Retargeting vesicular stomatitis virus glycoprotein pseudotyped lentiviral vectors with enhanced stability by in situ synthesized polymer shell.

The ability to introduce transgenes with precise specificity to the desired target cells or tissues is key to a more facile application of genetic therapy. Here, we describe a novel method using nanotechnology to generate lentiviral vectors with altered recognition of host cell receptor specificity. Briefly, the infectivity of the vesicular stomatitis virus glycoprotein (VSV-G) pseudotyped lentiviral vectors was shielded by a thin polymer shell synthesized in situ onto the viral envelope, and new binding ability was conferred to the shielded virus by introducing acrylamide-tailored cyclic arginine-glycine-aspartic acid (cRGD) peptide to the polymer shell. We termed the resulting virus "targeting nanovirus." The targeting nanovirus had similar titer with VSV-G pseudotypes and specifically transduced Hela cells with high transduction efficiency. In addition, the encapsulation of the VSV-G pseudotyped lentivirus by the polymer shell did not change the pathway that VSV-G pseudotypes enter and fuse with cells, as well as later events such as reverse transcription and gene expression. Furthermore, the targeting nanovirus possessed enhanced stability in the presence of human serum, indicating protection of the virus by the polymer shell from human serum complement inactivation. This novel use of nanotechnology demonstrates proof of concept for an approach that could be more generally applied for redirecting viral vectors for laboratory and clinical purposes.

Two distinct nanovirus species infecting faba bean in Morocco.

Using monoclonal antibodies raised against a Faba bean necrotic yellows virus (FBNYV) isolate from Egypt and a Faba bean necrotic stunt virus (FBNSV) isolate from Ethiopia, a striking serological variability among nanovirus isolates from faba bean in Morocco was revealed. To obtain a better understanding of this nanovirus variability in Morocco, the entire genomes of two serologically contrasting isolates referred to as Mor5 and Mor23 were sequenced. The eight circular ssDNA components, each identified from Mor5- and Mor23-infected tissues and thought to form the complete nanovirus genome, ranged in size from 952 to 1,005 nt for Mor5 and from 980 to 1,004 nt for Mor23 and were structurally similar to previously described nanovirus DNAs. However, Mor5 and Mor23 differed from each other in overall nucleotide and amino acid sequences by 25 and 26%, respectively. Mor23 was most closely related to typical FBNYV isolates described earlier from Egypt and Syria, with which it shared a mean amino acid sequence identity of about 94%. On the other hand, Mor5 most closely resembled a FBNSV isolate from Ethiopia, with which it shared a mean amino acid sequence identity of approximately 89%. The serological and genetic differences observed for Mor5 and Mor23 were comparable to those observed earlier for FBNYV, FBNSV, and Milk vetch dwarf virus. Following the guidelines on nanovirus species demarcation, this suggests that Mor23 and Mor5 represent isolates of FBNYV and FBNSV, respectively. This is the first report not only on the presence of FBNSV in a country other than Ethiopia but also on the occurrence and complete genome sequences of members of two nanovirus species in the same country, thus providing evidence for faba bean crops being infected by members of two distinct nanovirus species in a restricted geographic area.

Solution structure of the endonuclease domain from the master replication initiator protein of the nanovirus faba bean necrotic yellows virus and comparison with the corresponding geminivirus and circovirus structures.

Nanoviruses are a family of plant viruses that possess a genome of multiple circular single-stranded DNA (ssDNA) components and are strikingly similar in their replication mode to the plant geminiviruses and to the circoviruses that infect birds or mammals. These viruses multiply by rolling circle replication using virus-encoded multifunctional replication initiator proteins (Rep proteins) that catalyze the initiation of replication on a double-stranded DNA (dsDNA) intermediate and the resolution of the ssDNA into circles. Here we report the solution NMR three-dimensional structure of the endonuclease domain from the master Rep (M-Rep) protein of faba bean necrotic yellows virus (FBNYV), a representative of the nanoviruses. The domain comprises amino acids 2-95 (M-Rep2-95), and its global fold is similar to those previously described for the gemini- and circovirus Rep endonuclease domains, consisting of a central 5-stranded antiparallel beta-sheet covered on one side by an alpha-helix and irregular loops and on the other, more open side of the domain, by an alpha-helix containing the catalytic tyrosine residue (the catalytic helix). Longer domain constructs extending to amino acids 117 and 124 were also characterized. They contain an additional alpha-helix, are monomeric, and exhibit catalytic activity indistinguishable from that of M-Rep2-95. The binding site for the catalytic metal was identified by paramagnetic broadening and maps to residues on the exposed face of the central beta-sheet. A comparison with the previously determined Rep endonuclease domain structures of tomato yellow leaf curl Sardinia virus (TYLCSV), a geminivirus, and that of porcine circovirus type 2 (PCV2) Rep allows the identification of a positively charged surface that is most likely involved in dsDNA binding, and reveals common features shared by all endonuclease domains of nanovirus, geminivirus, and circovirus Rep proteins.

Nanoviruses: genome organisation and protein function.

Nanoviruses, single-stranded DNA (ssDNA) plant viruses with a multipartite genome, share similarities with members of the Circoviridae family that infect mammals or birds as well as with the Geminiviridae, the only other plant virus family with circular ssDNA genomes. Although the virions of the latter are unique and different from that of the circoviruses, the mode of replication of viruses with monopartite or multipartite circular ssDNA genomes is strikingly similar. They multiply by rolling circle replication using virus-encoded multifunctional replication initiator proteins (Rep proteins) that catalyse initiation of ssDNA replication and resolution of replicative ssDNA into circular single-stranded virion DNA. All these ssDNA viruses exploit host polymerases for DNA synthesis and code for proteins that modulate the host's cell cycle favourably for virus multiplication. Recent three-dimensional structure analyses of a geminivirus and a parvovirus Rep protein have revealed an intriguing similarity between the catalytic domains of their respective Rep proteins. Furthermore, these structural data revealed that ssDNA virus replication initiator proteins might represent evolutionary intermediates between certain RNA-binding proteins and some multifunctional origin-binding proteins of papovaviruses.

Characterisation of Rep-encoding components associated with banana bunchy top nanovirus in Vietnam.

We have analysed the sequence variability of the banana bunchy top nanovirus (BBTV) DNA-1 sequence from 17 isolates collected throughout Vietnam, and showed that the level of DNA-1 sequence variation within Vietnam was approximately double that previously reported for Asian BBTV isolates. Furthermore, the sequences separated into two geographical subgroups that generally correlated to the northern or southern regions of Vietnam. We have also characterised an additional putative Rep-encoding component associated with some BBTV isolates from Vietnam. This component, which we have named BBTV-S3, shared 47%, 69%, 56% and 65% nucleotide sequence identity with the previously reported Rep-encoding components BBTV DNA-1, S1, S2 and Y1 respectively.