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HIV-1 subtype C virus accounts for nearly 50% of the total HIV infections globally. Despite this high prevalence, our understanding of subtype C specific infections remains limited due to lack of an in vitro model system. This is the first report of construction and characterization of a full-length and infectious EGFP-tagged HIV-1 subtype C molecular clone. The EGFP gene was inserted in-frame between the Nef and Env sequence in the HIV genome. The recombinant virus displayed expression of viral genes, infectivity and replication kinetics similar to the parental virus. VSV-G pseudotyping of the recombinant virus led to enhancement of HIV infection. The presence of the EGFP gene provides a rapid, easy and quantitative measure of HIV infection by flow cytometry and fluorescence microscopy. This clone will serve as an extremely beneficial tool to study HIV-1 subtype C specific infections. • HIV-1 subtype C molecular clone (K3016-EGFP) expressing an EGFP tag was constructed. • The reporter clone expressed viral genes and was infectious. • The reporter clone replicated with similar kinetics like the parental clone. • This clone can serve as an important tool to develop models to study subtype C specific HIV infections. [ABSTRACT FROM AUTHOR]

Abstract Snakehead vesiculovirus (SHVV) is a new rhabdovirus isolated from diseased hybrid snakehead fish (Channa maculate ♀ x Channa argus ♂) and has caused serious economic losses in snakehead fish culture in China. To better understand the pathogenicity of SHVV, we developed a reverse genetics system for SHVV by using human and fish cells. In detail, human 293T cells were co-transfected with four plasmids encoding the full-length SHVV antigenomic RNA or the supporting proteins including nucleoprotein (N), phosphoprotein (P), and large polymerase (L), followed by the cultivation in Channel catfish ovary (CCO) cells. We also rescued a recombinant SHVV expressing enhanced green fluorescent protein (EGFP), which was inserted into the 3′ non-coding region (NCR) of the glycoprotein (G) gene of SHVV. Our study provides a potential tool for unveiling the pathogenicity of SHVV and a template for the rescue of other fish viruses by using both human 293T and fish cells. Highlights • Establishment of reverse genetics system for fish rhabdovirus SHVV; • Rescue of fish virus using human and fish cells; • Generating a recombinant virus expressing EGFP with impaired growth ability. [ABSTRACT FROM AUTHOR]

Abstract: An up to 10,000-fold resistance against the biocontrol agent Cydia pomonella granulovirus (CpGV) was observed in field populations of codling moth, C. pomonella, in Europe. Following different experimental approaches, a modified peritrophic membrane, a modified midgut receptor, or a change of the innate immune response could be excluded as possible resistance mechanisms. When CpGV replication was traced by quantitative PCR in different tissues of susceptible and resistant insects after oral and intra-hemocoelic infection, no virus replication could be detected in any of the tissues of resistant insects, suggesting a systemic block prior to viral DNA replication. This conclusion was corroborated by fluorescence microscopy using a modified CpGV (bacCpGVhsp-eGFP) carrying enhanced green fluorescent gene (eGFP), which showed that infection in resistant insects did not spread. In conclusion, the different lines of evidence indicate that CpGV can enter but not replicate in the cells of resistant codling moth larvae. [Copyright &y& Elsevier]

Abstract: During infection, human immunodeficiency virus type 1 integrase engages a number of molecules and mechanisms, both of viral and cellular origin. In one of such instances, integrase is thought to be degraded by the N-end rule proteasome pathway a process that targets the N-terminal residue of its substrates. Here we describe the properties of HIV-1 viruses in which the first amino acid residue of integrase has been substituted to render it resistant to the N-end rule pathway. As result of this exchange, we observe a set of class I and class II defects that result in a large decrease of viral replication efficiency. Specifically, reverse transcription and integration are the steps that appear to be affected. We propose that the severe deficiency of these mutants exert a strong selective pressure that leads to the near total conservation of the N-terminal residue of integrase in HIV-1, HIV-2 and SIV. [Copyright &y& Elsevier]

Abstract: The study of adenovirus could greatly benefit from diverse methods of virus detection. Recently, it has been demonstrated that carboxy-terminal EGFP fusions of adenovirus core proteins Mu, V, and VII properly localize to the nucleus and display novel function in the cell. Based on these observations, we hypothesized that the core proteins may serve as targets for labeling the adenovirus core with fluorescent proteins. To this end, we constructed various chimeric expression vectors with fusion core genes (Mu-EGFP, V-EGFP, preVII-EGFP, and matVII-EGFP) while maintaining expression of the native proteins. Expression of the fusion core proteins was suboptimal using E1 expression vectors with both conventional CMV and modified (with adenovirus tripartite leader sequence) CMV5 promoters, resulting in non-labeled viral particles. However, robust expression equivalent to the native protein was observed when the fusion genes were placed in the deleted E3 region. The efficient Ad-wt-E3-V-EGFP and Ad-wt-E3-preVII-EGFP expression vectors were labeled allowing visualization of purified virus and tracking of the viral core during early infection. The vectors maintained their viral function, including viral DNA replication, viral DNA encapsidation, cytopathic effect, and thermostability. Core labeling offers a means to track the adenovirus core in vector targeting studies as well as basic adenovirus virology. [Copyright &y& Elsevier]

Abstract: Zaire ebolavirus causes large outbreaks of severe and usually fatal hemorrhagic disease in humans for which there is no effective treatment or cure. To facilitate examination of early critical events in viral pathogenesis and to identify antiviral compounds, a recombinant Zaire ebolavirus was engineered to express a foreign protein, eGFP, to provide a rapid and sensitive means to monitor virus replication in infected cells. This genetically engineered virus represents the first insertion of a foreign gene into ebolavirus. We show that Ebola-eGFP virus (EboZ-eGFP) infects known early targets of human infections and serves as an ideal model to screen antiviral compounds in less time than any previously published assay. [Copyright &y& Elsevier]

We have previously discovered and characterized the nuclear import pathways for the E7 oncoproteins of mucosal alpha genus HPVs, type 16 and 11. Here we investigated the nuclear import of cutaneous beta genus HPV8 E7 protein using confocal microscopy after transfections of HeLa cells with EGFP-8E7 and mutant plasmids and nuclear import assays in digitonin-permeabilized HeLa cells. We determined that HPV8 E7 contains a nuclear localization signal (NLS) within its zinc-binding domain that mediates its nuclear import. Furthermore, we discovered that a mostly hydrophobic patch 65LRLFV69 within the zinc-binding domain is essential for the nuclear import and localization of HPV8 E7 via hydrophobic interactions with the FG nucleoporins Nup62 and Nup153. Substitution of the hydrophobic residues within the 65LRLFV69 patch to alanines, and not R66A mutation, disrupt the interactions between the 8E7 zinc-binding domain and Nup62 and Nup153 and consequently inhibit nuclear import of HPV8 E7., (© 2013 Published by Elsevier Inc.)

We previously discovered that nuclear import of high risk HPV16 E7 is mediated by a cNLS located within the zinc-binding domain via a pathway that is independent of karyopherins/importins (Angeline et al., 2003; Knapp et al., 2009). In this study we continued our characterization of the cNLS and nuclear import pathway of HPV16 E7. We find that an intact zinc-binding domain is essential for the cNLS function in mediating nuclear import of HPV16 E7. Mutagenesis of cysteine residues to alanine in each of the two CysXXCys motifs involved in zinc-binding changes the nuclear localization of the EGFP-16E7 and 2xEGFP-16E7 mutants. We further discover that a patch of hydrophobic residues, 65LRLCV69, within the zinc-binding domain of HPV16 E7 mediates its nuclear import via hydrophobic interactions with the FG domain of the central channel nucleoporin Nup62., (© 2013 Elsevier Inc. All rights reserved.)