Amino acid sequence divergence of Tat protein (exon1)of subtype B and C HIV-1 strains: Does it have implications for vaccine development?

Functional genes of HIV-1 like the tat express proteins essential for viral survival and propagation. There are variations reported in levels of Tat transactivation among the different subtypes of HIV-1. This study looked at the amino acid differences in the different regions of Tat protein (exon 1) of subtype B and C strains of HIV-1 and tried to observe a molecular basis for protein function. HIV-1 sequences of subtype B (n=30) and C (n=60) strains were downloaded from HIV-1 Los Alamos data base. Among the 60 subtype C strain sequences, 30 each were from India and Africa. A HIV-1 Tat protein (exon 1) sequence, the consensus B and C sequence was obtained from the 'sequence search interface' in the Los Alamos HIV-1 sequence data. The sequences were visualized using Weblogo and the RNA binding regions of the three consensus sequences were also determined using BindN software program. Compared to subtype B, there was a high level of divergence in the auxiliary domain of tat exon 1 (amino acid positions 58- 69). The net charge of the subtype C (Indian) Tat protein (exon 1) auxiliary domain was -1.9 at pH 7 and it had an isoelectric point of 4.1. The net charge of the subtype C (African) auxiliary domain was -2.9 at pH 7 and it had an isoelectric point of 3.7 while the net charge of same region in subtype B was -0.9 at pH 7 with an isoelectric point of 4.9. The ratio of the hydrophilic residues to the total number of residues was 60% in the in both the Indian and African subtype C in the auxiliary domain while this was 50% in subtype B. The consensus subtype B sequence was found to have 36 RNA binding sites while subtype C (India) had 33 and subtype C (Africa) had 32 RNA binding sites. The HIV-1 Tat-TAR interaction is a potential target for inhibitors and being considered for its potential use in HIV-1 vaccines. Development of such inhibitor/vaccines would have to take into consideration the variation in amino acid sequence analyzed in this study as this could determine epitope presentation on MHC class I antigen for afferent immune response.     

HIV-1 nef protein structures associated with brain infection and dementia pathogenesis

The difference between regional rates of HIV-associated dementia (HAD) in patients infected with different subtypes of HIV suggests that genetic determinants exist within HIV that influence the ability of the virus to replicate in the central nervous system (in Uganda, Africa, subtype D HAD rate is 89%, while subtype A HAD rate is 24%). HIV-1 nef is a multifunctional protein with known toxic effects in the brain compartment. The goal of the current study was to identify if specific three-dimensional nef structures may be linked to patients who developed HAD. HIV-1 nef structures were computationally derived for consensus brain and non-brain sequences from a panel of patients infected with subtype B who died due to varied disease pathologies and consensus subtype A and subtype D sequences from Uganda. Site directed mutation analysis identified signatures in brain structures that appear to change binding potentials and could affect folding conformations of brain-associated structures. Despite the large sequence variation between HIV subtypes, structural alignments confirmed that viral structures derived from patients with HAD were more similar to subtype D structures than to structures derived from patient sequences without HAD. Furthermore, structures derived from brain sequences of patients with HAD were more similar to subtype D structures than they were to their own non-brain structures. The potential finding of a brain-specific nef structure indicates that HAD may result from genetic alterations that alter the folding or binding potential of the protein.     

Dynamic evolution of the human immunodeficiency virus type 1 pathogenic factor, Nef

The human immunodeficiency virus type 1 (HIV-1) early gene product Nef is a multifunctional protein that alters numerous pathways of T-cell function, including endocytosis, signal transduction, vesicular trafficking, and immune modulation, and is a major determinant of pathogenesis. Individual Nef functions include PAK-2 activation, CD4 downregulation, major histocompatibility complex (MHC) class I downregulation, and enhancement of viral particle infectivity. How Nef accomplishes its multiple tasks presents a difficult problem of mechanistic analysis because of the complications associated with multiple, overlapping functional domains in the context of significant sequence variability. To address these issues we determined the conservation of each Nef residue based on 1,643 subtype B Nef sequences. Mutational analysis based on conservative substitutions and Nef sequence data allowed us to search for amino acids on the surface of Nef that are specifically required for PAK-2 activation. We found residues 85, 89, and 191 to be highly significant determinants for Nef's PAK-2 activation function but functionally unlinked to CD4 and MHC class I downregulation or enhancement of infectivity. These residues are not conserved across HIV-1 subtypes but are confined to separate sets of surface elements within a subtype. Thus, L85/H89/F191 and F85/F89/R191 are dominant in subtype B and subtype E or C, respectively. Our results provide support for developing subtype-specific interventions in HIV-1 disease.     

Human immunodeficiency virus type 1 env sequences from Calcutta in eastern India: identification of features that distinguish subtype C sequences in India from other subtype C sequences

India is experiencing a rapid spread of human immunodeficiency virus type 1 (HIV-1), primarily through heterosexual transmission of subtype C viruses. To delineate the molecular features of HIV-1 circulating in India, we sequenced the V3-V4 region of viral env from 21 individuals attending an HIV clinic in Calcutta, the most populous city in the eastern part of the country, and analyzed these and the other Indian sequences in the HIV database. Twenty individuals were infected with viruses having a subtype C env, and one had viruses with a subtype A env. Analyses of 192 subtype C sequences that included one sequence for each subject from this study and from the HIV database revealed that almost all sequences from India, along with a small number from other countries, form a phylogenetically distinct lineage within subtype C, which we designate C(IN). Overall, C(IN) lineage sequences were more closely related to each other (level of diversity, 10.2%) than to subtype C sequences from Botswana, Burundi, South Africa, Tanzania, and Zimbabwe (range, 15.3 to 20.7%). Of the three positions identified as signature amino acid substitution sites for C(IN) sequences (K340E, K350A, and G429E), 56% of the C(IN) sequences contained all three amino acids while 87% of the sequences contained at least two of these substitutions. Among the non-C(IN) sequences, all three amino acids were present in 2%, while 22% contained two or more of these amino acids. These results suggest that much of the current Indian epidemic is descended from a single introduction into the country. Identification of conserved signature amino acid positions could assist epidemiologic tracking and has implications for the development of a vaccine against subtype C HIV-1 in India.     

The inhibitory efficacy of RNA POL III-expressed long hairpin RNAs targeted to untranslated regions of the HIV-1 5' long terminal repeat

Human immunodeficiency virus type 1 (HIV-1) is a lentivirus that causes persistent infection resulting in the demise of immune regulatory cells, and ensuing diseases associated with acquired immune deficiency syndrome (AIDS). Although current therapeutic modalities have had a significant impact on mortality rates, novel therapies are constantly needed to prevent the emergence of resistant viral variants that escape the effects of antivirals. RNA Interference (RNAi) is a promising therapeutic modality for the inhibition of HIV-1 RNAs. Traditionally, RNAi effector sequences include expressed short hairpin RNAs (shRNAs) or short interfering RNAs (siRNAs). Recently, expressed long hairpin RNAs (lhRNAs) have been used with the aim of generating multiple independent siRNAs, which simultaneously target different susceptible sites on HIV-1. Here, modified lhRNAs expressed from U6 RNA Pol III promoters were targeted to sites within the first transcribed sequences of the HIV-1 5' long terminal repeat (LTR) region. Both Tat-dependent and independent suppressive efficacy was demonstrated against subtype B and C reporter sequences; however, lhRNAs complementary to the TAR stem-loop were refractory to silencing. None of the lhRNAs induced an unwanted interferon response as measured by interferon beta levels. Silencing by the lhRNAs was not equal across the extent of its cognate sequence, with the greatest efficacy observed for sequences located at the base of the stem. Nevertheless, direct antireplicative activity was seen when targeting lhRNAs to a subtype B HIV clone pNL4-3 Luc and a subtype C wild-type HIV-1 strain, FV5. These data highlight distinct target loci within the 5' LTR of HIV-1 that are susceptible to lhRNA targeting, and may prove to have an important advantage over other RNAi target sites within HIV-1. Although lhRNAs themselves require further manipulation to improve their overall efficacy in generating multiple functioning siRNAs, they may prove useful in any combinatorial-based approach to treating HIV-1 infection.     

Characterization of HIV Transmission in South-East Austria

To gain deeper insight into the epidemiology of HIV-1 transmission in South-East Austria we performed a retrospective analysis of 259 HIV-1 partial pol sequences obtained from unique individuals newly diagnosed with HIV infection in South-East Austria from 2008 through 2014. After quality filtering, putative transmission linkages were inferred when two sequences were ≤1.5% genetically different. Multiple linkages were resolved into putative transmission clusters. Further phylogenetic analyses were performed using BEAST v1.8.1. Finally, we investigated putative links between the 259 sequences from South-East Austria and all publicly available HIV polymerase sequences in the Los Alamos National Laboratory HIV sequence database. We found that 45.6% (118/259) of the sampled sequences were genetically linked with at least one other sequence from South-East Austria forming putative transmission clusters. Clustering individuals were more likely to be men who have sex with men (MSM; p<0.001), infected with subtype B (p<0.001) or subtype F (p = 0.02). Among clustered males who reported only heterosexual (HSX) sex as an HIV risk, 47% clustered closely with MSM (either as pairs or within larger MSM clusters). One hundred and seven of the 259 sequences (41.3%) from South-East Austria had at least one putative inferred linkage with sequences from a total of 69 other countries. In conclusion, analysis of HIV-1 sequences from newly diagnosed individuals residing in South-East Austria revealed a high degree of national and international clustering mainly within MSM. Interestingly, we found that a high number of heterosexual males clustered within MSM networks, suggesting either linkage between risk groups or misrepresentation of sexual risk behaviors by subjects.     

Constraints on HIV-1 diversity from protein structure

The high rate of HIV-1 evolution contributes to immune escape, enables the virus to escape drug therapy, and may underlie the difficulty of producing an effective vaccine. Identifying constraints on HIV evolution is therefore of prime importance. To investigate this problem, we examined the relationships between sequence diversity, selection, and protein structure. We found that while there was an increase in sequence diversity over time, this variation had a tendency to be limited to specific structural regions. When individual sites were analyzed, there was, in contrast, substantial and widespread evolutionary constraint over gag and env. This constraint was present even in the highly variable envelope proteins. The evolutionary significance of an individual site is indicated by the change in selection pressure along the time course: increasing entropy indicates that the site is evolving predominantly in a more "clock"-like manner, low entropy values with no increase indicate a high degree of constraint, and high entropy values indicate a lack of constraint. Few sites display high degrees of turnover. Mapping these sites onto the three-dimensional protein structure, we found a significant difference between evolutionary rates for regions buried in the core of the protein and those on the surface. This constraint did not change over the time period analyzed and was not subtype dependent, as similar results were found for subtypes B and C. This link between sequence and structure not only demonstrates the limits of recent HIV-1 evolution but also highlights the origins of evolutionary constraint on viral change.     

1992—1994年HIV—1国际流行株和云南株膜蛋白V3区氨基酸共有序列比较

对１９９２～１９９４年间１６个云南ＨＩＶ１株膜蛋白基因Ｖ３区进行了ＤＮＡ序列测定,经计算机ＤＮＡＳＩＳ及ＰＲＯＳＩＳ软件进行同源性分析,得出其相应的氨基酸共有序列ＹＮＶ３和两组共有序列ＹＮＶ３Ａ和ＹＮＶ３Ｂ,计算了ＹＮＶ３中每个氨基酸的保守性。分别将ＹＮＶ３Ａ和ＹＮＶ３Ｂ与世界各地的ＨＩＶ１代表株的相应序列进行了同源性比较。结果表明,ＨＩＶ１云南株膜蛋白Ｖ３区氨基酸共有序列ＹＮＶ３中每个氨基酸的平均变异度为７．６６％。两组共有序列ＹＮＶ３Ａ和ＹＮＶ３Ｂ,分别与ＨＩＶ１美欧株及泰国流行株Ｂ亚群相应序列有较高同源性。这一结果提示,在进化上云南瑞丽ＨＩＶ１流行毒株间有非常密切的关系,在这一时期该地区的流行毒株以ＨＩＶ１美欧株、泰国株Ｂ亚群及其衍生株为主。     

Phospholipid transfer protein: full-length cDNA and amino acid sequence in maize. Amino acid sequence homologies between plant phospholipid transfer proteins

We have determined the primary structure of a phospholipid transfer protein (PLTP) isolated from maize seeds. This protein consists of 93 amino acids and shows internal homology originating in the repetition of (do)decapeptides. By using antibodies against maize PLTP, we have isolated from a cDNA library one positive clone (6B6) which corresponds to the incomplete nucleotide sequence. Another cDNA clone (9C2) was obtained by screening a size-selected library with 6B6. Clone 9C2 (822 base pairs) corresponds to the full-length cDNA of the phospholipid-transfer protein whose mRNA contains 0.8 kilobase. Southern blot analysis shows that the maize genome may contain several PLTP genes. In addition, the deduced amino acid sequence of clone 9C2 reveals the presence of a signal peptide. The significance of this signal peptide (27 amino acids) might be related to the function of the phospholipid-transfer protein. The amino acid sequence of maize PLTP was compared to those isolated from spinach leaves or castor bean seeds which exhibit physicochemical properties close to those of the maize protein. A high homology was observed between the three sequences. Three domains can be distinguished: a highly charged central core (around 40-60), a very hydrophobic N-terminal sequence characteristic of polypeptide-membrane interaction, and a hydrophilic C terminus. A model for plant phospholipid-transfer proteins is proposed in which the phospholipid molecule is embedded within the protein with its polar moiety interacting with the central hydrophilic core of the protein, whereas the N-terminal region plunges within the membrane in the transfer process.     

Nucleotide sequence of the celC gene encoding endoglucanase C of Clostridium thermocellum

The nucleotide sequence of the cellulase gene celC, encoding endoglucanase C of Clostridium thermocellum, has been determined. The coding region of 1032 bp was identified by comparison with the N-terminal amino acid (aa) sequence of endoglucanase C purified from Escherichia coli. The ATG start codon is preceded by an AGGAGG sequence typical of ribosome-binding sites in Gram-positive bacteria. The derived amino acid sequence corresponds to a protein of Mr 40,439. Amino acid analysis and apparent Mr of endoglucanase C are consistent with the amino acid sequence as derived from the DNA sequencing data. A proposed N-terminal 21-aa residue leader (signal) sequence differs from other prokaryotic signal peptides and is non-functional in E. coli. Most of the protein bears no resemblance to the endoglucanases A, B, and D of the same organism. However, a short region of homology between endoglucanases A and C was identified, which is similar to the established active sites of lysozymes and to related sequences of fungal cellulases.     

山东省归国劳务人员中HIV—1感染毒株的序列特征和亚型分析

为了解自1992年以来,山东省归国劳务人员HIV－1感染毒株的分子流行病学特征。我们采集了11份确认为HIV－1感染者的僵血分离单核细胞（PBMC）,提取前病毒DNA,用套式PCR扩增HIV－1膜蛋白（env）基因,并对其envC2－V3区进行序列测定和分析。结果表明：10名归国劳工人员及1名归国劳工配偶的样本经序列测定和基因分析发现了HIV－1的4种基因亚型：A、B、C、E亚型,其中C亚型7名,B亚型2名、A和E亚型各1名。由此可见;山东省归国劳务人员中HIV－1毒株的感染情况较复杂,通过 些毒株间的基因离散率相差较大,提示应加强归国劳务人员的教育、检测和控制以及其它国家毒株传入。     

A putative signal peptidase recognition site and sequence in eukaryotic and prokaryotic signal peptides

Presecretory signal peptides of 39 proteins from diverse prokaryotic and eukaryotic sources have been compared. Although varying in length and amino acid composition, the labile peptides share a hydrophobic core of approximately 12 amino acids. A positively charged residue (Lys or Arg) usually precedes the hydrophobic core. Core termination is defined by the occurrence of a charged residue, a sequence of residues which may induce a beta-turn in a polypeptide, or an interruption in potential alpha-helix or beta-extended strand structure. The hydrophobic cores contain, by weight average, 37% Leu: 15% Ala: 10% Val: 10% Phe: 7% Ile plus 21% other hydrophobic amino acids arranged in a non-random sequence. Following the hydrophobic cores (aligned by their last residue) a highly non-random and localized distribution of Ala is apparent within the initial eight positions following the core: (formula; see text) Coincident with this observation, Ala-X-Ala is the most frequent sequence preceding signal peptidase cleavage. We propose the existence of a signal peptidase recognition sequence A-X-B with the preferred cleavage site located after the sixth amino acid following the core sequence. Twenty-two of the above 27 underlined Ala residues would participate as A or B in peptidase cleavage. Position A includes the larger aliphatic amino acids, Leu, Val and Ile, as well as the residues already found at B (principally Ala, Gly and Ser). Since a preferred cleavage site can be discerned from carboxyl and not amino terminal alignment of the hydrophobic cores it is proposed that the carboxyl ends are oriented inward toward the lumen of the endoplasmic reticulum where cleavage is thought to occur. This orientation coupled with the predicted beta-turn typically found between the core and the cleavage site implies reverse hairpin insertion of the signal sequence. The structural features which we describe should help identify signal peptides and cleavage sites in presumptive amino acid sequences derived from DNA sequences.     

Correlation of Functional Domains and Rates of Nucleotide Substitution in Cytochromeb

Distinguishing noise from signal presents a problem when DNA sequences are used for phylogeny reconstruction. Multiple substitutions at sites are a primary cause of noise and this is compounded by variation in substitution rates among sites. For protein-coding genes, one method used to determine if data are noisy is to assess levels of saturation of substitutions by codon position. However, this procedure may not be a fine enough filter for assessing noise. Variation in substitution rates may also be caused by constraints on change imposed by the function of the protein product. Using a structural model of the cytochromebprotein as a template, I divided cytbsequence data for species within the avian family Falconidae (falcons and caracaras) into three functional domains. Saturation of substitutions of sequences within these regions was assessed graphically. This qualitative determination of saturation was then used to differentially weight phylogenetic analysis, resulting in an hypothesis congruent with existing cladistic analyses and traditional morphology. These results demonstrate that saturation of substitutions is correlated with functional regions of cytochromeband that using this information improves phylogenetic inference.