Viruses2015, 7(3), 939-968; doi:10.3390/v7030939 (registering DOI) - published 4 March 2015 Show/Hide Abstract
Abstract: Human endogenous retrovirus (HERV-K (HML-2)) proviruses are among the few endogenous retroviral elements in the human genome that retain coding sequence. HML-2 expression has been widely associated with human disease states, including different types of cancers as well as with HIV-1 infection. Understanding of the potential impact of this expression requires that it be annotated at the proviral level. Here, we utilized the high throughput capabilities of next-generation sequencing to profile HML-2 expression at the level of individual proviruses and secreted virions in the teratocarcinoma cell line Tera-1. We identified well-defined expression patterns, with transcripts emanating primarily from two proviruses located on chromosome 22, only one of which was efficiently packaged. Interestingly, there was a preference for transcripts of recently integrated proviruses, over those from other highly expressed but older elements, to be packaged into virions. We also assessed the promoter competence of the 5’ long terminal repeats (LTRs) of expressed proviruses via a luciferase assay following transfection of Tera-1 cells. Consistent with the RNASeq results, we found that the activity of most LTRs corresponded to their transcript levels.
Viruses2015, 7(3), 915-938; doi:10.3390/v7030915 (registering DOI) - published 4 March 2015 Show/Hide Abstract
Abstract: Herpes simplex virus-1 (HSV-1), like all herpesviruses, is a large complex DNA virus containing up to 16 different viral membrane proteins in its envelope. The assembly of HSV-1 particles occurs by budding/wrapping at intracellular membranes producing infectious virions contained within the lumen of cytoplasmic membrane-bound compartments that are then released by secretion. To ensure incorporation of all viral membrane proteins into the envelope, they need to be localized to the appropriate intracellular membranes either via the endocytic pathway or by direct targeting to assembly sites from the biosynthetic secretory pathway. Many HSV-1 envelope proteins encode targeting motifs that direct their endocytosis and targeting, while others do not, including the essential entry proteins gD and the gH/gL complex, and so it has been unclear how these envelope proteins reach the appropriate assembly compartments. We now show that efficient endocytosis of gD and gH/gL and their incorporation into mature virions relies upon the presence of the HSV-1 envelope proteins gM and the gK/pUL20 complex. Our data demonstrate both redundant and synergistic roles for gM and gK/pUL20 in controlling the targeting of gD and gH/L to the appropriate intracellular virus assembly compartments.
Viruses2015, 7(3), 899-914; doi:10.3390/v7030899 - published 26 February 2015 Show/Hide Abstract
Abstract: Assembly of herpesvirus nucleocapsids shares significant similarities with the assembly of tailed dsDNA bacteriophages; however, important differences exist. A unique feature of herpesviruses is the presence of different mature capsid forms in the host cell nucleus during infection. These capsid forms, referred to as A-, B-, and C-capsids, represent empty capsids, scaffold containing capsids and viral DNA containing capsids, respectively. The C-capsids are the closest in form to those encapsidated into mature virions and are considered precursors to infectious virus. The evidence supporting A- and B-capsids as either abortive forms or assembly intermediates has been lacking. Interaction of specific capsid forms with viral tegument proteins has been proposed to be a mechanism for quality control at the point of nuclear egress of mature particles. Here, we will review the available literature on these capsid forms and present data to debate whether A- and B-capsids play an important or an extraneous role in the herpesvirus life cycle.
Viruses2015, 7(3), 887-898; doi:10.3390/v7030887 - published 25 February 2015 Show/Hide Abstract
Abstract: Infection of poultry with diverse lineages of H5N2 avian influenza viruses has been documented for over three decades in different parts of the world, with limited outbreaks caused by this highly pathogenic avian influenza virus. In the present study, three avian H5N2 influenza viruses, A/chicken/Shijiazhuang/1209/2013, A/chicken/Chiping/0321/2014, and A/chicken/Laiwu/0313/2014, were isolated from chickens with clinical symptoms of avian influenza. Complete genomic and phylogenetic analyses demonstrated that all three isolates are novel recombinant viruses with hemagglutinin (HA) and matrix (M) genes derived from H5N1, and remaining genes derived from H9N2-like viruses. The HA cleavage motif in all three strains (PQIEGRRRKR/GL) is characteristic of a highly pathogenic avian influenza virus strain. These results indicate the occurrence of H5N2 recombination and highlight the importance of continued surveillance of the H5N2 subtype virus and reformulation of vaccine strains.
Viruses2015, 7(3), 873-886; doi:10.3390/v7030873 - published 24 February 2015 Show/Hide Abstract
Abstract: Porcine reproductive and respiratory syndrome virus (PRRSV) is currently one of the most important viruses affecting the swine industry worldwide. Despite the large number of papers published each year, the participation of non-structural proteins (nsps) in the immune response is not completely clear. nsps have been involved in the host innate immune response, specifically, nsp1α/β, nsp2, nsp4 and nsp11 have been associated with the immunomodulation capability of the virus. To date, only participation by nsp1, nsp2, nsp4 and nsp7 in the humoral immune response has been reported, with the role of other nsps being overlooked. Furthermore, nsp1, nsp2, nsp5, nsp7 nsp9, nsp10, nsp11 have been implicated in the induction of IFN-γ and probably in the development of the cell-mediated immune response. This review discusses recent reports involving the participation of nsps in the modulation of the innate immune response and their role in the induction of both the humoral and cellular immune responses.
Viruses2015, 7(3), 857-872; doi:10.3390/v7030857 - published 20 February 2015 Show/Hide Abstract
Abstract: Development and evaluation of medical countermeasures for diagnostics, vaccines, and therapeutics requires production of standardized, reproducible, and well characterized virus preparations. For filoviruses this includes plaque assay for quantitation of infectious virus, transmission electron microscopy (TEM) for morphology and quantitation of virus particles, and real-time reverse transcription PCR for quantitation of viral RNA (qRT-PCR). The ViroCyt® Virus Counter (VC) 2100 (ViroCyt, Boulder, CO, USA) is a flow-based instrument capable of quantifying virus particles in solution. Using a proprietary combination of fluorescent dyes that stain both nucleic acid and protein in a single 30 min step, rapid, reproducible, and cost-effective quantification of filovirus particles was demonstrated. Using a seed stock of Ebola virus variant Kikwit, the linear range of the instrument was determined to be 2.8E+06 to 1.0E+09 virus particles per mL with coefficient of variation ranging from 9.4% to 31.5% for samples tested in triplicate. VC particle counts for various filovirus stocks were within one log of TEM particle counts. A linear relationship was established between the plaque assay, qRT-PCR, and the VC. VC results significantly correlated with both plaque assay and qRT-PCR. These results demonstrated that the VC is an easy, fast, and consistent method to quantify filoviruses in stock preparations.