitan areas.

Nucleolar localization of myc transcripts.

In situ hybridization has revealed a striking subnuclear distribution of c-myc RNA transcripts. A major fraction of the sense-strand nuclear c-myc transcripts was localized to the nucleoli. myc intron 1-containing RNAs were noticeably absent from nucleoli, accumulating instead in the nucleoplasm. The localization of myc RNA to nucleoli was shown to be common to a number of diverse cell types, including primary Sertoli cells and several cell lines. Furthermore, nucleolar localization was not restricted to c-myc and N-myc and myoD transcripts also displayed this phenomenon. In contrast, gamma-actin or lactate dehydrogenase transcripts did not display nucleolar localization. These observations suggest a new role for the nucleolus in transport and/or turnover of potential mRNAs.

High resolution two-dimensional protein gel evidence of differential gene expression during Entamoeba encystation.

As an initial step of investigation into the regulatory mechanisms of encystation in Entamoeba, we compared the protein profiles of newly formed cysts and trophozoites of E. invadens using high resolution two-dimensional PAGE and digitized video image analysis of silver stained gels. A total of 155 proteins unique to trophozoites and a total of 72 proteins unique to cysts were detected. Five of the most prominent cyst specific proteins were identified as candidates for further study. These results imply that extensive changes in gene expression accompany the progression of this parasite through its life cycle.

Expression of transfected vimentin genes in differentiating murine erythroleukemia cells reveals divergent cis-acting regulation of avian and mammalian vimentin sequences.

We studied the expression of transfected chicken and hamster vimentin genes in murine erythroleukemia (MEL) cells. MEL cells normally repress the levels of endogenous mouse vimentin mRNA during inducermediated differentiation, resulting in a subsequent loss of vimentin filaments. Expression of vimentin in differentiating MEL cells reflects the disappearance of vimentin filaments during mammalian erythropoiesis in vivo. In contrast, chicken erythroid cells express high levels of vimentin mRNA and vimentin filaments during terminal differentiation. We demonstrate here that chicken vimentin mRNA levels increase significantly in differentiating transfected MEL cells, whereas similarly transfected hamster vimentin genes are negatively regulated. In conjunction with in vitro nuclear run-on transcription experiments, these results suggest that the difference in vimentin expression in avian and mammalian erythropoiesis is due to a divergence of cis-linked vimentin sequences that are responsible for transcriptional and posttranscriptional regulation of vimentin gene expression. Transfected chicken vimentin genes produce functional vimentin protein and stable vimentin filaments during MEL cell differentiation, further demonstrating that the accumulation of vimentin filaments is determined by the abundance of newly synthesized vimentin.