Detection of viral sequence fragments of HIV-1 subfamilies yet unknown

<p>Abstract</p> <p>Background</p> <p>Methods of determining whether or not any particular HIV-1 sequence stems - completely or in part - from some unknown HIV-1 subtype are important for the design of vaccines and molecular detection systems, as well as for epidemiological monitoring. Nevertheless, a single algorithm only, the Branching Index (BI), has been developed for this task so far. Moving along the genome of a query sequence in a sliding window, the BI computes a ratio quantifying how closely the query sequence clusters with a subtype clade. In its current version, however, the BI does not provide predicted boundaries of unknown fragments.</p> <p>Results</p> <p>We have developed <it>Unknown Subtype Finder </it>(USF), an algorithm based on a probabilistic model, which automatically determines which parts of an input sequence originate from a subtype yet unknown. The underlying model is based on a simple profile hidden Markov model (pHMM) for each <it>known </it>subtype and an additional pHMM for an <it>unknown </it>subtype. The emission probabilities of the latter are estimated using the emission frequencies of the known subtypes by means of a (position-wise) probabilistic model for the emergence of new subtypes. We have applied USF to SIV and HIV-1 sequences formerly classified as having emerged from an unknown subtype. Moreover, we have evaluated its performance on artificial HIV-1 recombinants and non-recombinant HIV-1 sequences. The results have been compared with the corresponding results of the BI.</p> <p>Conclusions</p> <p>Our results demonstrate that USF is suitable for detecting segments in HIV-1 sequences stemming from yet unknown subtypes. Comparing USF with the BI shows that our algorithm performs as good as the BI or better.</p

Experimental evidence for splicing of intron-containing transcripts of plant LTR retrotransposon Ogre

Ogre elements are a distinct group of plant Ty3/gypsy-like retrotransposons characterized by several specific features, one of which is a separation of the gag-pol region into two non-overlapping open reading frames: ORF2 coding for Gag-Pro, and ORF3 coding for RT/RH-INT proteins. Previous characterization of Ogre elements from several plant species revealed that part of their transcripts lacks the region between ORF2 and ORF3, carrying one uninterrupted ORF instead. In this work, we investigated a hypothesis that this region represents an intron that is spliced out from part of the Ogre transcripts as a means for preferential production of ORF2-encoded proteins over those encoded by the complete ORF2–ORF3 region. The experiments involved analysis of transcription patterns of well-defined Ogre populations in a model plant Medicago truncatula and examination of transcripts carrying dissected pea Ogre intron expressed within a coding sequence of chimeric reporter gene. Both experimental approaches proved that the region between ORF2 and ORF3 is spliced from Ogre transcripts and showed that this process is only partial, probably due to weak splice signals. This is one of very few known cases of spliced LTR retrotransposons and the only one where splicing does not involve parts of the element’s coding sequences, thus resembling intron splicing found in most cellular genes

Differentially-Expressed Pseudogenes in HIV-1 Infection.

Not all pseudogenes are transcriptionally silent as previously thought. Pseudogene transcripts, although not translated, contribute to the non-coding RNA pool of the cell that regulates the expression of other genes. Pseudogene transcripts can also directly compete with the parent gene transcripts for mRNA stability and other cell factors, modulating their expression levels. Tissue-specific and cancer-specific differential expression of these "functional" pseudogenes has been reported. To ascertain potential pseudogene:gene interactions in HIV-1 infection, we analyzed transcriptomes from infected and uninfected T-cells and found that 21 pseudogenes are differentially expressed in HIV-1 infection. This is interesting because parent genes of one-third of these differentially-expressed pseudogenes are implicated in HIV-1 life cycle, and parent genes of half of these pseudogenes are involved in different viral infections. Our bioinformatics analysis identifies candidate pseudogene:gene interactions that may be of significance in HIV-1 infection. Experimental validation of these interactions would establish that retroviruses exploit this newly-discovered layer of host gene expression regulation for their own benefit

Effect of Major Splice Site deletions in Latency and Infectivity of 8 HIV Constructs

HonorsBiochemistryUniversity of Michiganhttp://deepblue.lib.umich.edu/bitstream/2027.42/167888/1/rnitsch.pd

HCV Proteins and Immunoglobulin Variable Gene (IgV) Subfamilies in HCV-Induced Type II Mixed Cryoglobulinemia: A Concurrent Pathogenetic Role

The association between hepatitis C virus (HCV) infection and type II mixed cryoglobulinemia (MCII) is well established, but the role played by distinct HCV proteins and by specific components of the anti-HCV humoral immune response remains to be clearly defined. It is widely accepted that HCV drives the expansion of few B-cell clones expressing a restricted pool of selected immunoglobulin variable (IgV) gene subfamilies frequently endowed with rheumatoid factor (RF) activity. Moreover, the same IgV subfamilies are frequently observed in HCV-transformed malignant B-cell clones occasionally complicating MCII. In this paper, we analyze both the humoral and viral counterparts at the basis of cryoglobulins production in HCV-induced MCII, with particular attention reserved to the single IgV subfamilies most frequently involved

Molecular signatures of hepatitis C virus (HCV)-induced type II mixed cryoglobulinemia (MCII)

The role of hepatitis C virus (HCV) infection in the induction of type II mixed cryoglobulinemia (MCII) and the possible establishment of related lymphoproliferative disorders, such as B-cell non-Hodgkin lymphoma (B-NHL), is well ascertained. However, the molecular pathways involved and the factors predisposing to the development of these HCV-related extrahepatic complications deserve further consideration and clarification. To date, several host- and virus-related factors have been implicated in the progression to MCII, such as the virus-induced expansion of selected subsets of B-cell clones expressing discrete immunoglobulin variable (IgV) gene subfamilies, the involvement of complement factors and the specific role of some HCV proteins. In this review, we will analyze the host and viral factors taking part in the development of MCII in order to give a general outlook of the molecular mechanisms implicated

Development of new molecular probes for imaging of cancer

The 35-amino acid long peptide pepR, derived from DENVC, presents interesting an-tibacterial and anti-HIV activity. However, peptides undergo proteolytic degradation there-fore, pepR in the presence of human serum, gives one main fragment with 17 amino acid res-idues (LKRWGTIKKSKAINVLR) named pepRF1. Interestingly, this human serum resistant fragment pepRF1 presents a more potent anti-HIV activity than its parent pepR (IC50 1.5 ± 0.4 pepRF1 and IC50 37 ± 3.8 pepR). pepRF1 prevents viral entry into target cells by binding to the viral co-receptor CXCR4, acting as an antagonist as proved in cytosolic calcium release assays. CXCR4, whose cognate ligand is chemokine CXCL12, is a G-protein-coupled receptor that is implicated in diverse pathophysiological processes, including various types of cancers (prostate, ovarian, brain), where it plays a key role in tumor development and metastization. Considering that CXCR4 is both an established druggable receptor and a relevant imaging target, we envisaged the possibility of exploring gallium-67(67Ga)-labeled pepRF1 and pepRF1-derived shorter peptide sequences (PepRF1_C2: LKRWGTIKKSKAINV; PepRF1_C4: LKRWGTIKKSKAI, PepRF1_C6: LKRWGTIKKSK; PepRF1_C8: LKRWGTIKK; and PepRF1_C10: LKRWGTI) as CXCR4-targeted imaging agents. To accomplish that goal, the short peptides were conjugated to the bifuncional chelator NODA-GA, giving the corresponding peptide conjugates of the type PepRF1_X_NOD (X = C2, C4, C6, C8 and C10), which were characterized by RP-HPLC and ESI-MS. Reaction of the peptide conjugates with 67GaCl3 gave radiopeptides of the type 67Ga-PepRF1_X_NOD in high radiochemical yield and purity >95%. The chemical identity of the 67Ga-labeled peptide con-jugates was confirmed by comparing their retention times in the RP-HPLC chromatograms (γ-detection) with those of the non-radioactive analogues (UV-detection). The cell uptake studies in cancer cell lines with different levels of CXCR4 expression have shown that there is no correlation between cellular uptake of the 67Ga-labeled peptide conjugates and CXCR4 expression. Moreover, low uptake values (ca. 1 - 2%) were obtained for all radiopeptides. The biodistribution studies in healthy CD-1 mice have shown that the pharmacokinetic profile of the radiopeptides present a similar trend, relatively fast blood clearance with rapid uptake by the kidneys and liver, and no relevant uptake in any other main organ. A moderate to rapid total excretion (32.6 ± 3.2 to 87.9 ± 5.8 % IA) from the whole animal body predomi-nantly through the urinary tract was observed.O pepR, um péptido com 35 resíduos de aminoácidos, derivado de DENVC, apresenta propriedades antibacteriana e anti-HIV interessantes. Contudo, os péptidos são propensos a degradação proteolítica, logo na presença de soro humano pepR, resulta em um fragmento maioritário com 17 resíduos de aminoácidos (LKRWGTIKKSKAINVLR), denominado pepRF1. Curiosamente, a espécie resistente a soro humano pepRF1 demonstra uma atividade anti-HIV mais potente que o seu progenitor pepR (IC50 1.5 ± 0.4 pepRF1 and IC50 37 ± 3.8 pepR). Este péptido previne a entrada do vírus em células alvo ao se ligar ao recetor viral, CXCR4 atuando como um antagonista como provado após o teste de libertação de cálcio citosólico. CXCR4, que tem como ligando natural único a quimiocina CXCL12, pertence à família dos recetores acoplados à proteína G e está implicado em diversos processos patofisiológicos, incluindo vários tipos de cancro (próstata, ovários, cérebro), onde desempenha um papel cru-cial no desenvolvimento de tumores e no seu processo de metastização. Tendo em conta que CXCR4 é um alvo estabelecido para o desenvolvimento de fármacos e agentes de imagem, esta Tese tem como objetivo explorar a possibilidade de se utilizarem os péptidos pepRF1 e os seus derivados mais curtos (PepRF1_C2: LKRWGTIKKSKAINV-NH2; PepRF1_C4: LKRWGTIKKSKAI-NH2, PepRF1_C6: LKRWGTIKKSK-NH2; PepRF1_C8: LKRWGTIKK-NH2 e PepRF1_C10: LKRWGTI-NH2), marcados com gálio-67 (67Ga), como agentes de imagem es-pecíficos para CXCR4 e imagiologia do cancro. De forma a atingir este objetivo, os péptidos foram conjugados com o quelato bifuncional NODA-GA, resultando respetivamente nos conjugados peptídicos do tipo PepRF1_X_NOD (X= C2, C4, C6, C8 e C10), os quais foram caracterizados por RP-HPLC e ESI-MS. A reação dos conjugados peptídicos com 67GaCl3, resultou em péptidos marcados do tipo 67Ga-PepRF1_X_NOD, com rendimentos e pureza radioquímica >95%. A identidade química dos conjugados peptídicos radioativos foi obtida através da comparação dos tempos de retenção em RP-HPLC nos cromatogramas (deteção-γ) com os dos análogos não radioativos (deteção UV). Os estudos de captação celular dos radiopéptidos em linhas celulares de cancro com diferentes níveis de expressão de CXCR4 demonstram que não correlação entre os baixos va-lores de captação obtidos (ca. 1-2%) e a expressão de CXCR4. Os estudos de biodistribuição em ratinhos CD-1 saudáveis mostram que os radiopépti-dos têm um perfil farmacocinético semelhante no que diz respeito à clearance sanguínea, uma rápida acumulação nos rins e no fígado e valores de acumulação irrelevantes em outros órgãos principais. A excreção total dos radiopéptidos do corpo do animal, predominantemente atra-vés do sistema urinário, varia entre níveis moderados a níveis rápidos (32.6 ± 3.2 até 87.9 ± 5.8 % IA)

The role of specific MHV-68 genes in persistent infection in the lung and virus pathogenesis

The gammaherpesvirus subfamily has long been the study of intensive investigation owing to the association between infection and development of lymphoproliferative disease. Well-known members ofthe Gammaherpesvirinae include Epstein-Barr virus (EBV) and Kaposi's Sarcoma-associated herpesvirus (KSHV). Common properties of gammaherpesviruses include a narrow host range of infection and limited productive growth in vitro, and these factors make the study of acute infection problematic. Murine gammaherpesvirus-68 (MHV-68) is able to undergo lytic replication in a range of cell types in vitro and can infect inbred strains of mice. These properties make MHV-68 an excellent model for the study of gammaherpesvirus pathogenesis.Herpesviruses have been indicated in development of diseases in the lung, including pneumonia and idiopathic pulmonary fibrosis. MHV-68 allows investigation of gammaherpesvirus infection of and persistence in the lung - following intranasal inoculation the virus establishes a life-long infection in this organ, with virus persisting in epithelial cells and/or B cells. Identification of key viral genes required for persistence may allow for development of vaccination and/or treatment strategies. Using real-time PCR the long-term viral load in the lungs was reduced following the deletion of key genes from the viral genome. Genes identified are the thymidine kinase gene, previously shown to play a role during acute infection of the lung and ORF73, a homologue of the KSHV LANA-1 gene. Initial data also suggests that the ORF72 and Mil genes, both involved in reactivation from latency, may play a role in maintaining viral load at late time points post-infection.In vivo investigation of the Ml gene of MHV-68 has demonstrated a potential role in control of viral reactivation from latency in the spleen. A novel MHV-68 mutant, MIA, lacking 1171 bp of the Ml ORF, was used to study the role of Ml in pathogenesis. Initial data suggests that in vivo infection with MIA results in increased viral titres during acute infection of the lung, indicating a potential role in control of initial infection. The major role of Ml appears to be during acute phase latency in the spleen, with the MIA virus failing to drive splenomegaly and establishing latency at lower levels. Despite the presence of fewer latently infected splenocytes, MIA reactivates at significantly higher levels, indicating that a function of Ml is to control viral reactivation from latency.A viral mutant (M4Inl) was created that carries a stop codon inserted at genome co¬ ordinate 8386 in the region between the M3 and M4 genes. The mutation is thought to be in an untranscribed region of the genome, potentially in the promoter region of the M3 or M4 genes. Studies demonstrated that the virus is attenuated following infection of both wild-type and IFNyR " mice with respect to lung pathology scores. The lethality of M4Inl in juvenile IFNyR." mice is reduced compared with wild-type MHV-68 infection. Despite the location of the mutation within potential promoter regions, M4Inl transcribes both M3 and M4 at wild type levels in vitro, and in vivo in the spleen. This evidence suggests an apparently untranscribed region of the MHV-68 genome is able to influence pathogenesis in the lung independent of the neighbouring genes

Evaluation of Broad Anti-Herpesviral Activity with α-Hydroxytropolones

Herpesviruses are ubiquitous in animals and cause economic losses concomitant with many diseases, including upper respiratory disease, keratitis, abortion, neonatal death, and neurologic disease. The majority of the domestic animal herpesviruses are within the subfamily Alphaherpesvirinae, along with the prototypical human herpes simplex virus 1 (HSV-1). Suppression of HSV-1 replication has been reported with α-hydroxytropolones (αHTs), which are aromatic ring compounds that have broad bioactivity due to potent chelating activity. It is postulated that αHTs inhibit enzymes within the nucleotidyltransferase superfamily (NTS), similarly structured enzymes that require divalent cations for nucleic acid cleavage activity. One potential herpesviral target includes the nuclease of the viral terminase, a highly conserved NTS-like enzyme that cleaves the viral genome for packaging into capsids. Inhibition of the nuclease activity of the viral terminase (pUL15C) by αHTs previously revealed variable potencies, ranging from negligible to marked. Interestingly, the most potent anti-terminase nuclease αHT compounds had limited effect on inhibiting HSV-1 replication. The aim of this study was to evaluate three different αHT molecules with varying in vitro anti-terminase nuclease activity against veterinary herpesviruses (BoHV-1, EHV-1, FHV-1) and HSV-1 to assess for broad inhibitory activity. Additionally, given the discordant potencies between anti-pUL15C and HSV-1 inhibition, a second objective was to elucidate the mechanism of action of these compounds. The results of this research show that αHTs broadly inhibit herpesviruses, with similar inhibitory effect among HSV-1, BoHV-1, EHV-1, and FHV-1 with IC50 values ranging from 30 to ≤ 5 μM. Based on immunoblotting, Southern blotting, and real-time qPCR, the compounds were found to specifically inhibit DNA replication. Thus, αHTs may represent a new class of anti-herpesviral compounds