Peptides derived from a distinct region of GB virus C glycoprotein E2 mediate strain-specific HIV-1 entry inhibition.

The nonpathogenic human GB virus C (GBV-C), a member of the Flaviviridae, is highly prevalent in individuals with HIV-1 infections or with parenteral and sexual risk factors. Long-term GBV-C viremia has been associated with better survival or improved diagnosis in several epidemiological studies. In a previous study we reported that the E2 glycoprotein of GBV-C interferes with HIV-1 entry in vitro. To address the question what region of the E2 protein is involved in suppression of HIV-1 replication, we performed an E2-derived peptide scanning and determined the HIV-inhibitory activity of each peptide in HIV replication assays. We demonstrate here that peptides representing the N-terminal part of the E2 protein from amino acids (aa) 29 to 72 are able to inhibit efficiently HIV-1 replication in vitro. In particular, the peptides P6-2 (representing the E2-region from aa 45 to 64) and P4762 (aa 37 to 64) showed the highest potency in HIV replication assays performed on TZM-bl cells with 50% inhibitory concentrations between 0.1 and 2 µM. However, primary HIV-1 isolates representing clades A to H showed a high variability in their sensitivity to E2 peptides. Pseudovirus inhibition assays revealed that the sensitivity is determined by the gp120/gp41 envelope proteins. Using HIV-1 BlaM-Vpr-based fusion assays, we demonstrate that the E2-derived peptides prevent HIV-1 binding or fusion, presumably via interaction with the HIV-1 particle. Together, these findings reveal a new mechanism of viral interference, suggesting that the envelope protein E2 of GBV-C target directly HIV-1 particles to avoid entry of these virions.

Clinical validation of a new triplex real-time polymerase chain reaction assay for the detection and discrimination of Herpes simplex virus types 1 and 2.

A new triplex real-time polymerase chain reaction (PCR) assay for Herpes simplex virus (HSV) (artus HSV-1/2 TM PCR kit, QIAGEN) was evaluated. This assay simultaneously uses three differently labeled probes targeted to HSV-1 (FAM), HSV-2 (NED), and to the manufacturer's Internal Control (VIC). HSV-1/2 typing capability and quantitation accuracy were determined using HSV stocks and quality control panels. Performance in routine clinical testing was compared with a nested HSV-1/2 PCR assay. Dilution series and quality control panel testing revealed an approximately 10-fold higher HSV-2 sensitivity in real-time PCR compared with an in-house nested PCR assay. The sensitivity for HSV-1 was comparable in both assays. All HSV-positive proficiency panel samples (n = 21) and virus stocks were typed correctly as HSV-1 or HSV-2 using real-time PCR. Quantitation correlated well with reference values (HSV-1, r = 0.98; HSV-2, r = 0.88), and 95% detection limits were determined as 9.4 HSV-2 copies/reaction and 18 HSV-1 copies/reaction. Based on C(t) values, the mean intra-assay coefficient of variation was 1%, whereas the interassay coefficient of variations were 2.7% and 2.5% for HSV-1 and -2, respectively. Testing of 309 clinical samples resulted in 100% specificity and 97% sensitivity. In conclusion, the artus HSV-1/2 TM PCR kit represents an excellent tool for the detection and differentiation of HSV-1 and -2 in clinical samples.

HIV entry inhibition by the envelope 2 glycoprotein of GB virus C.

Epidemiological studies have revealed an association between GB virus C (GBV-C) long-term viraemia and ameliorated HIV disease progression. We have provided evidence that a single protein of GBV-C, the glycoprotein E2, interferes with early HIV replication steps of both X4- and R5-tropic HIV strains. Preincubation with anti-E2 antibody specifically abrogates the inhibitory effect. Results were confirmed by the in-vitro expression of GBV-C E1/E2 encoding RNA.

Inhibition of HIV strains by GB virus C in cell culture can be mediated by CD4 and CD8 T-lymphocyte derived soluble factors.

OBJECTIVE: A number of studies concerning the pathogenesis of GB virus C (GBV-C) in HIV-infected people suggest a beneficial effect and improved survival for dually infected individuals. However there has remained controversy regarding the clinical relevance of these findings, as some studies have not confirmed these observations. To address the possibility of direct inhibitory mechanisms, we studied the impact of GBV-C on HIV-1 replication in vitro. METHODS: Peripheral blood mononuclear cells (PBMC) were infected with sera from GBV-C positive individuals or transfected with GBV-C specific RNA and superinfected with HIV. Replication kinetics of HIV were studied by quantification of HIV-p24 release. Induction of soluble antiretroviral factors were monitored with an HIV infection assay and by quantification of chemokine secretion. Changes in chemokine receptor expression were analysed by flow cytometry. RESULTS: We demonstrate that GBV-C infection of PBMC leads to significant replication inhibition of R5- and X4-HIV isolates representing eight HIV clades. The inhibitory effect is mediated by GBV-C infection and also by expression of GBV-C structural glycoproteins and/or of non-structural proteins NS2/NS3. Upon GBV-C infection CD4 and CD8 T lymphocytes produce soluble HIV-suppression factors. Induction of stromal cell-derived factor (SDF)-1 and subsequent internalization of CXCR4 was not observed. CONCLUSIONS: CD4 and CD8 T lymphocytes are stimulated by GBV-C to secrete antiretroviral factors, inhibiting R5- and X4-HIV strains. As no induction of SDF-1 and no down-regulation of the respective receptor CXCR4 could be observed, it is likely that additional unidentified factors causing inhibition of X4-HIV strains are induced by GBV-C.

HIV-1 fusion is blocked through binding of GB Virus C E2-derived peptides to the HIV-1 gp41 disulfide loop [corrected].

A strategy for antiviral drug discovery is the elucidation and imitation of viral interference mechanisms. HIV-1 patients benefit from a coinfection with GB Virus C (GBV-C), since HIV-positive individuals with long-term GBV-C viraemia show better survival rates than HIV-1 patients without persisting GBV-C. A direct influence of GBV-C on HIV-1 replication has been shown in coinfection experiments. GBV-C is a human non-pathogenic member of the flaviviridae family that can replicate in T and B cells. Therefore, GBV-C shares partly the same ecological niche with HIV-1. In earlier work we have demonstrated that recombinant glycoprotein E2 of GBV-C and peptides derived from the E2 N-terminus interfere with HIV entry. In this study we investigated the underlying mechanism. Performing a virus-cell fusion assay and temperature-arrested HIV-infection kinetics, we provide evidence that the HIV-inhibitory E2 peptides interfere with late HIV-1 entry steps after the engagement of gp120 with CD4 receptor and coreceptor. Binding and competition experiments revealed that the N-terminal E2 peptides bind to the disulfide loop region of HIV-1 transmembrane protein gp41. In conjunction with computational analyses, we identified sequence similarities between the N-termini of GBV-C E2 and the HIV-1 glycoprotein gp120. This similarity appears to enable the GBV-C E2 N-terminus to interact with the HIV-1 gp41 disulfide loop, a crucial domain involved in the gp120-gp41 interface. Furthermore, the results of the present study provide initial proof of concept that peptides targeted to the gp41 disulfide loop are able to inhibit HIV fusion and should inspire the development of this new class of HIV-1 entry inhibitors.

Quality control trial for human immunodeficiency virus type 1 drug resistance testing using clinical samples reveals problems with detecting minority species and interpretation of test results.

Between January and March 2000, a quality control panel for human immunodeficiency virus (HIV) drug resistance testing was analyzed by 20 laboratories in five countries. The panel consisted of three clinical samples with different drug resistance genotypes and phenotypes and one HIV-negative plasma. Participants were asked to report the methods used for amplification and sequencing, a list of drug resistance-associated mutations that were detected in the protease and reverse transcriptase of each sample, and an interpretation concerning the susceptibility or resistance to 14 antiretroviral drugs. A total of 22 genotypic data sets were generated, which showed an overall good technical quality except for three participants, who failed to report key mutations for drug resistance. Problems were encountered in three respects: (i). resistant minorities of L90M in the protease, which were determined to about 12% by real-time amplification, were only detected by one-fourth of the participants; (ii). newly described resistance mutations were frequently not reported; and (iii). interpretations of drug resistance-associated mutations varied widely, in particular for protease inhibitors. In some cases, different interpretations were caused by differences in the detection of resistant minorities, but even for the same genotypic profile, interpretations varied considerably. Similar discrepancies were revealed if current Web-based interpretation systems were used to predict drug resistance for samples of the proficiency panel. This indicates that a consensus for the interpretation of drug resistance-associated mutations is urgently needed.