Inhibition of porcine endogenous retrovirus in PK15 cell line by efficient multitargeting RNA interference.

To effectively suppress porcine endogenous retroviruses (PERV)s, RNAi technique was utilized. RNAi is the up-to-date skill for gene knockdown which simultaneously multitargets both gag and pol genes critical for replication of PERVs. Previously, two of the most effective siRNAs (gag2, pol2) were found to reduce the expression of PERVs. Concurrent treatment of these two siRNAs (gag2+pol2) showed knockdown efficiency of up to 88% compared to negative control. However, despite the high initial knockdown efficiency 48 h after transfection caused by siRNA, it may only be a transient effect of suppressing PERVs. The multitargeting vector was designed, containing both gag and pol genes and making use of POL II miR Expression Vector, which allowed for persistent and multiple targeting. This is the latest shRNA system technique expressing and targeting like miRNA. Through antibiotics resistance characteristics utilizing this vector, miRNA-transfected PK15 cells (gag2-pol2) were selected during 10 days. An 88.1% reduction in the level of mRNA expression was found. In addition, we performed RT-activity analysis and fluorescence in situ hybridization assay, and it demonstrated the highest knockdown efficiency in multitargeting (gag2+pol2) miRNA group. Therefore, according to the results above, gene knockdown system (siRNA and shRNA) through multitargeting strategy could effectively inhibit PERVs.

Scale-up of antiretroviral treatment in sub-Saharan Africa is accompanied by increasing HIV-1 drug resistance mutations in drug-naive patients.

OBJECTIVES: To evaluate the frequency and progression over time of the WHO-defined transmitted HIV-1 drug resistance mutations (DRMs) among antiretroviral treatment (ART)-naive HIV-1-infected patients in Cameroon. DESIGN: We analyzed HIV-1 DRM data generated from 369 ART-naive individuals consecutively recruited between 1996 and 2007 in urban and rural areas in Cameroon. METHODS: HIV-1 drug resistance genotyping was performed in the pol gene using plasma samples and surveillance DRMs were identified using the 2009 WHO-DRM list. RESULTS: We observed in Yaounde, the capital city, an increasing prevalence of DRMs over time: 0.0% (none of 61 participants) in 1996-1999; 1.9% (one of 53 participants) in 2001; 4.1% (two of 49 participants) in 2002; and 12.3% (10 of 81 participants) in 2007. In the rural areas with more recently implemented ART programs, we found DRMs in six of 125 (4.8%) ART-naive individuals recruited in 2006-2007. DRMs identified in both areas included resistance mutations to protease inhibitors, nucleoside reverse transcriptase inhibitors (NRTIs) and non-NRTIs (NNRTIs) that might impair the efficacy of available first-line and second-line treatments. CONCLUSION: This report showed an increase in transmitted DRMs in areas where antiretroviral drugs were introduced earlier, although other factors such as natural viral polymorphisms and acquired DRMs through exposure to antiretroviral cannot be totally excluded. Further surveillances are needed to confirm this evolution and inform public health policies on adequate actions to help limit the selection and transmission of drug-resistant HIV, while scaling up access to ART in developing countries.

Adherence and genotypic drug resistance mutations in HIV-1-infected patients failing current antiretroviral therapy.

The aim of this study was to assess the correlation between the estimated adherence of HIV-1 infected patients with antiretroviral (ARV) therapy failure and drug-resistant mutations. We studied 40 patients with virological and immunological ARV-therapy failure. In order to assess the adherence of patients we used the SERAD questionnaire. Genomic sequencing of the HIV-1 pol gene was performed. 100% adherence was reported by 27 patients (67.5%) (adherent patients). Multivariate analysis showed that only baseline and nadir CD4+ counts maintained a significant correlation with the adherence. For PR and NNRTI mutations, we did not find any difference between the two groups of patients. Baseline NRTI mutations were higher in adherent patients than in non-adherent patients (p<0.05). No differences were found between plasma mutations and PBMC mutations. The authors conclude that genotypic resistance mutations were found in the majority of patients with ARV-therapy failure despite a good self-reported adherence to therapy. Adequate adherence to therapy is not the only key factor in viral suppression.

Transmitted antiretroviral drug resistance among newly HIV-1 diagnosed young individuals in Kampala.

OBJECTIVE: To assess the emergence of transmitted HIV-1 drug resistance (TDR) in Kampala, Uganda, 10 years after the scale-up of antiretroviral treatment (ART) and to compare with a previous survey among antenatal clinic attendees in 2007 (reporting 0% TDR). DESIGN: A cross-sectional survey was conducted among newly HIV-1 diagnosed, antiretroviral-naive young adults attending two large voluntary counseling and testing centers within the geographic area of Kampala. METHODS: Proxy criteria for recent HIV-1 infection were used as defined by the WHO. Population sequencing of the pol gene was performed on plasma samples with HIV-1 RNA at least 1000 copies/ml. Surveillance drug resistance mutations (SDRMs) were identified according to the 2009 WHO list for surveillance of TDR. HIV-1 subtypes were designated using maximum likelihood phylogenetic reconstruction. RESULTS: : Genotypic test results were obtained for 70 of 77 (90.9%) participants. SDRMs were identified in six samples yielding a prevalence of TDR of 8.6% (95% confidence interval 3.2-17.7%). Two had SDRMs to nucleoside reverse-transcriptase inhibitors (D67G and L210W), three had SDRMs to nonnucleoside reverse transcriptase inhibitors (G190A, G190S, and K101E), and one had SDRMs to protease inhibitors (N88D). Frequencies of HIV-1 subtypes were A (36/70, 51.4%), C ( two of 70; 2.9%), D (23/70, 32.9%), and unique recombinant forms (nine of 70, 12.9%). CONCLUSION: This repeated survey suggests an increase in TDR in Kampala, compared with a previous survey. This finding justifies increased vigilance with respect to surveillance of TDR in areas in Africa where ART programs are rolled-out.

Production of HIV-1 integrase fusion protein-carrying lentiviral vectors for gene therapy and protein transduction.

Lentiviral vectors have broad target cell tropism and efficient machinery to integrate transgenes into the host genome. Modification of these vectors by incorporating heterologous proteins into virions has relied mostly on the fusion of proteins into the HIV-1 accessory protein Vpr. Vpr expression can be harmful for cells and its gene has been deleted from third-generation vector production plasmids. We therefore developed a direct integrase fusion protein strategy as an alternative way to package heterologous proteins into vectors. The method was tested by creating two different integrase fusion proteins, IN-p53 and IN-mCherry, cloned into the 3' end of pol in the packaging plasmid. Lentiviral vectors were produced by conventional methods, using the modified packaging plasmids. Vector-incorporated fusion proteins were correctly processed from Gag-Pol, retained the ability to catalyze transgene integration, and showed fusion protein-specific activity by being fluorescent or inducing apoptosis. Functional third-generation lentiviral vectors containing IN-fusion proteins can thus be produced by standard production protocols independent of Vpr expression. Our results suggest that this packaging method is useful for lentiviral vector-mediated protein transduction, such as intranuclear meganuclease, transposon, or zinc finger protein delivery, intracellular imaging of vector particles, and generation of modified lentiviral vectors that contain both toxic and nontoxic IN-fusion proteins.

HIV type 1 subtype diversity and drug resistance among HIV type 1-infected Kenyan patients initiating antiretroviral therapy.

The treatment of HIV-1 infection with antiretroviral drugs has greatly improved the survival of those who are infected. However, HIV-1 diversity and drug resistance are major challenges in patient management, especially in resource-poor countries. To evaluate HIV-1 genetic diversity and drug resistance-associated mutations among drug-naive patients in Kenya prior to antiretroviral therapy (ART), a genetic analysis of HIV-1 pol-RT and env-gp41 was performed on samples collected from 53 (18 males and 35 females) consenting patients between April and June 2005. The average age, baseline CD4(+) T cell counts, and viral loads were 38 (range, 24-62) years, 475 (range, 203-799) cells/mm(3), and 4.7 (range, 3.4-5.9) log(10) copies/ml, respectively. Phylogenetic analysis revealed that 40 samples (75.5%) were concordant subtypes for the two genes and 13 (24.5%) were discordant, suggesting possible recombination and/or dual infections. Prevalent subtypes included A1/A1(pol-RT/env-gp41), 31 (58.5%); D/D, 9 (16.9%); A1/C, 2 (3.8%); A1/D, 4 (7.5%); G/A1, 2 (3.8%); A1/A2, 1 (1.9%); C/A1, 2 (3.8%); D/A1, 1(1.9%); and D/A2, 1 (1.9%). Major reverse transcriptase inhibitor (RTI) resistance-associated mutations were found in four patients (7.5%). Of these patients, three had nucleoside RTI resistance mutations, such as M184V, K65R, D67N, K70R, and K219Q. Nonnucleoside RTI resistance-associated mutations K103N and Y181C were detected in three patients and one patient, respectively. Multiple drug resistance mutations were observed in this drug-naive population. With increasing numbers of patients that require treatment and the rapid upscaling of ART in Kenya, HIV-1 drug resistance testing is recommended before starting treatment in order to achieve better clinical outcomes.

Reappearance of an 11-year-old sequence in an HIV-1 infected patient during treatment interruption.

HIV-1 from a patient with multi-drug resistant virus was identified as wild type during treatment interruption. The aim of the study was to describe how the viral population is affected by treatment interruptions and use phylogeny to reconstruct the evolutionary pattern. 15 samples covering 13 y and 2 treatment interruptions were analysed in both pol and env. The wild type virus found in the sample from the second treatment interruption in 2002 had not been present as a dominant population since 1994. Phylogeny showed that the 2002 sample was more closely related to wild type sequences than to other sequences sampled in 2002. This indicated that the wild type virus was caused by recruitment from the viral archives rather than reversion of previously circulating resistant strains. A few weeks after re-initiated treatment, virus showed full resistance, indicating that resistant virus was present as a subpopulation and reselected due to higher fitness in the presence of drugs. Phylogeny of env showed that CCR5 and CXCR4 viruses coexist in the patient. In conclusion, the study showed that at all times during infection, virus is archived in the cells and can be recruited when the surrounding environment changes and the archived virus is more fit.

Ca2+-induced p38/SAPK signalling inhibited by the immunosuppressant cyclosporin A in human peripheral blood mononuclear cells.

To understand the effects of the immunosuppressant cyclosporin A (CsA) on Ca2+-mediated intracellular signalling pathways in human peripheral blood mononuclear cells (PBMCs), we investigated its effects on the activity profiles of mitogen-activated protein kinase (MAPK) cascades. PBMCs, or subpopulations thereof, were simultaneously stimulated with a phorbol ester and the calcium ionophore ionomycin, in the presence or absence of therapeutic concentrations of CsA. In these primary human cells, CsA significantly inhibited PMA/ionomycin-mediated and ionomycin-mediated activation of the MAPK kinase MKK6, as well as its downstream kinases SAPK2a (p38alpha) and MAPKAP-K2. PMA/ionomycin treatment also mediated activation of SAPK1 (JNKs) which was inhibited by CsA. Treatment with ionomycin alone also resulted in CsA-sensitive activation of SAPK1. With regard to transcription factors targeted by the Ca2+-induced MAPK signalling network, we found CsA to inhibit the ionomycin-mediated phosphorylation of ATF2 at Thr71. We identified the heterodimeric transcription factor ATF2/CREB as constitutively binding to the essential cAMP response element (CRE) site within the Ca2+-regulated DNA polymerase beta promoter and contributing to the activation of this promoter. Our data implicate ATF2 phosphorylation status as a nuclear sensor within PBMCs that monitors converging intracellular Ca2+-signalling pathways.

Characterization of rhesus cytomegalovirus genes associated with anti-viral susceptibility.

Studies were initiated to determine whether rhesus cytomegalovirus (RhCMV)-infected macaques could serve as an animal model for evaluating anti-CMV compounds, as macaques have a naturally occurring CMV that is similar to human CMV (HCMV). Utilizing plaque reduction assays, RhCMV was tested to anti-viral susceptibility. By these assays. RhCMV displayed anti-viral susceptibility to ganciclovir at a 50% effective dose (ED50) of 0.8 microM, acyclovir at an ED50 of 15 microM, and foscarnet at an ED50 of 250 microM. By Southern blot analysis with HCMV-UL97 (phosphotransferase) and DNA polymerase (pol) genes as probes, we isolated viral DNA fragments that strongly hybridized. DNA sequence analysis of these DNA fragments revealed two open reading frames with homology to HCMV UL97 and DNA polymerase. Steady-state RNA analysis revealed that the RhCMV UL97 homologue and pol genes are transcribed as early late and early genes, respectively. Comparison against HCMV showed the RhCMV UL97 homologue exhibits 54.4% amino acid (aa) sequence identity to HCMV UL97 and the RhCMV DNA polymerase 59.2% aa sequence identity to HCMV DNA polymerase. Results from anti-viral assays and molecular characterization of these two viral genes suggest that RhCMV-infected rhesus macaques should serve as an excellent animal model for evaluating future anti-CMV compounds.

Use of PCR to measure HIV viral changes in drug-resistant genes in genital fluids.

Our group has been studying HIV in genital fluids for several years. Our first findings demonstrated that both RNA PCR and proviral DNA PCR could be utilized to quantify virus within seminal interstitial fluid and non-spermatozoa mononuclear cells, respectively. It became clear that these techniques were more sensitive than viral culture and we also reported that both disease progression and treatment influenced sperm viral concentrations generally parallel to virus concentrations in blood. A more recent study from our group has demonstrated that mutations seen in the virus in the blood are seen in the seminal plasma viral as well as in the proviral forms within non-spermatozoa cells. We have also studied female patients for 2-month periods looking at RNA in plasma and cervical secretions, as well as proviral DNA in cervical and vaginal samples by polymerase chain reaction (PCR) amplification techniques. The HIV RNA levels again appears to be the most sensitive and well-related to systemic viral load. Thus, genital secretion of cell-free virus and cells containing proviral DNA in both sexes parallels systemic virus levels, is a site for measurement of transmission of drug-resistant virus and should be monitored in therapy as well as in pathogenesis studies.

Identification of drug-related genotypic changes in HIV-1 from serum using the selective polymerase chain reaction.

We attempted to detect drug-related HIV-1 pol gene mutations by selective polymerase chain reaction (PCR) using both proviral DNA and viral RNA isolated from patients (pts) with AIDS or ARC receiving antiretroviral therapy. Peripheral blood mononuclear cell (PBM)-associated proviral DNA and serum-derived viral RNA were obtained from eight patients before and after receiving an alternating regimen of AZT and ddC for 15-41 months or ddI monotherapy for 12-26 months. These specimens were examined for the presence of mutations at positions 70, 74, 215 and 219. We noted that selective PCR results can be ambiguous depending on the quantity of DNA template employed. We, therefore, used the minimal quantity of DNA templates that yielded evaluable PCR products in this study. For all the eight pairs of pre- and post-therapy proviral DNA samples, selective PCR results agreed with independently determined nucleotide sequences. Results of reverse transcription of serum-derived viral RNA followed by selective PCR differed in some cases from those using the proviral DNA. In particular, the use of serum viral RNA appeared to allow earlier detection of changes in drug-related mutations than the use of PBM-associated proviral DNA. We conclude that (i) selective PCR using the minimum and sufficient number of PBM-associated proviral DNA and serum viral RNA copies successfully detects the presence of known pol gene mutations; (ii) drug-related mutations may be distinguished earlier in virions in serum (or plasma) than in proviral DNA in PBM; and (iii) quantification of HIV-1 prior to selective PCR may be an important component in monitoring the therapy of HIV-1 infection.

Combination therapy with zidovudine and didanosine selects for drug-resistant human immunodeficiency virus type 1 strains with unique patterns of pol gene mutations.

Drug resistance conferred by specific human immunodeficiency virus type 1 (HIV-1) pol gene mutations has been associated with clinical progression in HIV-infected patients receiving anti-retroviral therapy. This study examined drug susceptibilities and pol mutations of HIV-1 strains from patients treated for 1 year with zidovudine, didanosine (ddI), or zidovudine and ddI. Ten (42%) of 24 patients receiving combination therapy versus 8/26 (31%) receiving only zidovudine had HIV-1 strains with phenotypic zidovudine resistance or a zidovudine resistance pol mutation at codon 215 (P = .6). In contrast, a ddI resistance mutation at codon 74 was less common among patients receiving combination therapy (2/24) than among those receiving ddI only (17/26; P < .001). Two patients receiving combination therapy developed resistance to zidovudine and ddI; they had HIV strains with amino acid mutations at codons 62, 75, 77, 116, and 151. Combination therapy with zidovudine and ddI selects for zidovudine-resistant HIV-1 strains lacking a ddI resistance mutation and for multidrug-resistant strains containing novel pol mutations.

Zidovudine susceptibility testing of human immunodeficiency virus type 1 (HIV) clinical isolates.

Traditional antiviral susceptibility testing methods using cell lines can be applied to no more than about 30% of clinical HIV isolates (Larder et al., 1989a; Fenyo et al., 1989). We tested the cell-free supernatant from low passage clinical HIV isolates using donor peripheral blood mononuclear cells (PBMC). Drug susceptibility was assessed by measuring the effect of increasing zidovudine (ZDV) concentrations on HIV P24 antigen production. Susceptibility results were obtained on 24/27 consecutive clinical isolates and 6/6 laboratory isolates. The mean IC90 of isolates from untreated patients was 0.008 microM ZDV (range: 0.002-0.038). The IC90s of isolates from ZDV-treated patients ranged from 0.007 to greater than 10 microM ZDV. All isolates with an IC90 < 0.1 microM ZDV had a wild type sequence at codon 215 of the HIV pol gene; 11/12 isolates with an IC90 > 0.1 microM ZDV had a mutation at codon 215 (P < 0.001). Among 16 ZDV-treated patients, there was a modest correlation between the change in CD4 count from the start of ZDV treatment and the IC90 of the patient's isolate following treatment (r = 0.51). Susceptibility testing using donor PBMC can be a sensitive means of testing a broad range of clinical HIV isolates.

A mutation in human immunodeficiency virus reverse transcriptase and decline in CD4 lymphocyte numbers in long-term zidovudine recipients.

A nested polymerase chain reaction assay was used to define the sequence of a specific codon, amino acid 215, of the human immunodeficiency virus (HIV) pol gene in DNA from peripheral blood mononuclear cells (PBMC) and viral RNA from serum from 38 patients treated with zidovudine for > or = 2 years. After treatment for a mean of 34 months, 17 patients with sequences with a codon 215 mutation had a mean 50% decrease in CD4 cells, compared with 21 patients with sequences wild-type at codon 215, who had a mean 11% increase in CD4 cells (P < .0001). Patients with a mutation at 215 had a ninefold higher provirus burden in PBMC. Detection of the codon 215 mutation in plasma viral RNA preceded detection of the mutation in DNA from PBMC and decline in CD4 cells. The appearance of a mutation at codon 215 in the HIV reverse transcriptase gene in patients receiving zidovudine may be a marker for impending immunologic decline.

Human immunodeficiency virus type 1 pol gene mutations which cause decreased susceptibility to 2',3'-dideoxycytidine.

To investigate whether human immunodeficiency virus type 1 pol gene mutations are selected during prolonged 2',3'-dideoxycytidine (ddC) therapy, we used the polymerase chain reaction to amplify a portion of the reverse transcriptase segment of the pol gene from the peripheral blood mononuclear cell DNA of a patient with AIDS before and after an 80-week course of ddC therapy. The consensus sequence from the second sample contained a unique double mutation (ACT to GAT) in the codon for reverse transcriptase amino acid 69, causing substitution of aspartic acid (Asp) for the wild-type threonine (Thr). A mutation (ACA to ATA) also occurred in the codon for position 165, causing substitution of isoleucine (Ile) for Thr. The GAT (Asp) codon was introduced into the pol gene of a molecular clone of human immunodeficiency virus via site-directed mutagenesis. Following transfection, mutant and wild-type viruses were tested for susceptibility to ddC by a plaque reduction assay. The mutant virus was fivefold less susceptible to ddC than the wild type; cross-resistance to 3'-azido-3'-deoxythymidine or 2'3'-dideoxyinosine was not found. The Ile-165 mutation did not confer additional ddC resistance. The Asp-69 substitution may have contributed to the generation of resistant virus in this patient.

A comparison of gag, pol and rev antisense oligodeoxynucleotides as inhibitors of HIV-1.

Sequences from the gag, pol and rev regions of the RF strain of HIV-1 (HIV-1RF) were chosen as targets for antisense phosphorothioate oligodeoxynucleotides (S-oligos). These sequences were the p18/p24 junction in gag, the active site of HIV protease in pol; a sequence from the first exon of the rev gene and S-oligodeoxycytidylic acid controls. Compounds were tested against HIV-1 in both acutely and chronically infected cells. The results show that these phosphorothioate analogues tested in acutely infected cells were active in the 0.1-2 microM range, were dependent on chain length but had no sequence specificity. To study the mechanism of action, the time of addition of S-oligos to acutely infected cells was delayed for up to 48 h post-infection. It was found that antiviral activity was lost when compounds were added to the cultures later than 10 h post-infection. With chronically infected cells only the antisense rev sequence showed activity at 30 microM and neither of the gag or pol antisense sequences has a significant effect on HIV replication at 50 microM. These results are consistent with previous in vitro studies which demonstrate that antisense S-oligodeoxynucleotides have several modes of action.

Effect of busulphan treatment and elevated temperature on the expression of the beta-pol gene in rat testis.

Changes in the expression pattern of the DNA polymerase beta gene during inhibition of spermatogenesis by busulphan and by temperature (artificial cryptorchidism) have been studied. Transient arrest of spermatogenesis in two-month-old rats after injection of a single dose of busulphan (10 mg/kg) resulted in parallel but transient decrease in the 1.4 kb of beta-pol mRNA level to an undetectable value, followed by its reappearance after resumption of spermatogenesis. An artificial cryptorchidism also caused a drastic decrease of beta-pol mRNA level. Both results as well as morphological examination of testis after busulphan injection and artificial cryptorchidism revealed that spermatocytes and spermatids represent the testicular cell fraction containing the elevated amount of beta-pol mRNA. Involvement of DNA polymerase beta in meiotic recombination is discussed.