Assembly of anthrax toxin pore: lethal-factor complexes into lipid nanodiscs.

We have devised a procedure to incorporate the anthrax protective antigen (PA) pore complexed with the N-terminal domain of anthrax lethal factor (LFN ) into lipid nanodiscs and analyzed the resulting complexes by negative-stain electron microscopy. Insertion into nanodiscs was performed without relying on primary and secondary detergent screens. The preparations were relatively pure, and the percentage of PA pore inserted into nanodiscs on EM grids was high (∼43%). Three-dimensional analysis of negatively stained single particles revealed the LFN -PA nanodisc complex mirroring the previous unliganded PA pore nanodisc structure, but with additional protein density consistent with multiple bound LFN molecules on the PA cap region. The assembly procedure will facilitate collection of higher resolution cryo-EM LFN -PA nanodisc structures and use of advanced automated particle selection methods.

Three-dimensional structure of the anthrax toxin pore inserted into lipid nanodiscs and lipid vesicles.

A major goal in understanding the pathogenesis of the anthrax bacillus is to determine how the protective antigen (PA) pore mediates translocation of the enzymatic components of anthrax toxin across membranes. To obtain structural insights into this mechanism, we constructed PA-pore membrane complexes and visualized them by using negative-stain electron microscopy. Two populations of PA pores were visualized in membranes, vesicle-inserted and nanodisc-inserted, allowing us to reconstruct two virtually identical PA-pore structures at 22-A resolution. Reconstruction of a domain 4-truncated PA pore inserted into nanodiscs showed that this domain does not significantly influence pore structure. Normal mode flexible fitting of the x-ray crystallographic coordinates of the PA prepore indicated that a prominent flange observed within the pore lumen is formed by the convergence of mobile loops carrying Phe427, a residue known to catalyze protein translocation. Our results have identified the location of a crucial functional element of the PA pore and documented the value of combining nanodisc technology with electron microscopy to examine the structures of membrane-interactive proteins.

Mitochondrial DNA sequence diversity in two groups of Italian Veneto speakers from Veneto.

Although frequencies of mitochondrial DNA (mtDNA) haplogroups in the different European populations are rather homogenous, there are a few European populations or linguistic isolates that show different mtDNA haplogroup distributions; examples are the Saami and Ladin speakers from the eastern Italian Alps. MtDNA sequence diversity was analysed from subjects from two villages in Veneto. The first, Posina, is situated in the Venetian Alps near Vicenza. The second, Barco di Pravisdomini is a village on the plains near Venice. In spite of their common Veneto dialect, the two group populations have not preserved a genetic homogeneity; particularly, they show differences in T and J haplogroups frequencies. MtDNA diversity in these two groups seems to depend more on their geographic situation.

Long-term evaluation of triple nucleoside therapy administered from primary HIV-1 infection.

OBJECTIVE: To study the long-term effect of triple-drug therapy initiated at the time of primary HIV-1 Infection and to evaluate the persistance of replication-competent virus in responding patients. METHODS: Prospective open-label pilot study. Patients received a combination of zidovudine, didanosine and lamivudine. Viral sequencing of the reverse transcriptase gene was performed before therapy and during follow-up. HIV-1 RNA and DNA as well as CD4 and CD8 T lymphocyte subsets were measured in blood and in lymph node biopsies during therapy. Isolated blood CD4 T cells were cultured in conditions that improved HIV isolation. Three patients received in vivo interleukin-2 and gamma-interferon in order to try to identify intracellular pools of replication-competent virus. SETTING: A tertiary care general hospital. PATIENTS: Fifteen patients observed within 28 days following the acute retroviral syndrome. RESULTS: After a mean follow-up of 27.5+/-2.9 months, plasma RNA remained < 20 copies/ml (four patients), fluctuated between 20 and 120 copies/ml (six patients) or rebounded (five patients). M184V and/or T215Y mutations were demonstrated in two of these last five patients. Proviral DNA in peripheral blood mononuclear cells (PBMC) decreased by an average of -1 log after 16+/-3 months, reaching undetectable levels in three patients. The culture of isolated CD4 T cells yielded virus in all but two patients. These last were characterized by a waning antibody reactivity on the Western blot, undetectable proviral DNA in PBMC and undetectable RNA in lymph nodes. Cytokine administration in vivo had no effect in one patient and unmasked plasma RNA in the other. Stopping therapy in the first patient led to a rebound in plasma RNA. CONCLUSION: Despite a lack of detectable plasma viral activity in some patients after 3 years of triple nucleoside therapy administered since the acute retroviral syndrome, replication-competent virus can still be demonstrated.

Residual human immunodeficiency virus type 1 RNA in lymphoid tissue of patients with sustained plasma RNA of <200 copies/mL.

Human immunodeficiency virus type 1 (HIV-1) RNA was measured in lymph node (LN) mononuclear cells of 50 patients with sustained plasma RNA of <200 copies/mL with therapy. Six patients had received a combination of three reverse transcriptase inhibitors (RTIs) since primary infection, 11 received this same combination during chronic disease, 21 received a combination of two RTIs plus a protease inhibitor (PI), and 12 received three RTIs plus a PI. The mean overall duration of therapy was 8.9 +/- 0.5 months (range, 5-24), with no significant difference between groups. LN HIV-1 RNA levels varied from undetectable to 1.7 million copies/10(6) cells according to cases. The mean LN HIV-1 RNA level was 2.99 +/- 0.42 log10 copies/10(6) cells in the 17 patients receiving three RTIs compared with 1.93 +/- 0.25 log10 copies/10(6) cells in the 33 patients receiving a PI (t test, P = .02). These data demonstrate that highly active antiretroviral regimens have unequivalent effects on LNs and invite redefinition of suboptimal therapy at this level.

A pilot study of a combination of three reverse transcriptase inhibitors in HIV-1 infection.

The objective of this study was to evaluate the efficacy and tolerability of a combination of three reverse transcriptase inhibitors in patients with human immunodeficiency virus type 1 (HIV-1) infection. The investigation was an open pilot study of 48 weeks duration. Forty-five patients with CD4 cell counts between 50 and 500 cells/mm3 received a combination of oral zidovudine (200 mg three times daily) plus didanosine (200 mg twice daily) and lamivudine (150 mg twice daily). Plasma HIV-1 RNA and CD4 cell levels were measured weekly during the first month, at weeks 6, 8 and monthly thereafter. HIV-1 RNA levels were also measured sequentially in the lymph nodes of five patients after the initiation of therapy, and after several months of undetectable plasma RNA in 10 additional cases. Sequencing was performed on virus from the peripheral blood mononuclear cells of a subset of 14 patients after a mean period of 11+/-1 months on therapy. The mean (+/-SE) plasma viral load was 5.04+/-0.09 log10 copies/ml and the mean CD4 cell count was 339+/-14 cells/mm3 at baseline. Plasma HIV-1 RNA levels decreased exponentially in each case and became undetectable in 36 out of 42 cases who continued therapy for 24 weeks. HIV-1 RNA levels were < 20 copies/ml in 73% of these cases with undetectable HIV RNA. HIV-1 RNA decreased exponentially in lymph nodes after the initiation of therapy. The mean residual lymph node HIV-1 RNA level was 3.06+/-0.58 log10 copies/10(6) cells in 10 patients evaluated after several months of having undetectable plasma HIV RNA levels. A mean gain of 212 and 237 CD4 cells/mm3 was observed at 24 and 48 weeks, respectively. Proviral DNA sequencing showed that the main resistance codon mutations were absent in each case. Only one patient presented with a mutation resulting in the K219Q substitution, and one other with a T200I substitution. We conclude that this combination can achieve a significant decrease in HIV-1 replication in both plasma and lymph nodes in most cases. It is safe, able to delay the selection of resistant mutants, and keeps open the option for the use of protease inhibitors in case of therapeutic failure.