HIV-1 CRF63_02A6 models as a tool for evaluating efficacy of developing antiretroviral drugs

Highly active antiretroviral therapy (HAART) allows not only to control the infection process in certain patient, but also to reduce a risk of HIV infection spreading in general, so that one of the goals for international community fighting against HIV-spread is to maximize coverage of infected subjects with HAART. Antiretroviral therapy in HIV infection is administered lifelong, so that therapeutic efficacy may be lowered due to emergence of resistant HIV-1 variants. Currently, development of new antiretroviral drugs is currently underway throughout the world, therefore standard HIV-1 models are demanded to evaluate antiviral efficacy of promising drugs. To reliably assess drug efficiency regarding Russiawide HIV-1 variants, HIV-1 genovariants widespread in Russia should be used as a virus model. A recently emerged recombinant form of CRF63_02A6 HIV-1 is spread in Russia being currently a dominant variant detected among HIV-infected individuals in an extended region of the Siberian Federal District: in the Novosibirsk, Tomsk, Omsk, Kemerovo Regions, Krasnoyarsk and Altai Krai. We have obtained CRF63_02A6 infectious isolates of HIV-1, one of which contains mutations, reducing the sensitivity to the applied inhibitors of the virus reverse transcriptase. In addition, we constructed infectious molecular clones based on HIV-1 CRF63_02A6 variants with an affinity for CCR5 coreceptors and CXCR4. Infectious isolates and molecular clones CRF63_02A6 tested as models for assessing efficacy of antiretroviral drugs using the example of the drug “Efavirenz”. The fifty percent inhibitory concentration determined on the models of HIV-1 infectious molecular clones and HIV-1 isolate 18RU7056 ranged from 0.00027 pg/ml to 0.00046 pg/ml being in agreement with data published elsewhere. Concentrations of “Efavirenz” used in the study did not suppress the replication of HIV-1 12RU6987, which is resistant to non-nucleoside reverse transcriptase inhibitors, which confirms the decrease in the sensitivity of HIV-1 12RU6987 to “Efavirenz” by no less than 10,000 times. Thus, our data demonstrate that CRF63_02A6 HIV-1 isolated strains and infectious molecular clones are relevant and complementary tools for assessing efficacy of developing drugs aimed at suppressing HIV-1, including non-nucleoside-resistant virus reverse transcriptase inhibitors

Retarded PDI diffusion and a reductive shift in poise of the calcium depleted endoplasmic reticulum

Background: Endoplasmic reticulum (ER) lumenal protein thiol redox balance resists dramatic variation in unfolded protein load imposed by diverse physiological challenges including compromise in the key upstream oxidases. Lumenal calcium depletion, incurred during normal cell signaling, stands out as a notable exception to this resilience, promoting a rapid and reversible shift towards a more reducing poise. Calcium depletion induced ER redox alterations are relevant to physiological conditions associated with calcium signaling, such as the response of pancreatic cells to secretagogues and neuronal activity. The core components of the ER redox machinery are well characterized; however, the molecular basis for the calcium-depletion induced shift in redox balance is presently obscure. Results: In vitro, the core machinery for generating disulfides, consisting of ERO1 and the oxidizing protein disulfide isomerase, PDI1A, was indifferent to variation in calcium concentration within the physiological range. However, ER calcium depletion in vivo led to a selective 2.5-fold decline in PDI1A mobility, whereas the mobility of the reducing PDI family member, ERdj5 was unaffected. In vivo, fluorescence resonance energy transfer measurements revealed that declining PDI1A mobility correlated with formation of a complex with the abundant ER chaperone calreticulin, whose mobility was also inhibited by calcium depletion and the calcium depletion-mediated reductive shift was attenuated in cells lacking calreticulin. Measurements with purified proteins confirmed that the PDI1A-calreticulin complex dissociated as Ca2+ concentrations approached those normally found in the ER lumen ([Ca2+] K-0.5max = 190 mu M). Conclusions: Our findings suggest that selective sequestration of PDI1A in a calcium depletion-mediated complex with the abundant chaperone calreticulin attenuates the effective concentration of this major lumenal thiol oxidant, providing a plausible and simple mechanism for the observed shift in ER lumenal redox poise upon physiological calcium depletion.Wellcome Trust [Wellcome 084812/Z/08/Z]; European Commission (EU FP7 Beta-Bat) [277713]; Fundacao para a Ciencia e Tecnologia, Portugal [PTDC/QUI-BIQ/119677/2010]info:eu-repo/semantics/publishedVersio

Выявление Вич-1, резистентных к антиретровирусным препаратам, среди жителей томской области с впервые диагностированной Вич-инфекцией

The purpose of this paper is to evaluate the spreading of HIV-1 resistant to antiretroviral drugs among Tomsk Oblast population with newly diagnosed HIV-infection.Materials and methods. It was collected 122 clinical samples of  peripheral blood of HIV-infected patients from Tomsk Oblast who did not take antiretroviral drugs. In HIV-1 isolated from clinical samples we studied nucleotide sequence of genome fragments encoding virus protease and reverse transcriptase. Complex analyses of epidemiologic data from patients and the presence in genome HIV-1 mutations associated with resistance development to protease inhibitors and virus reverse transcriptase were carried out.Results. Analysis of HIV-1 isolated from Tomsk Oblast naïve HIV-infected population made it possible to detect HIV-1 mutations associated with a decrease of virus sensitivity to antiretroviral drugs in 9,8% of cases. Among described mutations 50% were associated with resistance to virus protease inhibitors; 33,3% were resistance mutations to nonnuclease inhibitors of reverse transcriptase, and 16.7% were resistance mutations to nucleoside inhibitors of virus reverse transcriptase. Out of 9,8% of resistant viruses 7,3% of casesincluded mutations associated with the development of potentially low level of reduction of HIV-1 sensitivity to drugs. Main HIV-1 resistance mutations of high and average levels were registered only in 2,5% of genotyped HIV-1 isolated from people who inject drugs.Conclusion. Current study detected considerably low sampling rate of HIV-1 carrying mutations associated with resistance to antiretroviral drugs among Tomsk Oblast naïve HIV-infected population. It is believed to be caused by a relatively short period of extensive application of antiretroviral therapy in that territory. Analysis of epidemiologic data resulted in detection of factors negatively affecting prediction of further development of HIV-infection epidemic in the region including prevalence of risk behavior practice contributing to resistant HIV-1 transmission both among patients via heterosexual contacts and among people who inject drugs.Цель: оценка распространения ВИЧ-1, резистентных к антиретровирусным препаратам, среди жителей Томской области с впервые выявленной ВИЧ-инфекцией.Материалы и методы. Собрано 122 клинических образца периферической крови ВИЧ-инфицированных жителей Томской области, не принимавших антиретровирусные препараты. Для выделенных из клинических образцов ВИЧ-1 исследована нуклеотидная последовательность фрагментов генома, кодирующих протеазу и обратную транскриптазу вируса. Выполнен комплексный анализ эпидемиологических данных пациентов и наличия в геноме ВИЧ-1 мутаций, связанных с развитием резистентности к ингибиторам протеазы и обратной транскриптазы вируса.Результаты. Анализ ВИЧ-1, выделенных от наивных ВИЧ-инфицированных лиц Томской области, в 9,8% случаев выявил мутации ВИЧ-1, ассоциированные со снижением чувствительности вируса к антиретровирусным препаратам. Среди описанных мутаций 50%составляли мутации, связанные с резистентностью к ингибиторам протеазы вируса; в 33,3% – мутации резистентности к ненуклеозидным ингибиторам обратной транскриптазы и в 16,7% – к нуклеозидным ингибиторам обратной транскриптазы вируса. Из 9,8% резистентных вирусов в 7,3% случаев были выявлены мутации, ассоциированные с развитием потенциального/потенциально-низкого/низкого уровня снижения чувствительности ВИЧ-1 к препаратам. Основные мутации резистентности ВИЧ-1 высокого и среднего уровня были зарегистрированы лишь в 2,5% генотипированных ВИЧ-1, выделенных от лиц, потребляющих инъекционные наркотические препараты.Заключение. Выполненное исследование выявило среди наивных ВИЧ-инфицированных жителей Томской области достаточно низкую частоту регистрации ВИЧ-1, несущих мутации, ассоциированные с резистентностью к антиретровирусным препаратам. Вероятно это связано с относительно коротким периодом широкого применения в области  антиретровирусной терапии. Анализ эпидемиологических данных выявил факторы, негативно влияющие на прогноз дальнейшего развития эпидемии ВИЧ-инфекции в регионе – широкую распространенность практик рискованного поведения, способствующих передаче резистентных ВИЧ-1 как среди лиц, инфицированных при гетеросексуальных контактах, так и среди потребителей инъекционных наркотиков

Detection of HIV-1 resistant to antiretroviral drugs among tomsk oblast population with newly diagnosed HIV-infection

The purpose of this paper is to evaluate the spreading of HIV-1 resistant to antiretroviral drugs among Tomsk Oblast population with newly diagnosed HIV-infection.Materials and methods. It was collected 122 clinical samples of  peripheral blood of HIV-infected patients from Tomsk Oblast who did not take antiretroviral drugs. In HIV-1 isolated from clinical samples we studied nucleotide sequence of genome fragments encoding virus protease and reverse transcriptase. Complex analyses of epidemiologic data from patients and the presence in genome HIV-1 mutations associated with resistance development to protease inhibitors and virus reverse transcriptase were carried out.Results. Analysis of HIV-1 isolated from Tomsk Oblast naïve HIV-infected population made it possible to detect HIV-1 mutations associated with a decrease of virus sensitivity to antiretroviral drugs in 9,8% of cases. Among described mutations 50% were associated with resistance to virus protease inhibitors; 33,3% were resistance mutations to nonnuclease inhibitors of reverse transcriptase, and 16.7% were resistance mutations to nucleoside inhibitors of virus reverse transcriptase. Out of 9,8% of resistant viruses 7,3% of casesincluded mutations associated with the development of potentially low level of reduction of HIV-1 sensitivity to drugs. Main HIV-1 resistance mutations of high and average levels were registered only in 2,5% of genotyped HIV-1 isolated from people who inject drugs.Conclusion. Current study detected considerably low sampling rate of HIV-1 carrying mutations associated with resistance to antiretroviral drugs among Tomsk Oblast naïve HIV-infected population. It is believed to be caused by a relatively short period of extensive application of antiretroviral therapy in that territory. Analysis of epidemiologic data resulted in detection of factors negatively affecting prediction of further development of HIV-infection epidemic in the region including prevalence of risk behavior practice contributing to resistant HIV-1 transmission both among patients via heterosexual contacts and among people who inject drugs