Processing of Sesbania mosaic virus (SeMV) polyprotein 2a and 2ab was reanalyzed in the view of the new genome organization of sobemoviruses. Polyprotein 2a when expressed in E. coli, from the new cDNA clone, got cleaved at the earlier identified sites E325-T326, E402-T403 and E498-S499 to release protease, VPg, P10 and P8, respectively. Additionally, a novel cleavage was identified within the protease domain at position E132-S133, which was found to be essential for efficient polyprotein processing. Products, corresponding to cleavages identified in E. coli, were also detected in infected Sesbania leaves. Interestingly, though the sites are exactly the same in polyprotein 2ab, it got cleaved between Protease-VPg but not between VPg-RdRp. This indicates to a differential cleavage preference, governed probably by the conformation of 2ab. Also, the studies revealed that, in SeMV, processing is regulated by mode of cleavage and context of the cleavage site.
Elephants use vocalizations for both long and short distance communication. Whereas the acoustic repertoire of the African elephant (Loxodonta africana) has been extensively studied in its savannah habitat, very little is known about the structure and social context of the vocalizations of the Asian elephant (Elephas maximus), which is mostly found in forests. In this study, the vocal repertoire of wild Asian elephants in southern India was examined. The calls could be classified into four mutually exclusive categories, namely, trumpets, chirps, roars, and rumbles, based on quantitative analyses of their spectral and temporal features. One of the call types, the rumble, exhibited high structural diversity, particularly in the direction and extent of frequency modulation of calls. Juveniles produced three of the four call types, including trumpets, roars, and rumbles, in the context of play and distress. Adults produced trumpets and roars in the context of disturbance, aggression, and play. Chirps were typically produced in situations of confusion and alarm. Rumbles were used for contact calling within and among herds, by matriarchs to assemble the herd, in close-range social interactions, and during disturbance and aggression. Spectral and temporal features of the four call types were similar between Asian and African elephants.
Cytidine deaminases inhibit replication of a broad range of DNA viruses by deaminating cytidines on single-stranded DNA (ssDNA) to generate uracil. While several lines of evidence have revealed hepatitis B virus (HBV) genome editing by deamination, it is still unclear which nucleic acid intermediate of HBV is modified. Hepatitis B virus has a relaxed circular double-stranded DNA (rcDNA) genome that is reverse transcribed within virus cores from a RNA template. The HBV genome also persists as covalently closed circular DNA (cccDNA) in the nucleus of an infected cell. In the present study, we found that in HBV-producing HepAD38 and HepG2.2.15 cell lines, endogenous cytidine deaminases edited 10 to 25% of HBV rcDNA genomes, asymmetrically with almost all mutations on the 5' half of the minus strand. This region corresponds to the last half of the minus strand to be protected by plus-strand synthesis. Within this half of the genome, the number of mutations peaks in the middle. Overexpressed APOBEC3A and APOBEC3G could be packaged in HBV capsids but did not change the amount or distribution of mutations. We found no deamination on pregenomic RNA (pgRNA), indicating that an intact genome is encapsidated and deaminated during or after reverse transcription. The deamination pattern suggests a model of rcDNA synthesis in which pgRNA and then newly synthesized minus-sense single-stranded DNA are protected from deaminase by interaction with the virus capsid; during plus-strand synthesis, when enough dsDNA has been synthesized to displace the remaining minus strand from the capsid surface, the single-stranded DNA becomes deaminase sensitive. Host-induced mutation of the HBV genome by APOBEC proteins may be a path to clearing the virus. We examined cytidine-to-thymidine mutations in the genomes of HBV particles grown in the presence or absence of overexpressed APOBEC proteins. We found that genomes were subjected to deamination activity during reverse transcription, which takes place within the virus capsid. These observations provide a direct insight into the mechanics of reverse transcription, suggesting that newly synthesized dsDNA displaces ssDNA from the capsid walls, making the ssDNA accessible to deaminase activity.
Sesbania mosaic virus (SeMV) polyprotein is processed by its N-terminal serine protease domain. The crystal structure of the protease domain was determined to a resolution of 2.4 A using multiple isomorphous replacement and anomalous scattering. The SeMV protease domain exhibited the characteristic trypsin fold and was found to be closer to cellular serine proteases than to other viral proteases. The residues of the S1-binding pocket, H298, T279 and N308 were mutated to alanine in the DeltaN70-Protease-VPg polyprotein, and the cis-cleavage activity was examined. The H298A and T279A mutants were inactive, while the N308A mutant was partially active, suggesting that the interactions of H298 and T279 with P1-glutamate are crucial for the E-T/S cleavage. A region of exposed aromatic amino acids, probably essential for interaction with VPg, was identified on the protease domain, and this interaction could play a major role in modulating the function of the protease.
Many murine leukemia viruses (MLVs) are partially resistant to restriction by mouse APOBEC3 (mA3) and essentially fully resistant to induction of G-to-A mutations by mA3. In contrast, Vif-deficient HIV-1 (⌬Vif HIV-1) is profoundly restricted by mA3, and the restriction includes high levels of G-to-A mutation. Human APOBEC3G (hA3G), unlike mA3, is fully active against MLVs. We produced a glutathione S-transferase-mA3 fusion protein in insect cells and demonstrated that it possesses cytidine deaminase activity, as expected. This activity is localized within the N-terminal domain of this 2-domain protein; the C-terminal domain is enzymatically inactive but required for mA3 encapsidation into retrovirus particles. We found that a specific arginine residue and several aromatic residues, as well as the zinc-coordinating cysteines in the C-terminal domain, are necessary for mA3 packaging; a structural model of this domain suggests that these residues line a potential nucleic acid-binding interface. Mutation of a few potential phosphorylation sites in mA3 drastically reduces its antiviral activity by impairing either deaminase activity or its encapsidation. mA3 deaminates short single-stranded DNA oligonucleotides preferentially toward their 3= ends, whereas hA3G exhibits the opposite polarity. However, when packaged into infectious ⌬Vif HIV-1 virions, both mA3 and hA3G preferentially induce deaminations toward the 5= end of minus-strand viral DNA, presumably because of the sequence of events during reverse transcription in vivo. Despite the fact that mA3 in MLV particles does not induce detectable deaminations upon infection, its deaminase activity is easily detected in virus lysates. We still do not understand how MLV resists mA3-induced G-to-A mutation. IMPORTANCEOne way that mammalian cells defend themselves against infection by retroviruses is with APOBEC3 proteins. These proteins convert cytidine bases to uridine bases in retroviral DNA. However, mouse APOBEC3 protein blocks infection by murine leukemia viruses without catalyzing this base change, and the mechanism of inhibition is not understood in this case. We have produced recombinant mouse APOBEC3 protein for the first time and characterized it here in a number of ways. Our mutational studies shed light on the mechanism by which mouse APOBEC3 protein is incorporated into retrovirus particles. While mouse APOBEC3 does not catalyze base changes in murine leukemia virus DNA, it can be recovered from these virus particles in enzymatically active form; it is still not clear why it fails to induce base changes when these viruses infect new cells. M ammals possess several innate mechanisms for defense against retroviruses. One of these restriction factors is the APOBEC3 system. APOBEC3 proteins are cytidine deaminases (1). In many cases, they are encapsidated into assembling virions; when these virions infect a new cell and copy their RNA into DNA, the APOBEC3 protein from the virion binds to the singlestranded DNA (ssDNA; the initial DNA product) and deamin...
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