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Intrabody-based Mapping of Latency-associated Nuclear Antigen from Kaposi’s Sarcoma-associated Herpesvirus Show Conserved Epitopes for Viral Latency Inhibition
Sofia Corte-Real, Lídia Fonseca, Carlos Barbas III and Joao Goncalves
Virology: Research and Treatment , 2012, DOI: 10.4137/VRT.S975
Abstract: Kaposi’s sarcoma associated herpesvirus (KSHV or human herpesvirus 8 [HHV-8]) is a gammaherpesvirus highly associated with KS, primary effusion lymphoma (PEL), and multicentric Castleman’s disease, an aggressive lymphoproliferative disorder. KSHV, like other gammaherpesvirus latently infects predominantly B-cells and endothelial cells. Infected cells retain the virus from one generation to the next existing as a multicopy circular episomal DNA in the nucleus, expressing a limited subset of viral genes. Of these latently expressed genes, LANA1, the latency associated nuclear antigen is highly expressed in all forms of KS-associated malignancies. Various studies so far show that LANA1 tethers the viral episomes to host chromosomes and binds to specific sites within and close to the TR elements contributing to the stable maintenance of the viral episomes in successive daughter cells. Anti-LANA1 intrabody strategies might represent a new therapeutic approach to treatment of KSHV infections, since LANA1 is regained for KSHV latency. In addition, the use of intrabodies can help drug development by mapping LANA1 inhibiting regions. We report development of several LANA1 specific single chain antibodies from immunized rabbits that can be expressed intracellularly, bind to LANA1 epitopes and can be used for functional KSHV studies on viral latency.
Intrabody-based Mapping of Latency-associated Nuclear Antigen from Kaposi’s Sarcoma-associated Herpesvirus Show Conserved Epitopes for Viral Latency Inhibition
Sofia Corte-Real,Lídia Fonseca,Carlos Barbas III,Joao Goncalves
Virology: Research and Treatment , 2010,
Abstract: Kaposi’s sarcoma associated herpesvirus (KSHV or human herpesvirus 8 [HHV-8]) is a gammaherpesvirus highly associated with KS, primary effusion lymphoma (PEL), and multicentric Castleman’s disease, an aggressive lymphoproliferative disorder. KSHV, like other gammaherpesvirus latently infects predominantly B-cells and endothelial cells. Infected cells retain the virus from one generation to the next existing as a multicopy circular episomal DNA in the nucleus, expressing a limited subset of viral genes. Of these latently expressed genes, LANA1, the latency associated nuclear antigen is highly expressed in all forms of KS-associated malignancies. Various studies so far show that LANA1 tethers the viral episomes to host chromosomes and binds to specific sites within and close to the TR elements contributing to the stable maintenance of the viral episomes in successive daughter cells. Anti-LANA1 intrabody strategies might represent a new therapeutic approach to treatment of KSHV infections, since LANA1 is regained for KSHV latency. In addition, the use of intrabodies can help drug development by mapping LANA1 inhibiting regions. We report development of several LANA1 specific single chain antibodies from immunized rabbits that can be expressed intracellularly, bind to LANA1 epitopes and can be used for functional KSHV studies on viral latency.
Impact of Viral Factors on Subcellular Distribution and RNA Export Activity of HIV-1 Rev in Astrocytes 1321N1  [PDF]
Atoshi Banerjee, Ronald Benjamin, Sharmistha Banerjee
PLOS ONE , 2013, DOI: 10.1371/journal.pone.0072905
Abstract: CNS associated cells are permissive to HIV-1 infection, but poor in virus production due to attenuated Rev activity. The temporal and the spatial distribution of Rev in human astrocyte 1321N1 and glioblastoma GO-G-CCM were monitored for explaining the reduced Rev activity and low viral production during HIV-1 infection. Rev remained localized to the nuclei of these cells upon infection, attenuating its export activity, as manifested by low copy number of RRE-containing viral-mRNA in the cytoplasm of these cells. In contrast to infection, when Rev alone was transiently expressed, it localized in the cytoplasm of 1321N1. The localization changed to the nucleus when Rev was expressed in the presence of other viral proteins through pro-viral DNA pNL4-3. This study, for the first time, revealed the impact of other HIV-1 proteins apart from host factors in regulating the subcellular localization of Rev in astrocytes and hence the fate of HIV-1 infection in these cells.
PRMT6 diminishes HIV-1 Rev binding to and export of viral RNA
Cédric F Invernizzi, Baode Xie, Stéphane Richard, Mark A Wainberg
Retrovirology , 2006, DOI: 10.1186/1742-4690-3-93
Abstract: Here, we report the methylation of Rev due to a single arginine dimethylation in the N-terminal portion of its arginine rich motif and the association of Rev with PRMT6 in vivo. Further analysis demonstrated that the presence of increasing amounts of wild-type PRMT6, as well as a methylation-inactive mutant PRMT6, dramatically down-regulated Rev protein levels in concentration-dependent fashion, which was not dependent on the methyltransferase activity of PRMT6. Quantification of Rev mRNA revealed that attenuation of Rev protein levels was due to a posttranslational event, carried out by a not yet defined activity of PRMT6. However, no relevant protein attenuation was observed in subsequent chloramphenicol acetyltransferase (CAT) expression experiments that screened for RNA export and interaction with the RRE. Binding of the Rev arginine rich motif to the RRE was reduced in the presence of wild-type PRMT6, whereas mutant PRMT6 did not exert this negative effect. In addition, diminished interactions between viral RNA and mutant Rev proteins were observed, due to the introduction of single arginine to lysine substitutions in the Rev arginine rich motif. More importantly, wild-type PRMT6, but not mutant methyltransferase, significantly decreased Rev-mediated viral RNA export from the nucleus to the cytoplasm in a dose-dependent manner.These findings indicate that PRMT6 severely impairs the function of HIV-1 Rev.Human immunodeficiency virus type 1 (HIV-1) encodes a 116 amino acid regulator of viral protein expression termed Rev. This protein is found in the nucleolus, the perinuclear zone and the cytoplasm of infected cells [1,2]. A two-exon version of Rev is translated from fully spliced viral RNA during early stages of viral replication and mediates nuclear export of unspliced and partially spliced HIV-1 RNA [2]. Rev interacts with the cis-acting Rev response element (RRE) located in the env gene [3]. Shuttling of Rev between nucleus and cytoplasm is dependent on seve
Peptides Derived from HIV-1 Integrase that Bind Rev Stimulate Viral Genome Integration  [PDF]
Aviad Levin, Zvi Hayouka, Markus Helfer, Ruth Brack-Werner, Assaf Friedler, Abraham Loyter
PLOS ONE , 2009, DOI: 10.1371/journal.pone.0004155
Abstract: Background The human immunodeficiency virus type 1 (HIV-1) integrase protein (IN), catalyzes the integration of viral DNA into the host cell genome. IN catalyzes the first step of the integration process, namely the 3′-end processing in which IN removes a pGT dinucleotide from the 3′ end of each viral long terminal repeat (LTR). Following nuclear import of the viral preintegration complex, the host chromosomal DNA becomes accessible to the viral cDNA and the second step of the integration process, namely the strand-transfer step takes place. This ordered sequence of events, centered on integration, is mandatory for HIV replication. Methodology/Principal Findings Using an integrase peptide library, we selected two peptides, designated INr-1 and INr-2, which interact with the Rev protein and probably mediate the Rev-integrase interaction. Using an in-vitro assay system, we show that INr-1 and INr-2 are able to abrogate the inhibitory effects exerted by Rev and Rev-derived peptides on integrase activity. Both INr-1 and INr-2 were found to be cell-permeable and nontoxic, allowing a study of their effect in HIV-1-infected cultured cells. Interestingly, both INr peptides stimulated virus infectivity as estimated by production of the viral P24 protein, as well as by determination of the appearance of newly formed virus particles. Furthermore, kinetics studies revealed that the cell-permeable INr peptides enhance the integration process, as was indeed confirmed by direct determination of viral DNA integration by real-time PCR. Conclusions/Significance The results of the present study raise the possibility that in HIV-infected cells, the Rev protein may be involved in the integration of proviral DNA by controlling/regulating the activity of the integrase. Release from such inhibition leads to stimulation of IN activity and multiple viral DNA integration events.
The HIV-1 Rev/RRE system is required for HIV-1 5' UTR cis elements to augment encapsidation of heterologous RNA into HIV-1 viral particles
Adam S Cockrell, Henriette van Praag, Nicholas Santistevan, Hong Ma, Tal Kafri
Retrovirology , 2011, DOI: 10.1186/1742-4690-8-51
Abstract: We show for the first time that the Rev/RRE system can augment RNA encapsidation independent of all cis elements from the 5' UTR (R, U5, PBS, and ψ). Incorporation of all the 5' UTR cis elements did not enhance RNA encapsidation in the absence of the Rev/RRE system. In fact, we demonstrate that the Rev/RRE system is required for specific and efficient encapsidation commonly associated with the canonical packaging signal. The mechanism of Rev/RRE-mediated encapsidation is not a general phenomenon, since the combination of the Rev/RRE system and 5' UTR cis elements did not enhance encapsidation into MLV-derived viral particles. Lastly, we show that heterologous MLV RNAs conform to transduction properties commonly associated with HIV-1 viral particles, including in vivo transduction of non-dividing cells (i.e. mouse neurons); however, the cDNA forms are episomes predominantly in the 1-LTR circle form.Premised on encapsidation of a heterologous RNA into HIV-1 viral particles, our findings define a functional HIV-1 packaging system as comprising the 5' UTR cis elements, Gag, and the Rev/RRE system, in which the Rev/RRE system is required to make the RNA amenable to the ensuing interaction between Gag and the canonical packaging signal for subsequent encapsidation.Specific and efficient encapsidation of HIV-1 gRNA into viral particles is a multifaceted process of relocating the gRNA following transcription in the nucleus to sites of particle assembly at the plasma membrane. Cis packaging signals in the viral RNA confer specific selection among the milieu of host cell RNAs through interactions with trans factors encoded by the virus, and host cell. The conventional canonical cis packaging signal (ψ) is a ~120 bp fragment comprised of four stem-loop structures located in the HIV-1 5' untranslated region (UTR), and extending into the 5' end of the HIV-1 Gag coding sequence [1]. Interactions of the Gag polyprotein with stem-loops 2, 3, and 4 ensure efficient encapsidation of
Matrin 3 is a co-factor for HIV-1 Rev in regulating post-transcriptional viral gene expression
Venkat SRK Yedavalli, Kuan-Teh Jeang
Retrovirology , 2011, DOI: 10.1186/1742-4690-8-61
Abstract: The nucleus is a highly organized structure. Chromosomes occupy discrete regions, and specific proteins and nucleic acids are enriched in subnuclear structures such as nuclear lamina, nucleoli, Cajal bodies, nuclear speckles, and paraspeckles [1-6]. The nuclear matrix, a network of underlying filaments in the cell nucleus, shapes the nuclear architecture and functions in genome maintenance, transcription and RNA metabolism [7-17]. Accordingly, the nuclear matrix has important roles in tissue development and cellular proliferation; and the disruption of nuclear organization is often correlated with disease states such as the loss of subnuclear promyelocytic leukemia bodies in acute promyelocytic leukemia [18-21].HIV-1 gene expression and replication are regulated at transcriptional and post-transcriptional steps including the transactivation of the HIV-1 LTR by Tat [22] and the export of unspliced or partially spliced viral RNAs from the nucleus to the cytoplasm by Rev [23-26]. Rev is a trans-acting viral protein which binds to a cis-acting Rev responsive element (RRE) present in unspliced and partially spliced HIV transcripts. Rev has been shown to interact with cellular proteins CRM1, DDX3, PIMT and others to mediate the export of unspliced and singly spliced viral RNAs [27-30]. The mechanism of viral RNA export by Rev is discrete from the export pathways used by fully spliced HIV-1 mRNAs, CTE- (constitutive transport element) dependent RNAs, and cellular mRNAs [31-43].Recently, numerous studies have implicated the nuclear matrix in gene transcription, RNA splicing, and transport of cellular RNAs [5,7,9,44,45]; however, the role of the nuclear matrix in HIV-1 gene expression has been poorly explored [46-48]. Here, we identify Matrin 3 as a key component of factors that mediate the post-transcriptional regulation of HIV-1. Matrin 3 is a highly conserved inner nuclear matrix protein which has been previously shown to play a role in transcription [49-52]. It interacts
Viral Diversity and Diversification of Major Non-Structural Genes vif, vpr, vpu, tat exon 1 and rev exon 1 during Primary HIV-1 Subtype C Infection  [PDF]
Raabya Rossenkhan, Vladimir Novitsky, Theresa K. Sebunya, Rosemary Musonda, Berhanu A. Gashe, M. Essex
PLOS ONE , 2012, DOI: 10.1371/journal.pone.0035491
Abstract: To assess the level of intra-patient diversity and evolution of HIV-1C non-structural genes in primary infection, viral quasispecies obtained by single genome amplification (SGA) at multiple sampling timepoints up to 500 days post-seroconversion (p/s) were analyzed. The mean intra-patient diversity was 0.11% (95% CI; 0.02 to 0.20) for vif, 0.23% (95% CI; 0.08 to 0.38) for vpr, 0.35% (95% CI; ?0.05 to 0.75) for vpu, 0.18% (95% CI; 0.01 to 0.35) for tat exon 1 and 0.30% (95% CI; 0.02 to 0.58) for rev exon 1 during the time period 0 to 90 days p/s. The intra-patient diversity increased gradually in all non-structural genes over the first year of HIV-1 infection, which was evident from the vif mean intra-patient diversity of 0.46% (95% CI; 0.28 to 0.64), vpr 0.44% (95% CI; 0.24 to 0.64), vpu 0.84% (95% CI; 0.55 to 1.13), tat exon 1 0.35% (95% CI; 0.14 to 0.56 ) and rev exon 1 0.42% (95% CI; 0.18 to 0.66) during the time period of 181 to 500 days p/s. There was a statistically significant increase in viral diversity for vif (p = 0.013) and vpu (p = 0.002). No associations between levels of viral diversity within the non-structural genes and HIV-1 RNA load during primary infection were found. The study details the dynamics of the non-structural viral genes during the early stages of HIV-1C infection.
Multi-Faceted Post-Transcriptional Functions of HIV-1 Rev  [PDF]
Kuan-Teh Jeang
Biology , 2012, DOI: 10.3390/biology1020165
Abstract: Post-transcriptional regulation of HIV-1 gene expression is largely governed by the activities of the viral Rev protein. In this minireview, the multiple post-transcriptional activities of Rev in the export of partially spliced and unspliced HIV-1 RNAs from the nucleus to the cytoplasm, in the translation of HIV-1 transcripts, and in the packaging of viral genomic RNAs are reviewed in brief.
Characterization of the HIV-1 RNA associated proteome identifies Matrin 3 as a nuclear cofactor of Rev function
Anna Kula, Jessica Guerra, Anna Knezevich, Danijela Kleva, Michael P Myers, Alessandro Marcello
Retrovirology , 2011, DOI: 10.1186/1742-4690-8-60
Abstract: Here, we introduce a novel method to explore the proteome associated with the nuclear HIV-1 RNAs. At the core of the method is the generation of cell lines harboring an integrated provirus carrying RNA binding sites for the MS2 bacteriophage protein. Flag-tagged MS2 is then used for affinity purification of the viral RNA. By this approach we found that the viral RNA is associated with the host nuclear matrix component MATR3 (Matrin 3) and that its modulation affected Rev activity. Knockdown of MATR3 suppressed Rev/RRE function in the export of unspliced HIV-1 RNAs. However, MATR3 was able to associate with Rev only through the presence of RRE-containing viral RNA.In this work, we exploited a novel proteomic method to identify MATR3 as a cellular cofactor of Rev activity. MATR3 binds viral RNA and is required for the Rev/RRE mediated nuclear export of unspliced HIV-1 RNAs.Viruses have evolved to optimize their replication potential in the host cell. For this purpose, viruses take advantage of the molecular strategies of the infected host and, therefore, represent invaluable tools to identify novel cellular mechanisms that modulate gene expression [1].The primary viral transcription product is utilized in unspliced and alternatively spliced forms to direct the synthesis of all human immunodeficiency virus (HIV-1) proteins. Although nuclear export of pre-mRNA is restricted in mammalian cells, HIV-1 has evolved the viral Rev protein to overcome this restriction for viral transcripts [2,3], recently reviewed in [4]. Rev promotes the export of unspliced and partially spliced RNAs from the nucleus through the association with an RNA element called the Rev response element (RRE) that is present in the env gene [5-7]. In the cytoplasm, the RRE-containing HIV-1 transcripts serve as templates for the expression of viral structural proteins, and the full-length unspliced forms serve as genomic RNAs that are packaged into viral particles. In order to fulfill its function, Rev re
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