16,17 ✉ and The International AIDS Society (IAS) Global Scientific Strategy working group*Despite the success of antiretroviral therapy (ART) for people living with HIV, lifelong treatment is required and there is no cure. HIV can integrate in the host genome and persist for the life span of the infected cell. These latently infected cells are not recognized as foreign because they are largely transcriptionally silent, but contain replication-competent virus that drives resurgence of the infection once ART is stopped. With a combination of immune activators, neutralizing antibodies, and therapeutic vaccines, some nonhuman primate models have been cured, providing optimism for these approaches now being evaluated in human clinical trials. In vivo delivery of gene-editing tools to either target the virus, boost immunity or protect cells from infection, also holds promise for future HIV cure strategies. In this Review, we discuss advances related to HIV cure in the last 5 years, highlight remaining knowledge gaps and identify priority areas for research for the next 5 years.
Zinc-finger nucleases (ZFNs) are important tools for genome engineering. Despite intense interest by many academic groups, the lack of robust noncommercial methods has hindered their widespread use. The modular assembly (MA) of ZFNs from publicly available one-finger archives provides a rapid method to create proteins that can recognize a very broad spectrum of DNA sequences. However, three-and four-finger arrays often fail to produce active nucleases. Efforts to improve the specificity of the one-finger archives have not increased the success rate above 25%, suggesting that the MA method might be inherently inefficient due to its insensitivity to context-dependent effects. Here we present the first systematic study on the effect of array length on ZFN activity. ZFNs composed of six-finger MA arrays produced mutations at 15 of 21 (71%) targeted loci in human and mouse cells. A novel drop-out linker scheme was used to rapidly assess three-to six-finger combinations, demonstrating that shorter arrays could improve activity in some cases. Analysis of 268 array variants revealed that half of MA ZFNs of any array composition that exceed an ab initio B-score cutoff of 15 were active. These results suggest that, when used appropriately, MA ZFNs are able to target more DNA sequences with higher success rates than other current methods.
The Asian tiger mosquito, Aedes albopictus (Ae. albopictus), is an important vector that transmits arboviruses such as dengue (DENV), Zika (ZIKV) and Chikungunya virus (CHIKV). Long noncoding RNAs (lncRNAs) are known to regulate various biological processes. Knowledge on Ae. albopictus lncRNAs and their functional role in virus-host interactions are still limited. Here, we identified and characterized the lncRNAs in the genome of an arbovirus vector, Ae. albopictus, and evaluated their potential involvement in DENV and ZIKV infection. We used 148 public datasets, and identified a total of 10, 867 novel lncRNA transcripts, of which 5,809, 4,139, and 919 were intergenic, intronic and antisense respectively. The Ae. albopictus lncRNAs shared many characteristics with other species such as short length, low GC content, and low sequence conservation. RNA-sequencing of Ae. albopictus cells infected with DENV and ZIKV showed that the expression of lncRNAs was altered upon virus infection. Target prediction analysis revealed that Ae. albopictus lncRNAs may regulate the expression of genes involved in immunity and other metabolic and cellular processes. To verify the role of lncRNAs in virus infection, we generated mutations in lncRNA loci using CRISPR-Cas9, and discovered that two lncRNA loci mutations, namely XLOC_029733 (novel lncRNA transcript id: lncRNA_27639.2) and LOC115270134 (known lncRNA transcript id: XR_003899061.1) resulted in enhancement of DENV and ZIKV replication. The results presented here provide an important foundation for future studies of lncRNAs and their relationship with virus infection in Ae. albopictus.
There is a continuous search for an HIV cure as the success of ART in blocking HIV replication and the role of CD4 + T cells in HIV pathogenesis and immunity do not entirely eradicate HIV. The Berlin patient, who is virus-free, serves as the best model for a 'sterilizing cure' and many experts are trying to mimic this approach in other patients. Although failures were reported among Boston and Essen patients, the setbacks have provided valuable lessons to strengthen cure strategies. Following the Berlin patient, two more patients known as London and Düsseldorf patients might be the second and third person to be cured of HIV. In all the cases, the patients underwent chemotherapy regimen due to malignancy and hematopoietic stem cell transplantation (HSCT) which required matching donors for CCR5 32 mutation-an approach that may not always be feasible. The emergence of newer technologies, such as long-acting slow-effective release ART (LASER ART) and CRISPR/Cas9 could potentially overcome the barriers due to HIV latency and persistency and eliminate the need for CCR5 32 mutation donor. Appreciating the failure and success stories learned from these HIV breakthroughs would provide some insight for future HIV eradication and cure strategies.
26The Asian tiger mosquito, Aedes albopictus (Ae. albopictus), is an important vector that transmits 27 arboviruses such as dengue (DENV), Zika (ZIKV) and Chikungunya virus (CHIKV). On the other hand, 28 long noncoding RNAs (lncRNAs) are known to regulate various biological processes. Knowledge on Ae.29 albopictus lncRNAs and their functional role in virus-host interactions are still limited. Here, we 30 identified and characterized the lncRNAs in the genome of an arbovirus vector, Ae. albopictus, and 31 evaluated their potential involvement in DENV and ZIKV infection. We used 148 public datasets, and 32 identified a total of 10, 867 novel lncRNA transcripts, of which 5,809, 4,139, and 919 were intergenic, 33 intronic and antisense respectively. The Ae. albopictus lncRNAs shared many characteristics with other 34 species such as short length, low GC content, and low sequence conservation. RNA-sequencing of Ae.35 albopictus cells infected with DENV and ZIKV showed that the expression of lncRNAs was altered upon 36 virus infection. Target prediction analysis revealed that Ae. albopictus lncRNAs may regulate the 37 expression of genes involved in immunity and other metabolic and cellular processes. To verify the role 38 of lncRNAs in virus infection, we generated mutation in lncRNA loci using CRISPR-Cas9, and 39 discovered that two lncRNA loci mutations, namely XLOC_029733 (novel lncRNA transcript id: 40 lncRNA_27639.2) and LOC115270134 (known lncRNA transcript id: XR_003899061.1) resulted in 41 enhancement of DENV and ZIKV replication. The results presented here provide an important 42 foundation for future studies of lncRNAs and their relationship with virus infection in Ae. albopictus. 43 44 45 46 47 Author summary 48 49 Ae. albopictus is an important vector of arboviruses such as dengue and Zika. Studies on virus-50 host interaction at gene expression and molecular level are crucial especially in devising methods to 51 inhibit virus replication in Aedes mosquito. Previous reports showed that, besides protein-coding genes, 52 noncoding RNAs such as lncRNAs are also involved in virus-host interaction. In this study, we report a 53 comprehensive catalog of novel lncRNA transcripts in the genome of Ae. albopictus. We also show that 54 the expression of lncRNAs was altered upon infection with dengue and Zika. Additionally, depletion of 55 certain lncRNAs resulted in increased replication of dengue and Zika; hence, suggesting potential 56 association of lncRNAs in virus infection. Results of this study provide a new avenue to the investigation 57 of mosquito-virus interactions that may potentially pave way to the development of novel methods in 58 vector control. 59 60 Introduction 61 The Asian tiger mosquito, Aedes albopictus (Ae. albopictus) is an important vector of arboviruses 62 such as dengue virus (DENV), Chikungunya virus (CHIKV), and Zika virus (ZIKV). Due to its 63 invasiveness and aggressive spread, Ae. albopictus has widespread geographic distribution; hence, poses 64 serious health threat globally. Althou...
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