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The frequency of cardiovascular disorders is increasing in HIV-infected individuals despite a significant reduction in the viral load by antiretroviral therapies (ART). Since the CD4 + T-cells are responsible for the viral load as well as immunological responses, we hypothesized that chronic HIV-infection of T-cells produces novel proteins/enzymes that cause cardiac dysfunctions. To identify specific factors that might cause cardiac disorders without the influence of numerous cofactors produced by other pathogenic microorganisms that co-inhabit most HIV-infected individuals, we analyzed genome-wide proteomes of a CD4 + T-cell line at different stages of HIV replication and cell growth over > 6 months. Subtractive analyses of several hundred differentially regulated proteins from HIV-infected and uninfected counterpart cells and comparisons with proteins expressed from the same cells after treating with the antiviral drug Zidovudine/AZT and inhibiting virus replication, identified a well-coordinated network of 12 soluble/diffusible proteins in HIV-infected cells. Functional categorization, bioinformatics and statistical analyses of each protein predicted that the expression of cardiac-specific Ca2 + kinase together with multiple Ca2 + release channels causes a sustained overload of Ca2 + in the heart which induces fetal/cardiac myosin heavy chains (MYH6 and MYH7) and a myosin light-chain kinase. Each of these proteins has been shown to cause cardiac stress, arrhythmia, hypertrophic signaling, cardiomyopathy and heart failure (p = 8 × 10(- 11)). Translational studies using the newly discovered proteins produced by HIV infection alone would provide additional biomarkers that could be added to the conventional markers for an early diagnosis and/or development of specific therapeutic interventions for heart diseases in HIV-infected individuals.

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BACKGROUND: MicroRNAs (miRNAs) are highly conserved, short (18-22 nts), non-coding RNA molecules that regulate gene expression by binding to the 3' untranslated regions (3'UTRs) of mRNAs. While numerous cellular microRNAs have been associated with the progression of various diseases including cancer, miRNAs associated with retroviruses have not been well characterized. Herein we report identification of microRNA-like sequences in coding regions of several HIV-1 genomes. RESULTS: Based on our earlier proteomics and bioinformatics studies, we have identified 8 cellular miRNAs that are predicted to bind to the mRNAs of multiple proteins that are dysregulated during HIV-infection of CD4+ T-cells in vitro. In silico analysis of the full length and mature sequences of these 8 miRNAs and comparisons with all the genomic and subgenomic sequences of HIV-1 strains in global databases revealed that the first 18/18 sequences of the mature hsa-miR-195 sequence (including the short seed sequence), matched perfectly (100%), or with one nucleotide mismatch, within the envelope (env) genes of five HIV-1 genomes from Africa. In addition, we have identified 4 other miRNA-like sequences (hsa-miR-30d, hsa-miR-30e, hsa-miR-374a and hsa-miR-424) within the env and the gag-pol encoding regions of several HIV-1 strains, albeit with reduced homology. Mapping of the miRNA-homologues of env within HIV-1 genomes localized these sequence to the functionally significant variable regions of the env glycoprotein gp120 designated V1, V2, V4 and V5. CONCLUSIONS: We conclude that microRNA-like sequences are embedded within the protein-encoding regions of several HIV-1 genomes. Given that the V1 to V5 regions of HIV-1 envelopes contain specific, well-characterized domains that are critical for immune responses, virus neutralization and disease progression, we propose that the newly discovered miRNA-like sequences within the HIV-1 genomes may have evolved to self-regulate survival of the virus in the host by evading innate immune responses and therefore influencing persistence, replication and/or pathogenicity.

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BACKGROUND: Kaposi's sarcoma (KS), hemangioma, and other angioproliferative diseases are highly prevalent in HIV-infected individuals. While KS is etiologically linked to the human herpesvirus-8 (HHV8) infection, HIV-patients without HHV-8 and those infected with unrelated viruses also develop angiopathies. Further, HIV-Tat can activate protein-tyrosine-kinase (PTK-activity) of the vascular endothelial growth factor receptor involved in stimulating angiogenic processes. However, Tat by itself or HHV8-genes alone cannot induce angiogenesis in vivo unless specific proteins/enzymes are produced synchronously by different cell-types. We therefore tested a hypothesis that chronic HIV-replication in non-endothelial cells may produce novel factors that provoke angiogenic pathways. METHODS: Genome-wide proteins from HIV-infected and uninfected T-lymphocytes were tested by subtractive proteomics analyses at various stages of virus and cell growth in vitro over a period of two years. Several thousand differentially regulated proteins were identified by mass spectrometry (MS) and >200 proteins were confirmed in multiple gels. Each protein was scrutinized extensively by protein-interaction-pathways, bioinformatics, and statistical analyses. RESULTS: By functional categorization, 31 proteins were identified to be associated with various signaling events involved in angiogenesis. 88% proteins were located in the plasma membrane or extracellular matrix and >90% were found to be essential for regeneration, neovascularization and angiogenic processes during embryonic development. CONCLUSION: Chronic HIV-infection of T-cells produces membrane receptor-PTKs, serine-threonine kinases, growth factors, adhesion molecules and many diffusible signaling proteins that have not been previously reported in HIV-infected cells. Each protein has been associated with endothelial cell-growth, morphogenesis, sprouting, microvessel-formation and other biological processes involved in angiogenesis (p = 10-4 to 10-12). Bioinformatics analyses suggest that overproduction of PTKs and other kinases in HIV-infected cells has suppressed VEGF/VEGFR-PTK expression and promoted VEGFR-independent pathways. This unique mechanism is similar to that observed in neovascularization and angiogenesis during embryogenesis. Validation of clinically relevant proteins by gene-silencing and translational studies in vivo would identify specific targets that can be used for early diagnosis of angiogenic disorders and future development of inhibitors of angiopathies. This is the first comprehensive study to demonstrate that HIV-infection alone, without any co-infection or treatment, can induce numerous "embryonic" proteins and kinases capable of generating novel VEGF-independent angiogenic pathways.

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BACKGROUND: HIV-infected patients develop multiple metabolic abnormalities including insulin resistance, lipodystrophy and dyslipidemia. Although progression of these disorders has been associated with the use of various protease inhibitors and other antiretroviral drugs, HIV-infected individuals who have not received these treatments also develop lipid abnormalities albeit to a lesser extent. How HIV alters lipid metabolism in an infected cell and what molecular changes are affected through protein interaction pathways are not well-understood. RESULTS: Since many genetic, epigenetic, dietary and other factors influence lipid metabolism in vivo, we have chosen to study genome-wide changes in the proteomes of a human T-cell line before and after HIV infection in order to circumvent computational problems associated with multiple variables. Four separate experiments were conducted including one that compared 14 different time points over a period of >3 months. By subtractive analyses of protein profiles overtime, several hundred differentially expressed proteins were identified in HIV-infected cells by mass spectrometry and each protein was scrutinized for its biological functions by using various bioinformatics programs. Herein, we report 18 HIV-modulated proteins and their interaction pathways that enhance fatty acid synthesis, increase low density lipoproteins (triglycerides), dysregulate lipid transport, oxidize lipids, and alter cellular lipid metabolism. CONCLUSIONS: We conclude that HIV replication alone (i.e. without any influence of antiviral drugs, or other human genetic factors), can induce novel cellular enzymes and proteins that are significantly associated with biologically relevant processes involved in lipid synthesis, transport and metabolism (p = <0.0002-0.01). Translational and clinical studies on the newly discovered proteins may now shed light on how some of these proteins may be useful for early diagnosis of individuals who might be at high risk for developing lipid-related disorders. The target proteins could then be used for future studies in the development of inhibitors for preventing lipid-metabolic anomalies. This is the first direct evidence that HIV-modulates production of proteins that are significantly involved in disrupting the normal lipid-metabolic pathways.

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Present day approaches for the determination of protein-protein interaction networks are usually based on two hybrid experimental measurements. Here we consider a computational method that uses another type of experimental data: instead of direct information about protein-protein interactions, we consider data in the form of protein complexes. We propose a method for using these complexes to provide predictions of protein-protein interactions. When applied to a dataset obtained from a cat melanoma cell line we find that we are able to predict when a protein pair belongs to a complex with approximately 96% accuracy. Further, we are able to extrapolate the experimentally identified interaction pairs to the entire cat proteome.

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BACKGROUND: The amphotropic murine leukemia viruses (MuLV-A's) are naturally occurring, exogenously acquired gammaretroviruses that are indigenous to the Southern California wild mice. These viruses replicate in a wide range of cell types including human cells in vitro and they can cause both hematological and neurological disorders in feral as well as in the inbred laboratory mice. Since MuLV-A's also exhibit discrete interference and neutralization properties, the envelope proteins of these viruses have been extremely useful for studying virus-host cell interactions and as vehicles for transfer of foreign genes into a variety of hosts including human cells. However, the genomic structure of any of the several known MuLV-A's has not been established and the evolutionary relationship of amphotropic retroviruses to the numerous exogenous or endogenous MuLV strains remains elusive. Herein we present a complete genetic structure of a novel amphotropic virus designated MuLV-1313 and demonstrate that this retrovirus together with other MuLV-A's belongs to a distinct molecular, biological and phylogenetic class among the MuLV strains isolated from a large number of the laboratory inbred or feral mice. RESULTS: The host range of MuLV-1313 is similar to the previously isolated MuLV-A's except that this virus replicates efficiently in mammalian as well as in chicken cells. Compared to ENV proteins of other MuLV-A's (4070A, 1504A and 10A-1), the gp70 protein of MuLV-1313 exhibits differences in its signal peptides and the proline-rich hinge regions. However, the MuLV-1313 envelope protein is totally unrelated to those present in a broad range of murine retroviruses that have been isolated from various inbred and feral mice globally. Genetic analysis of the entire MuLV-1313 genome by dot plot analyses, which compares each nucleotide of one genome with the corresponding nucleotide of another, revealed that the genome of this virus, with the exception of the env gene, is more closely related to the biologically distinct wild mouse ecotropic retrovirus (Cas-Br-E) isolated from another region of the Southern California, than to any of the 15 MuLV strains whose full-length sequences are present in the GenBank. This finding was corroborated by phylogenetic analyses and hierarchical clustering of the entire genomic sequence of MuLV-1313, which also placed all MULV-A's in a genetically distinct category among the large family of retroviruses isolated from numerous mouse strains globally. Likewise, construction of separate dendrograms for each of the Gag, Pol and Env proteins of MuLV-1313 demonstrated that the amphotropic retroviruses belong to a phylogenetically exclusive group of gammaretroviruses compared to all known MuLV strains. CONCLUSION: The molecular, biological and phylogenetic properties of amphotropic retroviruses including MuLV-1313 are distinct compared to a large family of exogenously- or endogenously-transmitted ecotropic, polytropic and xenotropic MuLV strains of the laboratory and feral mice. Further, both the naturally occurring amphotropic and a biologically discrete ecotropic retrovirus of the Southern California wild mice are more closely related to each other on the evolutionary tree than any other mammalian gammaretrovirus indicating a common origin of these viruses. This is the first report of a complete genomic analysis of a unique group of phylogenetically distinct amphotropic virus.

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We have documented evidence of the human immunodeficiency virus (HIV) infection in four individuals including a blood donor and three members of a family (husband, wife, and a child) in Karachi, Pakistan. Our data indicate that HIV has been transmitted to the wife of the seropositive male, a drug abuser, in the recent past and that she has passed the virus to her newborn child perinatally/ transplacentally. The two seropositive males (blood donor and drug abuser) were diagnosed clinically as having persistent generalized lymphadenopathy, an AIDS-related condition (ARC), and the child presented with developmental disorders and neurologic manifestations. This is the first report of AIDS/ARC cases from Pakistan that can be directly related to HIV infection and its heterosexual transmission.

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BACKGROUND: Although several genes and proteins have been implicated in the development of melanomas, the molecular mechanisms involved in the development of these tumors are not well understood. To gain a better understanding of the relationship between the cell growth, tumorigenesis and differentiation, we have studied a highly malignant cat melanoma cell line that trans-differentiates into neuronal cells after exposure to a feline endogenous retrovirus RD114. METHODS: To define the repertoire of proteins responsible for the phenotypic differences between melanoma and its counterpart trans-differentiated neuronal cells we have applied proteomics technology and compared protein profiles of the two cell types and identified differentially expressed proteins by 2D-gel electrophoresis, image analyses and mass spectrometry. RESULTS: The melanoma and trans-differentiated neuronal cells could be distinguished by the presence of distinct sets of proteins in each. Although approximately 60-70% of the expressed proteins were shared between the two cell types, twelve proteins were induced de novo after infection of melanoma cells with RD114 virus in vitro. Expression of these proteins in trans-differentiated cells was significantly associated with concomitant down regulation of growth promoting proteins and up-regulation of neurogenic proteins (p = < 0.001). Based on their physiologic properties, >95% proteins expressed in trans-differentiated cells could be associated with the development, differentiation and regulation of nervous system cells. CONCLUSION: Our results indicate that the cat melanoma cells have the ability to differentiate into distinct neuronal cell types and they express proteins that are essential for self-renewal. Since melanocytes arise from the neural crest of the embryo, we conclude that this melanoma arose from embryonic precursor stem cells. This model system provides a unique opportunity to identify domains of interactions between the expressed proteins that halt the tumorigenic potential of melanoma cells and drive them toward neurogenerative pathways involved in early neurogenesis. A better understanding of these proteins in a well-coordinated signaling network would also help in developing novel approaches for suppression of highly malignant tumors that arise from stem-like embryonic cells.

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Los autores revisan y describe métodos moleculares para la detección de agentes etiológicos o secuencias genéticas involucradas en la patogénesis de varias enfermedades. Hay sondas moleculares ya disponibles para el diagnóstico rápido de enfermedades causadas por un gran número de virus, bacterias, hongos, espiroquetas, rickettsias y otros agentes infecciosos. Dado que las sondas de ácidos nucleicos pueden distinguir muy pequeñas diferencias indicativas de mutaciones genéticas o alteraciones, se pueden desarrollar sondas específicas aun para aquellas enfermedades con agentes etiológicos desconocidos. Además usando estas técnicas se han identificado cepas resistentes a medicamentos y cepas patógenas asociadas a epidemias en áreas ampliamente separadas de un país o de una ciudad. Así pues la sensibilidad y la especificidad de los métodods de detección molecular usando sondas radioactivas y no radioactivas están a tal punto que se pueden aplicar a especímenes clínicos para el diagnóstico rápido e identificación de agentes etiológicos responsables de la patogenia de una variedad de enfermedades

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