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...

Edited by Peter BrzezinskiKeywords: P10 P8 Natively unfolded Nucleic acid binding ATPase Sesbania mosaic virus a b s t r a c t Open reading frame (ORF) 2a of Sesbania mosaic virus (SeMV) codes for polyprotein 2a (Membrane anchor-protease-VPg-P10-P8). The C-terminal domain of SeMV polyprotein 2a was cloned, expressed and purified in order to functionally characterize it. The protein of size 8 kDa (P8) domain, like viral protein genome linked (VPg), was found to be natively unfolded and could bind to nucleic acids. Interestingly, P10-P8 but not P8 showed a novel Mg 2+ dependent ATPase activity that was inhibited in the presence of poly A. In the absence of P8, the ATPase activity of the protein of size 10 kDa (P10) domain was reduced suggesting that the natively unfolded P8 domain influenced the P10 ATPase.

N-terminal serine protease domain of Sesbania mosaic virus polyprotein, requires fused VPg for its activity. W43 of VPg mediates aromatic stacking interactions (characterized by 230 nm positive CD peak) with protease. A stretch of aromatic residues (F269, W271, Y315, and Y319) exposed in the protease domain were mutated to identify the interacting partner of W43. W271A Protease-VPg mutant showed absence of cleavage activity both in vivo and in trans, with concomitant loss of the 230 nm CD peak. F269A Protease-VPg mutant was partially active. Mutations of the tyrosines did not result in loss of protease activity or the CD peak. Interestingly, H275, though not a part of the exposed aromatic stretch, was shown to be essential for protease activity and contributed significantly to the CD peak. Hence, we conclude that W271 and H275 of the protease domain mediate aromatic stacking interactions with W43 of VPg thereby rendering the protease active.

Hepatitis B virus (HBV) core protein (Cp) can be found in the nucleus and cytoplasm of infected hepatocytes; however, it preferentially segregates to a specific compartment correlating with disease status. Regulation of this intracellular partitioning of Cp remains obscure. In this paper, we report that cellular compartments are filled and vacated by Cp in a time- and concentration-dependent manner in both transfections and infections. At early times after transfection, Cp, in a dimeric state, preferentially localizes to the nucleolus. Later, the nucleolar compartment is emptied and Cp progresses to being predominantly nuclear, with a large fraction of the protein in an assembled state. Nuclear localization is followed by cell-wide distribution, and then Cp becomes exclusively cytoplasmic. The same trend in Cp movement is seen during an infection. Putative nucleolar retention signals have been identified and appear to be structure dependent. Export of Cp from the nucleus involves the CRM1 exportin. Time-dependent flux can be recapitulated by modifying Cp concentration, suggesting transitions are regulated by reaching a threshold concentration. IMPORTANCE HBV is an endemic virus. More than 250 million people suffer from chronic HBV infection and about 800,000 die from HBV-associated disease each year. HBV is a pararetrovirus; in an infected cell, viral DNA in the nucleus is the template for viral RNA that is packaged in nascent viral capsids in the cytoplasm. Inside those capsids, while resident in cytoplasm, the linear viral RNA is reverse transcribed to form the circular double-stranded DNA (dsDNA) of the mature virus. The HBV core (or capsid) protein plays a role in almost every step of the viral life cycle. Here, we show the core protein appears to follow a programmed, sequential localization from cytoplasmic translation then into the nucleolus, to the nucleus, and back to the cytoplasm. Localization is primarily a function of time, core protein concentration, and assembly. This has important implications for our understanding of the mechanisms of antivirals that target HBV capsid assembly.