High mutation frequency during reverse transcription has a principal role in the genetic variation of primate lentiviral populations. It is the main driving force for the generation of drug resistance and the escape from immune surveillance. G to A hypermutation is one of the characteristics of primate lentiviruses, as well as other retroviruses, during replication in vivo and in cell culture 1-6 . The molecular mechanisms of this process, however, remain to be clarified. Here, we demonstrate that CEM15 (also known as apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like 3G; APOBEC3G) 7,8 , an endogenous inhibitor of human immunodeficiency virus type 1 (HIV-1) replication, is a cytidine deaminase and is able to induce G to A hypermutation in newly synthesized viral DNA. This effect can be counteracted by the HIV-1 virion infectivity factor (Vif). It seems that this viral DNA mutator is a viral defence mechanism in host cells that may induce either lethal hypermutation or instability of the incoming nascent viral reverse transcripts, which could account for the Vif-defective phenotype. Importantly, the accumulation of CEM15-mediated non-lethal hypermutation in the replicating viral genome could potently contribute to the genetic variation of primate lentiviral populations.HIV-1 Vif protein is required for viral replication in vivo and in some 'non-permissive' cells, such as peripheral blood mononuclear cells, macrophages and H9 T cells 9-11 . The vifdefective viruses (Δvif) from non-permissive cells cannot complete reverse transcription, or the newly synthesized DNA cannot exist in the target cells for a significant time period 12-14 . Recently, it has been demonstrated that CEM15 is an endogenous inhibitor of HIV-1 that exists only in non-permissive cells. Its inhibitory effect on HIV-1 replication can be counteracted by HIV-1 Vif protein 7 . As Vif binds to HIV-1 RNA in the cytoplasm of virusproducing cells 15-17 , we investigated whether CEM15, which shares significant homology with some other cytidine deaminases that edit RNA, could also edit HIV-1 genomic or spliced RNA. We have sequenced the nearly full-length genomic RNA (>98%) of HIV-1 from the Δvif virions generated from H9 T cells by polymerase chain reaction with reverse transcription (RT-PCR) techniques (primer pairs are listed in Supplementary Table S1). Compared with the sequence of pNL4-3Δvif DNA, the change of genomic RNA in the virions is not significant. We have found an A to G change at positions 2257 and 3608, and a G to T change at position 9418 (data not shown). We have also sequenced several spliced HIV-1(NL4-3Δvif) RNA in H9 cells ( Supplementary Fig. S1 and Table S1); however, no mutations were demonstrated.Correspondence and requests for materials should be addressed to H.Z (hui.zhang@jefferson.edu).. Supplementary Information accompanies the paper on www.nature.com/nature. Competing interests statementThe authors declare that they have no competing financial interests. Table S1) that we have analysed (Fig. 1a, row 2 ...

HIV-1 Rev escorts unspliced viral mRNAs out of the nucleus of infected cells, which allows formation of infectious HIV-1 virions. We have identified a putative DEAD box (Asp-Glu-Ala-Asp) RNA helicase, DDX1, as a cellular co-factor of Rev, through yeast and mammalian two-hybrid systems using the N-terminal motif of Rev as "bait". DDX1 is not a functional homolog of HIV-1 Rev, but down-regulation of DDX1 resulted in an alternative splicing pattern of Rev-responsive element (RRE)-containing mRNA, and attenuation of Gag p24 antigen production from HLfb rev- cells rescued by exogenous Rev. Co-transfection of a DDX1 expression vector with HIV-1 significantly increased viral production. DDX1 binding to Rev, as well as to the RRE, strongly suggest that DDX1 affects Rev function through the Rev-RRE axis. Moreover, down-regulation of DDX1 altered the steady state subcellular distribution of Rev, from nuclear/nucleolar to cytoplasmic dominance. These findings indicate that DDX1 is a critical cellular co-factor for Rev function, which maintains the proper subcellular distribution of this lentiviral regulatory protein. Therefore, alterations in DDX1-Rev interactions could induce HIV-1 persistence and targeting DDX1 may lead to rationally designed and novel anti-HIV-1 strategies and therapeutics.

Cellular cytidine deaminases APOBEC3 family is a group of potent inhibitors for many exogenous and endogenous retroviruses. It has been demonstrated that they induce G to A hypermutations in the nascent retroviral DNA, resulting from the cytosine (C) to uracil (U) conversions in minus-stranded viral DNA. In this report, we have demonstrated that the result of C to U conversion in minus-stranded DNA of human immunodeficiency virus type 1 (HIV-1) could trigger a degradation of nascent viral DNA mediated by uracil DNA glycosylases-2 (UNG2) and apurinic/apyrimidinic endonuclease (APE). Since antiviral activity of APOBEC3G is partially affected by UNG2 inhibitor Ugi or UNG2-specific short-interfering RNA in virus-producing cells but not target cells, the virion-associated UNG2 most likely mediates this process. Interestingly, as APE-specific short-interfering RNA can also partially inhibit the anti-HIV-1 activity of APOBEC3G in virus-producing cells but not in target cells and APE molecules can be detected within HIV-1 virions, it seems that the required APE is also virion-associated. Furthermore, the in vitro cleavage experiment using uracil-containing single-stranded DNA as a template has demonstrated that the uracil-excising catalytic activity of virion-associated UNG2 can remove dU from the uracil-containing viral DNA and leave an abasic site, which could be further cleaved by virion-associated APE. Based upon our observations, we propose that the degradation of APOBEC3G-edited viral DNA mediated by virion-associated UNG2 and APE during or after reverse transcription could be partially responsible for the potent anti-HIV-1 effect by APOBEC3G in the absence of vif.APOBEC3 is a family of cytidine deaminases that has been identified as the host factor to restrict various retroviruses, endogenous retroviruses, and long interspersed nucleotide element (LINE) elements (1-11). It is closely related to APOBEC1, a cytidine deaminase that causes a specific cytosine to uracil change in the apolipoprotein B mRNA, and to an activationinduced deaminase (AID) enzyme that causes hypermutation of immunoglobulin genes (12). The similarities of the catalytic domains in these proteins strongly suggest that APOBEC3 edits the nucleic acids of various retrotransposons. Among these cytidine deaminases, APOBEC3G is the most extensively studied enzyme. It can be efficiently incorporated into human immunodeficiency virus type 1 (HIV-1) 4 particles and causes extensive cytosine to uracil conversion in the viral minus-stranded DNA during reverse transcription (2, 3, 13). The significant C to U conversion in minus-stranded DNA is apparently correlated with the decreased viral infectivity. Two consequences could occur after the C to U conversion. First, uracil in minusstranded DNA could be excised by recruited uracil DNA glycosylase-2 (UNG2), a host DNA repair enzyme. The resulting abasic site could be further cleaved by apurinic/apyrimidinic endonucleases (APE). The vif-defective viruses generated from the restrictive cells are unable to effe...

The apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like 3G (APOBEC3G or A3G) and its fellow cytidine deaminase family members are potent restrictive factors for human immunodeficiency virus type 1 (HIV-1) and many other retroviruses. A3G interacts with a vast spectrum of RNAbinding proteins and is located in processing bodies and stress granules. However, its cellular function remains to be further clarified. Using a luciferase reporter gene and green fluorescent protein reporter gene, we demonstrate that A3G and other APOBEC family members can counteract the inhibition of protein synthesis by various microRNAs (miRNAs) such as mir-10b, mir-16, mir-25, and let-7a. A3G could also enhance the expression level of miRNA-targeted mRNA. Further, A3G facilitated the association of microRNA-targeted mRNA with polysomes rather than with processing bodies. Intriguingly, experiments with a C288A/C291A A3G mutant indicated that this function of A3G is separable from its cytidine deaminase activity. Our findings suggest that the major cellular function of A3G, in addition to inhibiting the mobility of retrotransposons and replication of endogenous retroviruses, is most likely to prevent the decay of miRNA-targeted mRNA in processing bodies. MicroRNAs (miRNAs)2 are 20 -22-nt regulatory RNAs that participate in the regulation of various biological functions in numerous eukaryotic lineages, including plants, insects, vertebrate, and mammals (1-3). More than 474 miRNAs have been identified in humans so far, and ϳ30% of the genes in the human genome are predicted to be subject to miRNA regulation (4). The expression of many miRNAs is usually specific to a tissue or developmental stage, and the miRNA expression pattern is altered during the development of many diseases (3). Mature miRNAs are generated from RNA polymerase II-transcribed primary miRNAs that are processed sequentially by the nucleases Drosha and Dicer. Although miRNA can guide mRNA cleavage, the basic function of miRNA is to mediate inhibition of protein translation (1, 5-8) through miRNA-induced silencing complexes (miRISCs). The guiding strand of miRNA in a miRISC interacts with a complementary sequence in the 3Ј-untranslated region (3Ј-UTR) of its target mRNA by partial sequence complementarities, resulting in translational inhibition (1). A 7-nucleotide "seed" sequence (at positions 2-8 from the 5Ј-end) in miRNAs seems to be essential for this action (4). The composition of the miRISC is similar to that of the RNA-induced silencing complex (RISC), which is responsible for mRNA cleavage guided by small interfering RNAs (siRNAs) (1, 3, 7). Nevertheless, some differences exist between miRISCs and siRNA RISCs. For example, the major Argonaute protein in siRNA RISC is Ago-2, whereas all four of the Ago proteins (Ago1-4) are found in miRISC (3,8). Further, the siRNA RISC may be associated with various RNA-binding proteins such as fragile-X mental retardation protein (FMRP), TAR RNA-binding protein (TRBP), and the human homolog of the Drosophila helicase Arm...

BackgroundThere is great potential for using transgenic technology to improve the quality of cow milk and to produce biopharmaceuticals within the mammary gland. Lysozyme, a bactericidal protein that protects human infants from microbial infections, is highly expressed in human milk but is found in only trace amounts in cow milk.Methodology/Principal FindingsWe have produced 17 healthy cloned cattle expressing recombinant human lysozyme using somatic cell nuclear transfer. In this study, we just focus on four transgenic cattle which were natural lactation. The expression level of the recombinant lysozyme was up to 25.96 mg/L, as measured by radioimmunoassay. Purified recombinant human lysozyme showed the same physicochemical properties, such as molecular mass and bacterial lysis, as its natural counterpart. Moreover, both recombinant and natural lysozyme had similar conditions for reactivity as well as for pH and temperature stability during in vitro simulations. The gross composition of transgenic and non-transgenic milk, including levels of lactose, total protein, total fat, and total solids were not found significant differences.Conclusions/SignificanceThus, our study not only describes transgenic cattle whose milk offers the similar nutritional benefits as human milk but also reports techniques that could be further refined for production of active human lysozyme on a large scale.

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In lepidopteran insects, odorant receptors are involved in the perception of sex pheromones and general odorants. In the Asian corn borer, Ostrinia furnacalis, although several pheromone receptors have been identified, no general odorant receptor has been reported. In this study, an RNA sequencing analysis was carried out to identify the whole repertoire of the odorant receptors expressed in the antennae of O. furnacalis. Among 12 million reads obtained from the antennae of male and female moths, 52 candidate odorant receptors were identified, including 45 novel ones. Expression levels of candidate odorant receptors were estimated by read mapping and quantitative reverse transcription PCR. These analyses confirmed that the expression of the previously identified pheromone receptors was highly male biased. In contrast, none of the newly identified odorant receptors showed male-biased expression. Three of the newly identified odorant receptors showed female-biased expression. Two of them were the most highly expressed odorant receptors in the female antennae, suggesting that they may be involved in the detection of odorants important for the induction of female-specific behaviors such as oviposition site selection. In addition, candidate genes of 21 ionotropic receptors, 5 gustatory receptors, 2 sensory neuron membrane proteins, and 26 odorant degrading enzymes were identified. Our results provide a basis for further analysis of the chemosensory system in the Ostrinia species.

Background: APOBEC3G (A3G), which is part of cytidine deaminase family, significantly inhibits miRNA-mediated repression of translation, but the mechanism remains unknown. Results: A3G competitively binds to the C terminus of MOV10 to inhibit the interaction between AGO2 and MOV10. Conclusion: A3G affects the normal assembly or maturation of miRISC. Significance: Our data demonstrate a novel mechanism of the regulation of miRISC activity.