Mature reverse transcriptase (p66/p51) is responsible for low levels of viral DNA found in human immunodeficiency virus type 1 (HIV-1)

Reverse transcription of the HIV RNA genome is thought to occur in the host cell cytoplasm after viral adsorption. However, viral DNA has been isolated in cell-free virus particles. We have quantitated by polymerase chain reaction (PCR) amplification the amount of viral DNA in virions as compared to RNA. Virus produced by proviral DNA transfections of cos-7 cells or by chronically-infected H9 cells; neither of which express the cell surface CD4 receptor, contained at least 1000 times more viral RNA than DNA. In contrast, only 60 times more RNA than DNA was present in virus particles produced by transfection of Jurkat cells, which were CD4-positive and thus potentially susceptible to superinfection. Protease-defective virus, carrying only the precursor of reverse transcriptase (RT) p160gag-pol, contained virtually no detectable DNA. These results indicate that only mature RT (p66/p51) and not its precursor (p160gag-pol) is responsible for the presence of viral DNA in HIV.

Oral administration of iron-saturated bovine lactoferrin-loaded ceramic nanocapsules for breast cancer therapy and influence on iron and calcium metabolism

We determined the anticancer efficacy and internalization mechanism of our polymeric-ceramic nanoparticle system (calcium phosphate nanocores, enclosed in biodegradable polymers chitosan and alginate nanocapsules/nanocarriers [ACSC NCs]) loaded with iron-saturated bovine lactoferrin (Fe-bLf) in a breast cancer xenograft model. ACSC-Fe-bLf NCs with an overall size of 322±27.2 nm were synthesized. In vitro internalization and anticancer efficacy were evaluated in the MDA-MB-231 cells using multicellular tumor spheroids, CyQUANT and MTT assays. These NCs were orally delivered in a breast cancer xenograft mice model, and their internalization, cytotoxicity, biodistribution, and anticancer efficacy were evaluated. Chitosan-coated calcium phosphate Fe-bLf NCs effectively (59%, P≤0.005) internalized in a 1-hour period using clathrin-mediated endocytosis (P≤0.05) and energy-mediated pathways (P≤0.05) for internalization; 3.3 mg/mL of ACSC-Fe-bLf NCs completely disintegrated (~130-fold reduction, P≤0.0005) the tumor spheroids in 72 hours and 96 hours. The IC50 values determined for ACSC-Fe-bLf NCs were 1.69 mg/mL at 10 hours and 1.62 mg/mL after 20 hours. We found that Fe-bLf-NCs effectively (P≤0.05) decreased the tumor size (4.8-fold) compared to the void NCs diet and prevented tumor recurrence when compared to intraperitoneal injection of Taxol and Doxorubicin. Receptor gene expression and micro-RNA analysis confirmed upregulation of low-density lipoprotein receptor and transferrin receptor (liver, intestine, and brain). Several micro-RNAs responsible for iron metabolism upregulated with NCs were identified. Taken together, orally delivered Fe-bLf NCs offer enhanced antitumor activity in breast cancer by internalizing via low-density lipoprotein receptor and transferrin receptor and regulating the micro-RNA expression. These NCs also restored the body iron and calcium levels and increased the hematologic counts.

Analysis of the contribution of reverse transcriptase and integrase proteins to retroviral RNA dimer conformation

All retroviruses contain two copies of genomic RNA that are linked noncovalently. The dimeric RNA of human immunodeficiency virus type 1 (HIV-1) undergoes rearrangement during virion maturation, whereby the dimeric RNA genome assumes a more stable conformation. Previously, we have shown that the packaging of the HIV-1 polymerase (Pol) proteins reverse transcriptase (RT) and integrase (IN) is essential for the generation of the mature RNA dimer conformation. Analysis of HIV-1 mutants that are defective in processing of Pol showed that these mutant virions contained altered dimeric RNA conformation, indicating that the mature RNA dimer conformation in HIV-1 requires the correct proteolytic processing of Pol. The HIV-1 Pol proteins are multimeric in their mature enzymatically active forms; RT forms a heterodimer, and IN appears to form a homotetramer. Using RT and IN multimerization defective mutants, we have found that dimeric RNA from these mutant virions has the same stability and conformation as wild-type RNA dimers, showing that the mature enzymatically active RT and IN proteins are dispensable for the generation of mature RNA dimer conformation. This also indicated that formation of the mature RNA dimer structure occurs prior to RT or IN maturation. We have also investigated the requirement of Pol for RNA dimerization in both Mason-Pfizer monkey virus (M-PMV) and Moloney murine leukemia virus (MoMuLV) and found that in contrast to HIV-1, Pol is dispensable for RNA dimer maturation in M-PMV and MoMuLV, demonstrating that the requirement of Pol in retroviral RNA dimer maturation is not conserved among all retroviruses.

Transcriptomics-based analysis using RNA-Seq of the coconut (Cocos nucifera) leaf in response to yellow decline phytoplasma infection

Invasive phytoplasmas wreak havoc on coconut palms worldwide, leading to high loss of income, food insecurity and extreme poverty of farmers in producing countries. Phytoplasmas as strictly biotrophic insect-transmitted bacterial pathogens instigate distinct changes in developmental processes and defence responses of the infected plants and manipulate plants to their own advantage; however, little is known about the cellular and molecular mechanisms underlying host–phytoplasma interactions. Further, phytoplasma-mediated transcriptional alterations in coconut palm genes have not yet been identified. This study evaluated the whole transcriptome profiles of naturally infected leaves of Cocos nucifera ecotype Malayan Red Dwarf in response to yellow decline phytoplasma from group 16SrXIV, using RNA-Seq technique. Transcriptomics-based analysis reported here identified genes involved in coconut innate immunity. The number of down-regulated genes in response to phytoplasma infection exceeded the number of genes up-regulated. Of the 39,873 differentially expressed unigenes, 21,860 unigenes were suppressed and 18,013 were induced following infection. Comparative analysis revealed that genes associated with defence signalling against biotic stimuli were significantly overexpressed in phytoplasma-infected leaves versus healthy coconut leaves. Genes involving cell rescue and defence, cellular transport, oxidative stress, hormone stimulus and metabolism, photosynthesis reduction, transcription and biosynthesis of secondary metabolites were differentially represented. Our transcriptome analysis unveiled a core set of genes associated with defence of coconut in response to phytoplasma attack, although several novel defence response candidate genes with unknown function have also been identified. This study constitutes valuable sequence resource for uncovering the resistance genes and/or susceptibility genes which can be used as genetic tools in disease resistance breeding.

Functional analysis of yeast snoRNA and snRNA 3' end formation mediated by uncoupling of cleavage and polyadenylation

Many nuclear and nucleolar small RNAs are accumulated as nonpolyadenylated species and require 3′-end processing for maturation. Here, we show that several genes coding for box C/D and H/ACA snoRNAs and for the U5 and U2 snRNAs contain sequences in their 3′ portions which direct cleavage of primary transcripts without being polyadenylated. Genetic analysis of yeasts with mutations in different components of the pre-mRNA cleavage and polyadenylation machinery suggests that this mechanism of 3"-end formation requires cleavage factor IA (CF IA) but not cleavage and polyadenylation factor activity. However, in vitro results indicate that other factors participate in the reaction besides CF IA. Sequence analysis of snoRNA genes indicated that they contain conserved motifs in their 3" noncoding regions, and mutational studies demonstrated their essential role in 3"-end formation. We propose a model in which CF IA functions in cleavage and polyadenylation of pre-mRNAs and, in combination with a different set of factors, in 3"-end formation of nonpolyadenylated polymerase II transcripts.

Yeast Rnt1p is required for cleavage of the pre-ribosomal RNA in the 3' ETS but not the 5' ETS

We have reexamined the role of yeast RNase III (Rnt1p) in ribosome synthesis. Analysis of pre-rRNA processing in a strain carrying a complete deletion of the RNT1 gene demonstrated that the absence of Rnt1p does not block cleavage at site A0 in the 5' external transcribed spacers (ETS), although the early pre-rRNA cleavages at sites A0, A1, and A2 are kinetically delayed. In contrast, cleavage in the 3' ETS is completely inhibited in the absence of Rnt1p, leading to the synthesis of a reduced level of a 3' extended form of the 25S rRNA. The 3' extended forms of the pre-rRNAs are consistent with the major termination at site T2 (+210). We conclude that Rnt1p is required for cleavage in the 3' ETS but not for cleavage at site A0. The sites of in vivo cleavage in the 3' ETS were mapped by primer extension. Two sites of Rnt1p-dependent cleavage were identified that lie on opposite sides of a predicted stem loop structure, at +14 and +49. These are in good agreement with the consensus Rnt1p cleavage site. Processing of the 3' end of the mature 25S rRNA sequence in wild-type cells was found to occur concomitantly with processing of the 5' end of the 5.8S rRNA, supporting previous proposals that processing in ITS1 and the 3' ETS is coupled.

Yhh1p/Cft1p directly links poly(A) site recognition and RNA polymerase II transcription termination

RNA polymerase II (pol II) transcription termination requires co-transcriptional recognition of a functional polyadenylation signal, but the molecular mechanisms that transduce this signal to pol II remain unclear. We show that Yhh1p/Cft1p, the yeast homologue of the mammalian AAUAAA interacting protein CPSF 160, is an RNA-binding protein and provide evidence that it participates in poly(A) site recognition. Interestingly, RNA binding is mediated by a central domain composed of predicted -propeller-forming repeats, which occurs in proteins of diverse cellular functions. We also found that Yhh1p/Cft1p bound specifically to the phosphorylated C-terminal domain (CTD) of pol II in vitro and in a two-hybrid test in vivo. Furthermore, transcriptional run-on analysis demonstrated that yhh1 mutants were defective in transcription termination, suggesting that Yhh1p/Cft1p functions in the coupling of transcription and 3'-end formation. We propose that direct interactions of Yhh1p/Cft1p with both the RNA transcript and the CTD are required to communicate poly(A) site recognition to elongating pol II to initiate transcription termination.

Proteolytic processing of the P2/nucleocapsid cleavage site is critical for human immunodeficiency virus type 1 RNA dimer maturation

Differences in virion RNA dimer stability between mature and protease-defective (immature) forms of human immunodeficiency virus type 1 (HIV-1) suggest that maturation of the viral RNA dimer is regulated by the proteolytic processing of the HIV-1 Gag and Gag-Pol precursor proteins. However, the proteolytic processing of these proteins occurs in several steps denoted primary, secondary, and tertiary cleavage events and, to date, the processing step associated with formation of stable HIV-1 RNA dimers has not been identified. We show here that a mutation in the primary cleavage site (p2/nucleocapsid [NC]) hinders formation of stable virion RNA dimers, while dimer stability is unaffected by mutations in the secondary (matrix/capsid [CA], p1/p6) or a tertiary cleavage site (CA/p2). By introducing mutations in a shared cleavage site of either Gag or Gag-Pol, we also show that the cleavage of the p2/NC site in Gag is more important for dimer formation and stability than p2/NC cleavage in Gag-Pol. Electron microscopy analysis of viral particles shows that mutations in the primary cleavage site in Gag but not in Gag-Pol inhibit viral particle maturation. We conclude that virion RNA dimer maturation is dependent on proteolytic processing of the primary cleavage site and is associated with virion core formation.

The A-rich RNA sequences of HIV-1 pol are important for the synthesis of viral cDNA

The bias of A-rich codons in HIV-1 pol is thought to be a record of hypermutations in viral genomes that lack biological functions. Bioinformatic analysis predicted that A-rich sequences are generally associated with minimal local RNA structures. Using codon modifications to reduce the amount of A-rich sequences within HIV-1 genomes, we have reduced the flexibility of RNA sequences in pol to analyze the functional significance of these A-rich ‘structurally poor’ RNA elements in HIV-1 pol. Our data showed that codon modification of HIV-1 sequences led to a suppression of virus infectivity by 5–100-fold, and this defect does not correlate with, viral entry, viral protein expression levels, viral protein profiles or virion packaging of genomic RNA. Codon modification of HIV-1 pol correlated with an enhanced dimer stability of the viral RNA genome, which was associated with a reduction of viral cDNA synthesis both during HIV-1 infection and in a cell free reverse transcription assay. Our data provided direct evidence that the HIV-1 A-rich pol sequence is not merely an evolutionary artifact of enzyme-induced hypermutations, and that HIV-1 has adapted to rely on A-rich RNA sequences to support the synthesis of viral cDNA during reverse transcription, highlighting the utility of using ‘structurally poor’ RNA domains in regulating biological process.

Integrated RNA extraction and RT-PCR for semi-quantitative gene expression studies on a microfluidic device

This paper describes the development of a microfluidic methodology, using RNA extraction and reverse transcription PCR, for investigating expression levels of cytochrome P450 genes. Cytochrome P450 enzymes are involved in the metabolism of xenobiotics, including many commonly prescribed drugs, therefore information on their expression is useful in both pharmaceutical and clinical settings. RNA extraction, from rat liver tissue or primary rat hepatocytes, was performed using a silica-based solid-phase extraction technique. Following elution of the purified RNA, amplification of target sequences for the housekeeping gene, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and the cytochrome P450 gene CYP1A2, was carried out using a one-step reverse transcription PCR. Once the microfluidic methodology had been optimized, analysis of control and 3-methylcholanthrene-induced primary rat hepatocytes were used to evaluate the system. As expected, GAPDH was consistently expressed, whereas CYP1A2 levels were found to be raised in the drug-treated samples. The proposed system offers an initial platform for development of both rapid throughput analyzers for pharmaceutical drug screening and point-of-care diagnostic tests to aid provision of drug regimens, which can be tailor-made to the individual patient.

Comparative analysis of caveolins in mouse and tammar wallaby: role in regulating mammary gland function.

Recent studies using the mouse showed an inverse correlation between the Caveolin 1 gene expression and lactation, and this was regulated by prolactin. However, current study using mammary explants from pregnant mice showed that while insulin (I), cortisol (F) and prolactin (P) resulted in maximum induction of the β-casein gene, FP and IFP resulted in the downregulation of Caveolin 1. Additionally, IF, FP and IFP resulted in the downregulation of Caveolin 2. Immunohistochemistry confirmed localisation of Caveolin 1 specific to myoepithelial cells and adipocytes. Comparative studies with the tammar wallaby showed Caveolin 1 and 2 had 70-80% homology with the mouse proteins. However, in contrast to the mouse, Caveolin 1 and 2 genes showed a significantly increased level of expression in the mammary gland during lactation. The regulation of tammar Caveolin 1 and 2 gene expression was examined in mammary explants from pregnant tammars, and no significant difference was observed either in the absence or in the presence of IFP.

Specific recognition of the HIV-1 genomic RNA by the Gag precursor

During assembly of HIV-1 particles in infected cells, the viral Pr55(Gag) protein (or Gag precursor) must select the viral genomic RNA (gRNA) from a variety of cellular and viral spliced RNAs. However, there is no consensus on how Pr55(Gag) achieves this selection. Here, by using RNA binding and footprinting assays, we demonstrate that the primary Pr55(Gag) binding site on the gRNA consists of the internal loop and the lower part of stem-loop 1 (SL1), the upper part of which initiates gRNA dimerization. A double regulation ensures specific binding of Pr55(Gag) to the gRNA despite the fact that SL1 is also present in spliced viral RNAs. The region upstream of SL1, which is present in all HIV-1 RNAs, prevents binding to SL1, but this negative effect is counteracted by sequences downstream of SL4, which are unique to the gRNA.

Comparative analysis of microRNA expression in mouse and human brown adipose tissue

BACKGROUND: In small mammals brown adipose tissue (BAT) plays a predominant role in regulating energy expenditure (EE) via adaptive thermogenesis. New-born babies require BAT to control their body temperature, however its relevance in adults has been questioned. Active BAT has recently been observed in adult humans, albeit in much lower relative quantities than small mammals. Comparing and contrasting the molecular mechanisms controlling BAT growth and development in mice and humans will increase our understanding or how human BAT is developed and may identify potential therapeutic targets to increase EE. MicroRNAs are molecular mechanisms involved in mouse BAT development however, little is known about the miRNA profile in human BAT. The aims of this study were to establish a mouse BAT-enriched miRNA profile and compare this with miRNAs measured in human BAT. To achieve this we firstly established a mouse BAT enriched-miRNA profile by comparing miRNAs expressed in mouse BAT, white adipose tissue and skeletal muscle. Following this the BAT-enriched miRNAs predicted to target genes potentially involved in growth and development were identified.

METHODS: MiRNA levels were measured using PCR-based miRNA arrays. Results were analysed using ExpressionSuite software with the global mean expression value of all expressed miRNAs in a givensample used as the normalisation factor. Bio-informatic analyses was used to predict gene targets followed by Ingenuity Pathway Analysis.

RESULTS: We identified 35 mouse BAT-enriched miRNAs that were predicted to target genes potentially involved in growth and development. We also identified 145 miRNAs expressed in both mouse and human BAT, of which 25 were enriched in mouse BAT. Of these 25 miRNAs, miR-20a was predicted to target MYF5 and PPARγ, two important genes involved in brown adipogenesis, as well as BMP2 and BMPR2, genes involved in white adipogenesis. For the first time, 69 miRNAs were identified in human BAT but absent in mouse BAT, and 181 miRNAs were expressed in mouse but not in human BAT.

CONCLUSION: The present study has identified a small sub-set of miRNAs common to both mouse and human BAT. From this sub-set bioinformatics analysis suggested a potential role of miR-20a in the control of cell fate and this warrants further investigation. The large number of miRNAs found only in mouse BAT or only in human BAT highlights the differing molecular profile between species that is likely to influence the functional role of BAT across species. Nevertheless the BAT-enriched miRNA profiles established in the present study suggest targets to investigate in the control BAT development and EE.