Abstract:a b s t r a c tHuman immunodeficiency virus (HIV) infection yields a high level of non-integrated viral DNA in the infected cells and up to 99% of total viral DNA can be capable of transcription. This capability of non-integrated viral DNA is reducing the efficacy of anti-HIV drug development approaches that solely focus on the integration reactions of viral replication. Using kinetic modeling, we show the transient coordinated regulation of viral DNA production by retrovirus-encoded interacting enzymes revers… Show more
“… 37 Similarly, previous studies have reported that mutations of retroviral integrase affected reverse transcription. 38 , 39 , 40 Figure 4 Genome Integrations of Retroviral Vectors When Having Residual Primer-Binding Site Sequences (A) Schematic overview of retroviral genomic DNA processing during integration. Left panel: if RNA processing during reverse transcription is incomplete, residual primer-binding site sequences can form in the 3′ LTR end of the provirus (refer to Figure S2 ).…”
The unique ability of retroviruses to integrate genes into host genomes is of great value for long-term expression in gene therapy, but only when integrations occur at safe genomic sites. To reap the benefit of using retroviruses without severe detrimental effects, we developed several murine leukemia virus (MLV)-based gammaretroviral vectors with safer integration patterns by perturbing the structure of the integrase via insertion of DNA-binding zinc-finger domains (ZFDs) into an internal position of the enzyme. ZFD insertion significantly reduced the inherent, strong MLV integration preference for genomic regions near transcriptional start sites (TSSs), which are the most dangerous spots. The altered retroviral integration pattern was related to increased formation of residual primer-binding site sequences at the 3′ end of proviruses. Several ZFD insertion mutants showed lower frequencies of integrations into the TSS genome regions when having the residual primer-binding site sequences in the proviruses. Our findings not only can extend the use of retroviruses in biomedical applications, but also provide a glimpse into the mechanisms underlying retroviral integration.
“… 37 Similarly, previous studies have reported that mutations of retroviral integrase affected reverse transcription. 38 , 39 , 40 Figure 4 Genome Integrations of Retroviral Vectors When Having Residual Primer-Binding Site Sequences (A) Schematic overview of retroviral genomic DNA processing during integration. Left panel: if RNA processing during reverse transcription is incomplete, residual primer-binding site sequences can form in the 3′ LTR end of the provirus (refer to Figure S2 ).…”
The unique ability of retroviruses to integrate genes into host genomes is of great value for long-term expression in gene therapy, but only when integrations occur at safe genomic sites. To reap the benefit of using retroviruses without severe detrimental effects, we developed several murine leukemia virus (MLV)-based gammaretroviral vectors with safer integration patterns by perturbing the structure of the integrase via insertion of DNA-binding zinc-finger domains (ZFDs) into an internal position of the enzyme. ZFD insertion significantly reduced the inherent, strong MLV integration preference for genomic regions near transcriptional start sites (TSSs), which are the most dangerous spots. The altered retroviral integration pattern was related to increased formation of residual primer-binding site sequences at the 3′ end of proviruses. Several ZFD insertion mutants showed lower frequencies of integrations into the TSS genome regions when having the residual primer-binding site sequences in the proviruses. Our findings not only can extend the use of retroviruses in biomedical applications, but also provide a glimpse into the mechanisms underlying retroviral integration.
“…HIV-1 RT has been identified to physically interact with viral integrase by using GST pulldown assays, coimmunoprecipitation assays, dot blot assays, NMR spectroscopy analyses, and surface plasmon resonance analyses (101)(102)(103)(104)(105)(106)(107)(108). The binding of integrase to RT does not require multimeric integrase or an integrase with complete enzymatic activity (108).…”
Section: Rt-integrase Interactionmentioning
confidence: 99%
“…HIV integrase has been determined to physically interact with RT by using GST pulldown assays, coimmunoprecipitation assays, dot blot assays, NMR spectroscopy, and surface plasmon resonance analyses (101)(102)(103)(104)(105)(106)(107). Two functions of the integrase-RT interaction have been proposed.…”
SUMMARYThe HIV genome encodes a small number of viral proteins (i.e., 16), invariably establishing cooperative associations among HIV proteins and between HIV and host proteins, to invade host cells and hijack their internal machineries. As a known example, the HIV envelope glycoprotein GP120 is closely associated with GP41 for viral entry. From a genome-wide perspective, a hypothesis can be worked out to determine whether 16 HIV proteins could develop 120 possible pairwise associations either by physical interactions or by functional associations mediated via HIV or host molecules. Here, we present the first systematic review of experimental evidence on HIV genome-wide protein associations using a large body of publications accumulated over the past 3 decades. Of 120 possible pairwise associations between 16 HIV proteins, at least 34 physical interactions and 17 functional associations have been identified. To achieve efficient viral replication and infection, HIV protein associations play essential roles (e.g., cleavage, inhibition, and activation) during the HIV life cycle. In either a dispensable or an indispensable manner, each HIV protein collaborates with another viral protein to accomplish specific activities that precisely take place at the proper stages of the HIV life cycle. In addition, HIV genome-wide protein associations have an impact on anti-HIV inhibitors due to the extensive cross talk between drug-inhibited proteins and other HIV proteins. Overall, this study presents for the first time a comprehensive overview of HIV genome-wide protein associations, highlighting meticulous collaborations between all viral proteins during the HIV life cycle.
“…RT and IN proteins play central roles in the HIV-1 life cycle. The replication of HIV-1 upon successful penetration of the host cell requires the reverse transcription of viral RNA genome and the integration of newly synthesized pro-viral DNA into the host genome by RT and IN for reverse transcription of the RNA genome and integration, respectively ( Chakraborty et al. 2013 ).…”
The replication of human immunodeficiency virus-1 (HIV-1) requires reverse transcription of the viral RNA genome and integration of newly synthesized pro-viral DNA into the host genome. This is mediated by the viral proteins reverse transcriptase (RT) and integrase (IN). The formation and stabilization of the pre-integration complex (PIC), which is an essential step for reverse transcription, nuclear import, chromatin targeting, and subsequent integration, involves direct and indirect modes of interaction between RT and IN proteins. While epitope-based treatments targeting IN–viral DNA and IN–RT complexes appear to be a promising combination for an anti-HIV treatment, the mechanisms of IN-RT interactions within the PIC are not well understood due to the transient nature of the protein complex and the intrinsic flexibility of its components. Here, we identify potentially interacting regions between the IN and RT proteins within the PIC through the coevolutionary analysis of amino acid sequences of the two proteins. Our results show that specific regions in the two proteins have strong coevolutionary signatures, suggesting that these regions either experience direct and prolonged interactions between them that require high affinity and/or specificity or that the regions are involved in interactions mediated by dynamic conformational changes and, hence, may involve both direct and indirect interactions. Other regions were found to exhibit weak, but positive correlations, implying interactions that are likely transient and/or have low affinity. We identified a series of specific regions of potential interactions between the IN and RT proteins (e.g., specific peptide regions within the C-terminal domain of IN were identified as potentially interacting with the Connection domain of RT). Coevolutionary analysis can serve as an important step in predicting potential interactions, thus informing experimental studies. These studies can be integrated with structural data to gain a better understanding of the mechanisms of HIV protein interactions.
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