[Analysis of HIV-1 (Human immunodeficiency virus-1, Lentivirus, Orthoretrovirinae, Retroviridae) Nef protein polymorphism of variants circulating in the former USSR countries.]

INTRODUCTION: The human immunodeficiency virus (HIV) Nef protein is one of the key factors determining the infectivity and replicative properties of HIV. With the ability to interact with numerous proteins of the host cell, this protein provides the maximum level of virus production and protects it from the immune system. The main activities of Nef are associated with a decrease in the expression of the CD4 receptor and major histocompatibility complex class I molecules (MHC-I), as well as the rearrangement of the cytoskeleton. These properties of the protein are determined by the structure of several motifs in the structure of the nef gene encoding it, which is quite variable. OBJECTIVES: The main goal of the work was to analyze the characteristics of Nef protein of HIV-1 variant A6, which dominates in the countries of the former USSR. The objective of the work was a comparative analysis of natural polymorphisms in the nef gene of HIV-1 sub-subtypes A6 and A1 and subtype B. MATERIAL AND METHODS: The sequences of the HIV-1 genome obtained during the previous work of the laboratory were used, as well as the reference sequence from GenBank. In this work, Sanger sequencing and new generation sequencing methods, as well as bioinformation analysis methods were used. RESULTS AND DISCUSSION: The existence of noticeable differences in the prevalence of Nef natural polymorphisms (A32P, E38D, I43V, A54D, Q104K, H116N, Y120F, Y143F, V168M, H192T, V194R, R35Q, D108E, Y135F, E155K, E182M, R184K and F191L), some of which are characteristic mutations for variant A6, was shown. CONCLUSION: Characteristic substitutions were found in the Nef structure, potentially capable of weakening the replicative properties of HIV-1 variant A6.

Evaluating the accuracy and sensitivity of detecting minority HIV-1 populations by Illumina next-generation sequencing.

The accuracy and sensitivity of deep sequencing were assessed using viral standards (pNL4-3 and pLAI.2) of both DNA and RNA. The sequencing accuracy did not depend on the type of nucleic acid, but critically depended on the number of reads and threshold of sensitivity to minor viral populations. With coverage of more than 236 reads, the accuracy of viral RNA sequencing was equal to or exceeded 99.9%, with a sensitivity threshold to minor nucleotides of 20%. When the sensitivity threshold was below 1%, reduced accuracy dynamics were clearly visible even when the coverage was massive (more than 9.000 reads). It was found that the floating sensitivity threshold allowed the sequencing accuracy to be maintained at an acceptable level in cases of low coverage (less than 1.500-2.000) of reads. These results indicate the quality that can be expected with a specific number of reads and sensitivity threshold. Deep sequencing is a very powerful tool that can significantly improve the value of study results, but despite its superior performance, it should be used with caution regarding its sensitivity to minor populations below 1%.

[EFFECT OF ANTI-CANCER DRUG DOXORUBICINE ON CYTOMEGALOVIRUS INFECTED HUMAN FIBROBLASTS].

The anticancer antibiotic doxorubicine (DOX) is highly toxic and induces functional complications in vital organs. The effect of DOX on normal cells has not been examined in sufficient detail, and the search for compounds reducing DOX toxicity did not lead to success so far. It has been suggested that DOX induces death of cancer cells via p53-dependent apoptosis, however, the information regarding the role of p73 protein, a member of p53 tumor suppressor family, is scanty. Cytomegalovirus (CMV) induces an antiapoptosis program that allows its replication until death of the target cell. Our objectives were to examine the effect of DOX on normal cells (human fibroblasts), analyze the ability of CMV-induced antiapoptosis program to reduce DOX toxicity, and to evaluate the involvement of p73 protein and its isoforms in the regulation of death of CMV-infected and DOX-treated cells. Within a 24-h time period DOX caused death of about 70% human embryonic lung fibroblasts (HELF) in cell culture, this parameter decreased significantly in CMV-infected DOX-treated HELF cells. TUNEL has shown that the number of cells with DNA fragmentation decreases from 5.2% under the effect of DOX to 3.2% (P < 0.05) after combined CMV-DOX treatment. Analysis of mitotic figures revealed that DOX causes accumulation of mitotic cells, which was not observed in CMV-infected DOX-treated cells. PCR analysis of mRNA of two p73 protein isoforms (TAp73 and dNp73) has shown that in uninfected cells the expression of TAp73 isoform was low, while in CMV-infected cells level of TAp73 was significant and expression of dNp73 was demonstrated for the first time. Expression of TAp73 associated with lack of mitosis block. The activation of caspases 8, 9 and 3 in CMV-infected cells was registered but cell death was not, however, as massive as that caused by DOX. From these findings it can be concluded that CMV attenuates DOX-related damage to normal cells. It can be suggested that induction of TAp73 and dNp73 isoforms provides conditions for reduction of DOX effect which leads to DNA damage and death of normal cells.