Rtt105 stimulates Rad51-ssDNA assembly and orchestrates Rad51 and RPA actions to promote homologous recombination repair.

Homologous recombination (HR) is essential for the maintenance of genome stability. During HR, Replication Protein A (RPA) rapidly coats the 3'-tailed single-strand DNA (ssDNA) generated by end resection. Then, the ssDNA-bound RPA must be timely replaced by Rad51 recombinase to form Rad51 nucleoprotein filaments that drive homology search and HR repair. How cells regulate Rad51 assembly dynamics and coordinate RPA and Rad51 actions to ensure proper HR remains poorly understood. Here, we identified that Rtt105, a Ty1 transposon regulator, acts to stimulate Rad51 assembly and orchestrate RPA and Rad51 actions during HR. We found that Rtt105 interacts with Rad51 in vitro and in vivo and restrains the adenosine 5' triphosphate (ATP) hydrolysis activity of Rad51. We showed that Rtt105 directly stimulates dynamic Rad51-ssDNA assembly, strand exchange, and D-loop formation in vitro. Notably, we found that Rtt105 physically regulates the binding of Rad51 and RPA to ssDNA via different motifs and that both regulations are necessary and epistatic in promoting Rad51 nucleation, strand exchange, and HR repair. Consequently, disrupting either of the interactions impaired HR and conferred DNA damage sensitivity, underscoring the importance of Rtt105 in orchestrating the actions of Rad51 and RPA. Our work reveals additional layers of mechanisms regulating Rad51 filament dynamics and the coordination of HR.

Real-world dosing of regorafenib and outcomes among patients with metastatic colorectal cancer: a retrospective analysis using US claims data.

BACKGROUND: The randomized, dose-optimization, open-label ReDOS study in US patients with metastatic colorectal cancer (CRC) showed that, compared with a standard dosing approach, initiating regorafenib at 80 mg/day and escalating to 160 mg/day depending on tolerability increased the proportion of patients reaching their third treatment cycle and reduced the incidence of adverse events without compromising efficacy. Subsequently, the ReDOS dose-escalation strategy was included as an alternative regorafenib dosing option in the National Comprehensive Cancer Network (NCCN) Clinical Practice Guidelines. A retrospective analysis was conducted using a US claims database to assess whether inclusion of this dose-escalation strategy in NCCN Guidelines has influenced the use of flexible dosing in routine US clinical practice, and to describe clinical outcomes pre- and post-inclusion in NCCN Guidelines. METHODS: Patients with CRC in the Optum's de-identified Clinformatics® Data Mart database initiating regorafenib for the first time between January 2016 and June 2020 were stratified based on whether they initiated regorafenib pre- or post-inclusion of ReDOS in NCCN Guidelines, and in two groups: flexible dosing (< 160 mg/day; < 84 tablets in the first treatment cycle) and standard dosing (160 mg/day; ≥ 84 tablets in the first treatment cycle). The primary endpoints were the proportion of patients who initiated their third treatment cycle and the mean number of treatment cycles per group. RESULTS: 703 patients initiated regorafenib during the study period, of whom 310 (44%) initiated before and 393 (56%) initiated after inclusion of ReDOS in NCCN Guidelines. After inclusion in the guidelines, the proportion of patients who received flexible dosing increased from 21% (n = 66/310) to 45% (n = 178/393), the proportion who received standard dosing decreased from 79% (n = 244/310) to 55% (n = 215/393), the proportion who initiated their third treatment cycle increased from 36% (n = 113/310) to 46% (n = 179/393), and the mean (standard deviation) number of treatment cycles increased from 2.6 (2.9) to 3.2 (3.1). CONCLUSIONS: Following inclusion of ReDOS in NCCN Guidelines, real-world data suggest that US clinicians have markedly increased use of flexible dosing in clinical practice, potentially maximizing clinical benefits and safety outcomes for patients with metastatic CRC receiving regorafenib.

Tumor-associated antigen-specific cell imaging based on upconversion luminescence and nucleic acid rolling circle amplification.

Tumor-associated antigen (TAA)-based diagnosis has gained prominence for early tumor screening, treatment monitoring, prognostic assessment, and minimal residual disease detection. However, limitations such as low sensitivity and difficulty in extracting non-specific binding membrane proteins still exist in traditional detection methods. Upconversion luminescence (UCL) exhibits unique physical and chemical properties under wavelength near-infrared light excitation. Rolling circle amplification (RCA) is an efficient DNA amplification technique with amplification factors as high as 105. Therefore, the above two excellent techniques can be employed for highly accurate imaging analysis of tumor cells. Herein, we developed a novel nanoplatform for TAA-specific cell imaging based on UCL and RCA technology. An aptamer-primer complex selectively binds to Mucin 1 (MUC1), one of TAA on cell surface, to trigger RCA reaction, generating a large number of repetitive sequences. These sequences provide lots of binding sites for complementary signal probes, producing UCL from lanthanide-doped upconversion nanoparticles (UCNPs) after releasing quencher group. The experimental results demonstrate the specific attachment of upconversion nanomaterials to cancer cells which express a high level of MUC1, indicating the potential of UCNPs and RCA in tumor imaging.

Development of highly accurate digital PCR method and reference material for monkeypox virus detection.

Human monkeypox has attracted attention recently. Monkeypox virus (MPXV) keeps evolving as it spreading around the world rapidly, which may threaten the health of more and more people. Here, we have developed a high order reference method based on digital PCR (dPCR) for MPXV detection, of which the limits of quantification (LoQ) and detection (LoD) are 38 and 6 copies/reaction, respectively. Pseudovirus reference materials (RM) containing the conserved F3L gene has been developed, and the homogeneity assessment showed that the RM was homogeneous. The reference value with its expanded uncertainty determined by the established dPCR is (2.74 ± 0.46) × 103 copies/µL. Six different MPXV test kits were accessed by the RM. Four out of six test kits cannot reach their claimed LoDs. The poor analytical sensitivity might cause false-negative results, which lead to incorrect diagnosis and treatment. The establishment of a high order reference method of dPCR and pseudovirus RM is very useful for improving the accuracy and reliability of MPXV detection.

Establishment of Digital PCR Method and Reference Material for Adenoviruses 40 and 41.

Coinfection with human adenovirus (HAdV) and SARS-CoV-2 has been associated with acute hepatitis in children with unknown etiology. Similar cases have been reported in many countries, and HAdV 40 and HAdV 41 have been identified. The quantification method is established based on digital PCR (dPCR) for HAdV 40/41, which is more convenient for low-concentration virus detection. The limit of detections of HAdV 40/41 dPCR were 4 and 5 copies/µL. Pseudovirus reference material (RM) that contains the highly conserved HEXON gene was developed and quantified with the dPCR method. The assigned values with expanded uncertainty were (1.43 ± 0.35) × 103 copies/µL for HAdV 40 RM and (1.21 ± 0.28) × 103 copies/µL for HAdV 41 RM. The values could be reproduced on multiple platforms. The dPCR method and pseudovirus RMs contribute to the improved accuracy of HAdV 40/41 detection, which is crucial for clinical diagnosis.

Computer-Aided Design of DNA Self-Limited Assembly for Relative Quantification of Membrane Proteins.

Immunofluorescence imaging of cells plays a vital role in biomedical research and clinical diagnosis. However, when it is applied to relative quantification of proteins, it suffers from insufficient fluorescence intensity or partial overexposure, resulting in inaccurate relative quantification. Herein, we report a computer-aided design of DNA self-limited assembly (CAD-SLA) technology and apply it for relative quantification of membrane proteins, a concept proposed for the first time. CAD-SLA can achieve exponential cascade signal amplification in one pot and terminate at any desired level. By conjugating CAD-SLA with immunofluorescence, in situ imaging of cell membrane proteins is achieved with a controllable amplification level. Besides, comprehensive fluorescence intensity information from fluorescent images can be obtained, accurately showing relative quantitative information. Slight protein expression differences previously indistinguishable by immunofluorescence or Western blotting can now be discriminated, making fluorescence imaging-based relative quantification a promising tool for membrane protein analysis. From the perspectives of both DNA self-assembly technology and immunofluorescence technology, this work has solved difficult problems and provided important reference for future development.

Series of TM-OFs as a Platform for Efficient Catalysis and Multifunctional Luminescence Sensing.

Three novel porous transition-metal-organic frameworks (TM-OFs), formulated as [Co3(DCPN)2(µ2-OH2)4(H2O)4](DMF)2 (1), [Cd3(DCPN)2(µ2-OH2)4(H2O)4](DMF)2 (2), and [CdK(DCPN)(DMA)] (3), have been successfully prepared via solvothermal conditions based on a 5-(3',6'-dicarboxylic phenyl) nicotinic carboxylic acid (H3DCPN) ligand. 1 and 2 both have the same porous 3D network structure with the point symbol of {410·614·84}·{45·6}2 based on trinuclear ({Co3} or {Cd3}) clusters, indicating a one-dimensional porous channel, and possess excellent water and thermal stability; 3 also displays a porous 3D network structure with a 4-connected sra topology based on the heteronuclear metal cluster {CdK}. Complex 1 can be used to load Pd nanoparticles (Pd NPs) via a wetness impregnation strategy to obtain Pd@1. The reduction of nitrophenols (2-NP, 3-NP, 4-NP) by Pd@1 in aqueous solution shows outstanding conversion, excellent rate constants (k), and remarkable cycling stability due to the synergistic effect of complex 1 and Pd NPs. Luminescence sensing tests confirmed that 2 is a reliable multifunctional chemical sensor with high selectivity and sensitivity for low concentrations of Fe3+, Cr2O72-, CPFX, and NFX. Specifically, 2 shows a fluorescence enhancement behavior toward fluoroquinolone antibiotics (CPFX and NFX), which has not been reported previously in the literature. Moreover, the rational mechanism of fluorescence sensing was also systematically investigated by various detection means and theoretical calculations.

Paper-based netlike rolling circle amplification (NRCA) for ultrasensitive and visual detection of SARS-CoV-2.

COVID-19 is a highly diffuse respiratory infection caused by severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2). Currently, quantitative real-time polymerase chain reaction (qRT-PCR) technology is commonly used in clinical diagnosis of COVID-19. However, this method is time-consuming and labor-intensive, which is limited in clinical application. Here, we propose a new method for the ultrasensitive and visual detection of SARS-CoV-2 viral nucleic acid. The assay integrates with a paper device and highly efficient isothermal amplification technology - Netlike rolling circle amplification (NRCA), which can reach a limit of detection of 4.12 aM. The paper-based NRCA owns advantages of specificity, portability, visualization and low-cost. Therefore, this method can effectively meet the requirements of point-of-care testing, providing a novel molecular detection technology for clinical diagnosis of COVID-19 and promoting the development of NRCA devices.

A double-stranded RNA platform is required for the interaction between a host restriction factor and the NS1 protein of influenza A virus.

Influenza A viruses cause widespread human respiratory disease. The viral multifunctional NS1 protein inhibits host antiviral responses. This inhibition results from the binding of specific cellular antiviral proteins at various positions on the NS1 protein. Remarkably, binding of several proteins also requires the two amino-acid residues in the NS1 N-terminal RNA-binding domain (RBD) that are required for binding double-stranded RNA (dsRNA). Here we focus on the host restriction factor DHX30 helicase that is countered by the NS1 protein, and establish why the dsRNA-binding activity of NS1 is required for its binding to DHX30. We show that the N-terminal 152 amino-acid residue segment of DHX30, denoted DHX30N, possesses all the antiviral activity of DHX30 and contains a dsRNA-binding domain, and that the NS1-DHX30 interaction in vivo requires the dsRNA-binding activity of both DHX30N and the NS1 RBD. We demonstrate why this is the case using bacteria-expressed proteins: the DHX30N-NS1 RBD interaction in vitro requires the presence of a dsRNA platform that binds both NS1 RBD and DHX30N. We propose that a similar dsRNA platform functions in interactions of the NS1 protein with other proteins that requires these same two amino-acid residues required for NS1 RBD dsRNA-binding activity.

Types and Applications of Nicking Enzyme-Combined Isothermal Amplification.

Due to the sudden outbreak of COVID-19 at the end of 2019, rapid detection has become an urgent need for community clinics and hospitals. The rapid development of isothermal amplification detection technology for nucleic acids in the field of molecular diagnostic point-of-care testing (POCT) has gained a great deal of attention in recent years. Thanks to intensive research on nicking enzymes, nicking enzyme-combined isothermal amplification has become a promising platform for rapid detection. This is a novel technique that uses nicking enzymes to improve ordinary isothermal amplification. It has garnered significant interest as it overcomes the complexity of traditional molecular diagnostics and is not subject to temperature limitations, relying on cleavage enzymes to efficiently amplify targets in a very short time to provide a high level of amplification efficiency. In recent years, several types of nicking enzyme-combined isothermal amplification have been developed and they have shown great potential in molecular diagnosis, immunodiagnosis, biochemical identification, and other fields. However, this kind of amplification has some disadvantages. In this review, the principles, advantages and disadvantages, and applications of several nicking enzyme-combined isothermal amplification techniques are reviewed and the prospects for the development of these techniques are also considered.

Recovery of CaO from CaSO4 via CO reduction decomposition under different atmospheres.

CaSO4 reduction decomposition for CaO preparation provides a theoretical basis for the utilization of the industrial byproduct, gypsum. In this study, the effects of temperature (950 °C-1150 °C), CO2/CO partial-pressure ratio (1-15), CO concentration (1%-5%), and O2 concentration (1%-7%) on the preparation of CaO from CaSO4 by CO reduction decomposition under different reaction atmospheres were investigated. The physical properties of CaO prepared by the decomposition of CaSO4 and CaCO3 were analyzed and compared. Finally, the reaction mechanism of the reduction decomposition of CaSO4 to CaO by CO was studied. The findings reveal that CaSO4 can be completely decomposed into CaO when the reaction temperature exceeds 1000 °C, CO% ≥ 2%, and P(CO2)/P(CO) ≥ 8. Furthermore, the addition of an appropriate amount of O2 can improve the yield of CaO in the products. In an O2-CO-N2 atmosphere, where O2% = 7% and CO% = 16%, CaSO4 can be completely decomposed into CaO without the addition of CO2. The physical properties of CaO prepared by the reduction and decomposition of CaSO4 are better than those prepared by the calcination of CaCO3. An analysis of the reaction mechanism of the reduction decomposition of CaSO4 by CO reveals that CaSO4 generates CaO and CaS simultaneously. In addition, CaS can react with unreacted CaSO4 to form CaO. Furthermore, it can react with CO2 to produce CaO if an appropriate amount of CO2 is added to the reaction atmosphere. The secondary interactions of CaS with CaSO4 and CO2 can significantly improve the yield of CaO in the product.

Single-Cell Analysis of Highly Metastatic Circulating Tumor Cells by Combining a Self-Folding Induced Release Reaction with a Cell Capture Microchip.

Nondestructive analysis of the single-cell molecular phenotype of circulating tumor cells (CTCs) is of great significance to the precise diagnosis and treatment of cancer but is also a huge challenge. To address this issue, here, we develop a facile analysis system that integrates CTCs' capture and molecular phenotype analysis. An isothermal nucleic acid amplification technique named self-folding induced release reaction (sFiR), which has high-efficiency signal amplification capabilities and can run under physiological conditions, is first developed to meet the high requirements for sensitivity and nondestructivity. By combining the sFiR with immune recognition and a single cell capture microchip, the molecular phenotype analysis of a single CTC is realized. As a model, nondestructive analysis of junction plakoglobin (JUP), an overexpressed membrane protein that is closely related to the metastasis of CTCs, is successfully achieved. Results reveal that this sFiR-based analysis system can clearly distinguish the expression of JUP in different cancer cell lines and can present quantitative information on the expression of JUP. Furthermore, the captured and analyzed CTCs maintain their basic physiological activity and can be used for drug sensitivity testing. Considering the excellent performance and ease of operation of the system, it can provide technical support for CTC-based cancer liquid biopsy and drug development.

mRNA Delivery by a pH-Responsive DNA Nano-Hydrogel.

The delivery of mRNA to manipulate protein expression has attracted widespread attention, since that mRNA overcomes the problem of infection and mutation risks in transgenes and can work as drugs for the treatment of diseases. Although there are currently some vehicles that deliver mRNA into cells, they have not yet reached a good balance in terms of expression efficiency and biocompatibility. Here, a DNA nano-hydrogel system for mRNA delivery is developed. The nano-hydrogel is all composed of DNA except the target mRNA, so it has superior biocompatibility compared with those chemical vehicles. In parallel, the nano-hydrogel can be compacted into a nanosphere under the crosslinking by well-designed "X"-shaped DNA scaffolds and DNA linkers, facilitating the delivery into cells through endocytosis. In addition, smart intracellular release of the mRNA is achieved by incorporating a pH-responsive i-motif structure into the nano-hydrogel. Thus, taking the efficient delivery and release together, mRNA can be translated into the corresponding protein with a high efficiency, which is comparable to that of the commercial liposome but with a much better biocompatibility. Due to the excellent biocompatibility and efficiency, this nano-hydrogel system is expected to become a competitive alternative for delivering functional mRNA in vivo.

Anti-arthritic activity of D-carvone against complete Freund's adjuvant-induced arthritis in rats through modulation of inflammatory cytokines.

Chronic joint pain due to loss of cartilage function, degradation of subchondral bone, and related conditions are common plights of an arthritis patient. Antioxidant compounds could solve the problems in arthritic condition. The objective of this study was to evaluate the anti-arthritic activity of D-carvone against complete Freund's adjuvant (CFA)-induced arthritis in rats. D-carvone was orally administered for 25 days at the doses of 30 and 60 mg/kg against CFA-induced arthritic rats. Changes in body weight, paw swelling, organ index, hematological parameters, oxidative stress markers, inflammatory cytokines, and histopathology were recorded. Oral treatment of D-carvone significantly improved the body weight, reduced the paw swelling, edema formation, and organ index in arthritic rats. The levels of white blood cells were reduced, red blood cells and hemoglobin levels were improved in D-carvone treated arthritic rats. Lipid peroxidation levels were lowered whereas enzymatic and non-enzymatic antioxidants were significantly elevated by D-carvone administration against arthritic rats. D-carvone significantly modulated inflammatory cytokine levels and improved the ankle joint pathology against CFA-induced arthritic inflammation. In conclusion, D-carvone proved significant anti-arthritic activity against CFA-induced arthritis in rats.

pH-Based immunoassay: explosive generation of hydrogen ions through an immuno-triggered nucleic acid exponential amplification reaction.

In this work, we propose a novel concept and a proof-of-concept strategy for the fabrication of a pH-based immunoassay platform with a certain degree of universality and scalability to make it adaptable for different application scenarios. The immunoreactions for the target detection are converted to pH changes through an engineered and optimized isothermal nucleic acid amplification, named exponential amplification reaction (EXPAR). Thus, a variety of well-developed methods for pH analysis, e.g. pH indicators, pH-strips and pH meters, can be applied for immunoassay directly. Here, we show that this proof-of-concept strategy is applicable for both macromolecular and micromolecular antigens by adopting human platelet-derived growth factor-BB (PDGF-BB) and chloramphenicol (CAP) as the model targets, respectively. The detection can be achieved using a colorimetric pH indicator after a 15 min reaction of the immuno-triggered isothermal nucleic acid amplification. In addition, compared with the traditional enzyme-linked immunosorbent assay (ELISA), the performance of our strategy, especially the detection limits, is improved to varying degrees for different targets, making the strategy a promising alternative for diverse application scenarios of immunoassay.

New method for detection of T4 polynucleotide kinase phosphatase activity through isothermal EXPonential amplification reaction.

As a bifunctional enzyme, T4 polynucleotide kinase phosphatase (T4 PNKP) catalyzes the phosphorylation of 5'-hydroxyl, and also removes the terminal 3'-phosphate group. This is closely related to the restructuring, replication, and damage repair of nucleic acid. In this paper, we describe a new method for the sensitive detection of T4 PNKP activity based on the isothermal EXPonential amplification reaction (EXPAR). T4 PNKP can be linearly assayed in the range from 0.001 to 0.01 U mL-1 with a detection limit of 7.9 × 10-4 U mL-1. Moreover, the method exhibits high specificity and sensitivity and can be applied in the enzyme analysis of complex serum samples. In view of its simplicity and moderate experimental conditions, the method may suitable for use in a commercial kit for the analysis of T4 PNKP activity.

A nanoflow cytometric strategy for sensitive ctDNA detection via magnetic separation and DNA self-assembly.

Circulating tumor DNA (ctDNA) is a tumor-derived fragmented DNA in the bloodstream that is not associated with cells. It has been greatly focused in the recent decade because of its potential clinical utility for liquid biopsies. Development of ctDNA analytical techniques with high sensitivity and cost-efficiency will undoubtedly promote the clinical spread of ctDNA testing. In this paper, we propose a novel flow cytometry-based ctDNA sensing strategy which combines enzyme-free amplification and magnetic separation. The target DNA is capable of triggering a hybridization chain reaction, producing a fluorescent long linear assembly of DNA, which can be further captured by magnetic beads to present fluorescent signals using flow cytometry. In comparison with some conventional methods, our strategy has the advantages of easy operation and cost-efficiency, and thereby shows a promising application in clinical diagnosis. Graphical abstract.

Micropterus salmoides rhabdovirus (MSRV) infection induced apoptosis and activated interferon signaling pathway in largemouth bass skin cells.

Largemouth bass (Micropterus salmoides) rhabdovirus (MSRV) was isolated from infected juveniles of largemouth bass, and the infected fish exhibited corkscrew, irregular swimming, and crooked body. To our knowledge, the potential molecular mechanisms underlying the pathogenesis of MSRV infection remain largely unknown. In the current study, we found that MSRV infection in largemouth bass skin (LBS) cells induced typical apoptosis, evidenced by the presence of apoptotic bodies and caspase-3 activation. To further analyze the host factors involved in MSRV infection in LBS cells, the transcriptomic profiles during MSRV infection were uncovered using deep RNA sequencing technique, and several differentially expressed genes (DEGs) were validated by quantitative PCR. Our results showed that a total of 124483 unigenes were assembled. Among them, 34465 and 27273 had significant hits to those in the NR and SwissProt databases. After MSRV infection, a total of 2432 and 2480 genes which involved in multiples pathways including TNF signaling, NF-κB signaling, Toll-like receptor signaling and RIG-I signaling pathway were differentially expressed in MSRV infected LBS cells compared to mock-infected cells at 12 h, respectively. Furthermore, quantitative PCR showed that the expression levels of 9 differentially expressed genes (DEGs) related to apoptosis and interferon signaling pathway was consistent with that from transcriptomic profiles. Together, our results not only demonstrated that interferon signaling pathway and apoptosis pathway might exerted crucial roles during MSRV infection, but also provided a useful resource for subsequent investigation of other immune-related genes related to virus infection.

Battle between influenza A virus and a newly identified antiviral activity of the PARP-containing ZAPL protein.

Previous studies showed that ZAPL (PARP-13.1) exerts its antiviral activity via its N-terminal zinc fingers that bind the mRNAs of some viruses, leading to mRNA degradation. Here we identify a different antiviral activity of ZAPL that is directed against influenza A virus. This ZAPL antiviral activity involves its C-terminal PARP domain, which binds the viral PB2 and PA polymerase proteins, leading to their proteasomal degradation. After the PB2 and PA proteins are poly(ADP-ribosylated), they are associated with the region of ZAPL that includes both the PARP domain and the adjacent WWE domain that is known to bind poly(ADP-ribose) chains. These ZAPL-associated PB2 and PA proteins are then ubiquitinated, followed by proteasomal degradation. This antiviral activity is counteracted by the viral PB1 polymerase protein, which binds close to the PARP domain and causes PB2 and PA to dissociate from ZAPL and escape degradation, explaining why ZAPL only moderately inhibits influenza A virus replication. Hence influenza A virus has partially won the battle against this newly identified ZAPL antiviral activity. Eliminating PB1 binding to ZAPL would be expected to substantially increase the inhibition of influenza A virus replication, so that the PB1 interface with ZAPL is a potential target for antiviral development.

Zinc-finger antiviral protein inhibits HIV-1 infection by selectively targeting multiply spliced viral mRNAs for degradation.

The zinc-finger antiviral protein (ZAP) was originally identified as a host factor that inhibits the replication of Moloney murine leukemia virus. Here we report that ZAP inhibits HIV-1 infection by promoting the degradation of specific viral mRNAs. Overexpression of ZAP rendered cells resistant to HIV-1 infection in a ZAP expression level-dependent manner, whereas depletion of endogenous ZAP enhanced HIV-1 infection. Both human and rat ZAP inhibited the propagation of replication-competent HIV-1. ZAP specifically targeted the multiply spliced but not unspliced or singly spliced HIV-1 mRNAs for degradation. We provide evidence indicating that ZAP selectively recruits cellular poly(A)-specific ribonuclease (PARN) to shorten the poly(A) tail of target viral mRNA and recruits the RNA exosome to degrade the RNA body from the 3' end. In addition, ZAP recruits cellular decapping complex through its cofactor RNA helicase p72 to initiate degradation of the target viral mRNA from the 5' end. Depletion of each of these mRNA degradation enzymes reduced ZAP's activity. Our results indicate that ZAP inhibits HIV-1 by recruiting both the 5' and 3' mRNA degradation machinery to specifically promote the degradation of multiply spliced HIV-1 mRNAs.