Consensus Integrase of a New HIV-1 Genetic Variant CRF63_02A1.

The high genetic variability of the human immunodeficiency virus (HIV-1) leads to a constant emergence of new genetic variants, including the recombinant virus CRF63_02A1, which is widespread in the Siberian Federal District of Russia. We studied HIV-1 CRF63_02A1 integrase (IN_CRF) catalyzing the incorporation of viral DNA into the genome of an infected cell. The consensus sequence was designed, recombinant integrase was obtained, and its DNA-binding and catalytic activities were characterized. The stability of the IN_CRF complex with the DNA substrate did not differ from the complex stability for subtype A and B integrases; however, the rate of complex formation was significantly higher. The rates and efficiencies of 3'-processing and strand transfer reactions catalyzed by IN_CRF were found to be higher, too. Apparently, all these distinctive features of IN_CRF may result from specific amino acid substitutions in its N-terminal domain, which plays an important role in enzyme multimerization and binding to the DNA substrate. It was also found that the drug resistance mutations Q148K/G140S and G118R/E138K significantly reduce the catalytic activity of IN_CRF and its sensitivity to the strand transfer inhibitor raltegravir. Reduction in sensitivity to raltegravir was found to be much stronger in the case of double-mutation Q148K/G140S.

[Molecular-genetic characteristic of HIV-1 A and B subtypes variants isolated in Novosibirsk region].

AIM: Study phylogenetic interconnections of HIV-1 subtype A and B variants circulating in Novosibirsk region (NSR). MATERIALS AND METHODS: 268 HIV-1 variants isolated in 2007 - 2010 from blood samples of HIV infected patients in NSR, Samara, Congo and Moscow. HIV-1 variant genotyping was performed by analysis of 1.3 kb long pol gene nucleotide sequences. Phylogenetic analysis of nucleotide sequences was carried out by program Mega version 4.1 by constructing phylogenetic trees by nearest neighbor method. Nucleotide distances were calculated by Kimura method. RESULTS: The studied HIV-1 subtype B variants form separate phylogenetic groups with a low HIV-1 nucleotide sequence homology level combined based on territorial principle and/or time of HIV infection in a territory but not possessing interconnection with a specific population risk group. Subtype A HIV-1 is a fairly homogenous monophyletic group. Phylogenetic differences during studies of HIV-1 isolated from risk group patients - injection drugs users and individuals infected through sexual contacts were not detected. HIV variants isolated from patients infected in Moscow and Samara generally grouped with HIV variants circulating in the European part of Russia. CONCLUSION: An independent circulation of genetically separate HIV-1 subtype B groups is observed on the territory of siberian region which is a result of multiple independent introductions of distant variants of the virus. The confirmed limited spread of this HIV-1 genetic variant with a subsequent territorial separateness creates a possibility of formation of genetically different virus populations. The studies of subtype A viruses performed confirm the high level of homogeneity detected earlier in other Russia territories of HIV-1 belonging to this genetic variant. Monophyly of subtype A HIV variants is explained by imposition of 2 factors - territorial mobility of the population inside the country and lack of specific transmission routes for HIV-1 subtype A.

[Properties of CRF02_AG HIV-1 isolates circulating in Novosibirsk region].

AIM: Study of circulating 02_AG recombinant form HIV-1 isolates that have been rapidly spreading in Novosibirsk region during 3 recent years. MATERIALS AND METHODS: WHO protocol for primary HIV isolation was used, automatic sequencer was used for genetic characterization of isolates. Virus specific RNA were isolated and env HIV-1 region DNA fragments were processed. Phylogenetic analysis was also performed. RESULTS: CRF_02AG HIV-1 isolated from peripheral blood of HIV-1 positive patients belonged to CCR5 tropic viruses and had various reproduction characteristics. Most of the HIV isolated were rapidly replicating virus variants characterized by an ability to accumulate high levels of virus protein p24 in cultural fluid. Infectivity and reproductive properties of HIV isolates were confirmed in experimental infection by using clarified cultural liquid of mononuclear cells from healthy donors. Phylogenetic analysis of CRF02_AG HIV-1 variants isolated in Novosibirsk region in 2007 - 2010 showed the formation of a separate outbreak in the area caused by emergence of CRF02_AG HIV-1 in human population. CONCLUSION: A collection of genetically and biologically characterized CRF02_AG HIV-1 isolates that has not been spreading previously in Russia.

[Construction of DNA-fragments' libraries with complete genomes of different variola strains].

Libraries of hybrid plasmids carrying DNA fragments of complete genomes of 8 variola virus strain from the Russian Collection belonging to 2 epidemical types and isolated in various geographic regions of the world were obtained. Genomic sequences of variola virus can be thus preserved for a long time in a biologically safe form and provide the research work on studying the genetic organization of this unique virus and on developing modern methods for rapid detection of variola virus and other orthopoxviruses.

[Expression of genes for orthopoxviral TNF-binding proteins and study resulted recombinant proteins].

Genes for TNF-binding proteins (CrmBs) of variola virus (VARV), monkeypox virus (MPXV) or cowpox (CPXV) were isolated with PCR from viral genomes and expressed within baculovirus DNAs in Sf21 insect cell line. Properties of resulted recombinant proteins were studied with physical-chemical and immunological methods. It was shown with solid phase enzyme-linked immunoassay that viral proteins inhibited hTNF binding with polyclonal hTNF-antibodies. The strongest inhibitor was VARV-CrmB, the less one was MPXV-CrmB. Biological activity of recombinant protein preparations was studied in the test of neutralization of TNF cytotoxicity for L929 murine fibroblast cells. It was shown that recombinant CrmBs neutralized cytotoxicity of hTNF, mTNF or rTNF in species-specific manner. It was shown also that effectiveness of hTNF cytotoxicity inhibition in vitro with VARV-CrmB exceeded the same effect of polyclonal hTNF-antibody. A possibility of the elaboration of new therapeutics for anti-TNF therapy on the base of CrmB-like proteins is discussed.

[New recombinant variant of human immunodeficiency virus of type 1, subtype envB/envA, isolated in Novosibirsk]. / Novyi rekombinantnyi variant subtipa envB/envA VICH-1, vydelennyi v Novosibirske.

The nucleotide sequence of the variant of human immunodeficiency virus of type 1 (HIV-1), mostly widespread on the territory of the Novosibirsk region, was determined. The analysis of the nucleotide sequence confirmed that this variant belonged to HIV-1 of subtype A. The HIV-1 recombinant variant of subtype envB/envA with the recombination area within the second conservative region C2 of gene env, so far unknown, was detected and characterized. In HIV-1 the area at the beginning of gene env (5'-env) was found to belong to subtype B and the sequence at the end of gene env (3'-env), to subtype A. The analysis of the amino acid sequence of the third variable region of gene env demonstrated that the viruses under study belonged to macrophagotropic "slow/low" variants, characterized by low replication speed. The analysis of nucleotide sequences of the isolated variants of HIV-1 revealed their close genetic relationship with HIV-1 isolates circulating on the territory of Ukraine.

[Orthopoxvirus genes for Kelch-like proteins. I. Analysis of species specific differences by gene structure and organization]. / Izuchenie ortopoksvirusnykh genov, kodiruiushchikh kelch-podobnye belki. I. Analiz vidospetsifichnykh razlichii po strukturnoi organizatsii.

Genes and proteins of the kelch superfamily were structurally analyzed in the smallpox (SPV), monkeypox (MPV), cowpox (CPV), and vaccinia (VV) viruses. Genes potentially coding for the kelch-like proteins were found only in the variable terminal regions of the orthopoxvirus genome. The set and sizes of their protein products varied with species. All genes of the superfamily proved to be disrupted by mutations in SPV, which is highly pathogenic for its only host, man. The largest set of kelch-like proteins was observed for CPV, which is low-pathogenic for humans and has the broadest animal host range. The kelch-like proteins of one virus showed low homology to each other, whereas isologs of different viruses were highly homologous. The results testified to the earlier assumption that CPV is the most ancient and an ancestor of the other orthopoxviruses pathogenic for humans.

Analysis of the monkeypox virus genome.

Monkeypox virus (MPV) belongs to the orthopoxvirus genus of the family Poxviridae, is endemic in parts of Africa, and causes a human disease that resembles smallpox. The 196,858-bp MPV genome was analyzed with regard to structural features and open reading frames. Each end of the genome contains an identical but oppositely oriented 6379-bp terminal inverted repetition, which similar to that of other orthopoxviruses, includes a putative telomere resolution sequence and short tandem repeats. Computer-assisted analysis was used to identify 190 open reading frames containing >/=60 amino acid residues. Of these, four were present within the inverted terminal repetition. MPV contained the known essential orthopoxvirus genes but only a subset of the putative immunomodulatory and host range genes. Sequence comparisons confirmed the assignment of MPV as a distinct species of orthopoxvirus that is not a direct ancestor or a direct descendent of variola virus, the causative agent of smallpox.

Human monkeypox and smallpox viruses: genomic comparison.

Monkeypox virus (MPV) causes a human disease which resembles smallpox but with a lower person-to-person transmission rate. To determine the genetic relationship between the orthopoxviruses causing these two diseases, we sequenced the 197-kb genome of MPV isolated from a patient during a large human monkeypox outbreak in Zaire in 1996. The nucleotide sequence within the central region of the MPV genome, which encodes essential enzymes and structural proteins, was 96.3% identical with that of variola (smallpox) virus (VAR). In contrast, there were considerable differences between MPV and VAR in the regions encoding virulence and host-range factors near the ends of the genome. Our data indicate that MPV is not the direct ancestor of VAR and is unlikely to naturally acquire all properties of VAR.

Conserved surface-exposed K/R-X-K/R motifs and net positive charge on poxvirus complement control proteins serve as putative heparin binding sites and contribute to inhibition of molecular interactions with human endothelial cells: a novel mechanism for evasion of host defense.

Vaccinia virus complement control protein (VCP) has been shown to possess the ability to inhibit both classical and alternative complement pathway activation. The newly found ability of this protein to bind to heparin has been shown in previous studies to result in uptake by mast cells, possibly promoting tissue persistence. It has also been shown to reduce chemotactic migration of leukocytes by blocking chemokine binding. In addition, this study shows that VCP-through its ability to bind to glycosaminoglycans (heparin-like molecules) on the surface of human endothelial cells-is able to block antibody binding to surface major histocompatibility complex class I molecules. Since heparin binding is critical for many functions of this protein, we have attempted to characterize the molecular basis for this interaction. Segments of this protein, generated by genetic engineering of the DNA encoding VCP into the Pichia pastoris expression system, were used to localize the regions with heparin binding activity. These regions were then analyzed to more specifically define their properties for binding. It was found that the number of putative binding sites (K/R-X-K/R), the overall positive charge, and the percentage of positively charged amino acids within the protein were responsible for this interaction.

Alastrim smallpox variola minor virus genome DNA sequences.

Alastrim variola minor virus, which causes mild smallpox, was first recognized in Florida and South America in the late 19th century. Genome linear double-stranded DNA sequences (186,986 bp) of the alastrim virus Garcia-1966, a laboratory reference strain from an outbreak associated with 0.8% case fatalities in Brazil in 1966, were determined except for a 530-bp fragment of hairpin-loop sequences at each terminus. The DNA sequences (EMBL Accession No. Y16780) showed 206 potential open reading frames for proteins containing >/=60 amino acids. The amino acid sequences of the putative proteins were compared with those reported for vaccinia virus strain Copenhagen and the Asian variola major strains India-1967 and Bangladesh-1975. About one-third of the alastrim viral proteins were 100% identical to correlates in the variola major strains and the remainder were >/=95% identical. Compared with variola major virus DNA, alastrim virus DNA has additional segments of 898 and 627 bp, respectively, within the left and right terminal regions. The former segment aligns well with sequences in other orthopoxviruses, particularly cowpox and vaccinia viruses, and the latter is apparently alastrim-specific.

Molecular mimicry of the inflammation modulatory proteins (IMPs) of poxviruses: evasion of the inflammatory response to preserve viral habitat.

Microorganisms encode numerous immunomodulators that resemble, in structure and function, molecules captured over the millennia from their hosts [G. J. Kotwal J. Leukoc. Biol. 62, 415-429]. The vaccinia virus complement control protein (VCP) was the first soluble microbial protein to have a postulated role in the immunomodulation and evasion of host defense [G. J. Kotwal and B. Moss Nature 355, 176-179]. Purified bioactive VCP has been shown to bind to C3 and C4, block the complement cascade at multiple sites [G. J. Kotwal et al. Science 250, 827-830; R. Mckenzie, G. J. Kotwal et al. J. Infect. Dis. 166, 1245-1250] and exhibit a greater potency than the human complement 4b binding protein, C4b-BP [G. J. Kotwal, Am. Biotech. Lab. 9, 76]. The importance of this protein to poxviruses was further demonstrated in rabbits and guinea pigs through the use of recombinant virus lacking an intact DNA coding for VCP [Isaacs, G. J. Kotwal, and B. Moss Proc. Natl. Acad. Sci. 89, 628-672]. Studies in mice have shown that the homolog of VCP in cowpox virus (CPV), referred to as the inflammation modulatory protein (IMP) can, in a mouse model, significantly diminish the specific footpad swelling response [C. G. Miller, S. N. Shchelkunov, and G. J. Kotwal Virol. 229, 126-133]. To determine the precise cellular changes at the site of infection, BALB/c mice were subcutaneously injected (in the backs) with CPV or a recombinant virus lacking IMP, CPV-IMP. Differences in histology were observed by staining the adjoining skin tissue sections with hematoxylin & eosin or by removal of the connective tissue and staining with May-Grunwald-Geimsa. All mice that were injected with the CPV-IMP experienced severe tissue destruction and formation of nodular lesions compared with the mice injected with CPV. Microscopic examination indicated significantly greater cellular infiltration and destruction of skeletal muscle cells in the sections of connective tissue and adjoining skin tissue, respectively, of the mice injected with the CPV-IMP [G. J. Kotwal et al. Mol. Cell. Biochem. in press]. Thus IMP preserves the tissue at the site of infection (viral habitat). In this review, we present evidence for molecular mimicry and evolutionary relationship to other homologs of IMP and discuss their relationships with other IMPs such as the poxviral chemokine and cytokine receptor-like proteins.