Evolutionary Selection Against Short Nucleotide Sequences in Viruses and Their Related Hosts

Viruses are under constant evolutionary pressure to effectively interact with the host intracellular factors, while evading its immune system. Understanding how viruses co-evolve with their hosts is a fundamental topic in molecular evolution and may also aid in developing novel viral based applications such as vaccines, oncologic therapies, and anti-bacterial treatments. Here, based on a novel statistical framework and a large-scale genomic analysis of 2,625 viruses from all classes infecting 439 host organisms from all kingdoms of life, we identify short nucleotide sequences that are under-represented in the coding regions of viruses and their hosts. These sequences cannot be explained by the coding regions’ amino acid content, codon, and dinucleotide frequencies. We specifically show that short homooligonucleotide and palindromic sequences tend to be under-represented in many viruses probably due to their effect on gene expression regulation and the interaction with the host immune system. In addition, we show that more sequences tend to be under-represented in dsDNA viruses than in other viral groups. Finally, we demonstrate, based on in vitro and in vivo experiments, how under-represented sequences can be used to attenuated Zika virus strains

Structural insights into RNA processing by the human RISC-loading complex.

Targeted gene silencing by RNA interference (RNAi) requires loading of a short guide RNA (small interfering RNA (siRNA) or microRNA (miRNA)) onto an Argonaute protein to form the functional center of an RNA-induced silencing complex (RISC). In humans, Argonaute2 (AGO2) assembles with the guide RNA-generating enzyme Dicer and the RNA-binding protein TRBP to form a RISC-loading complex (RLC), which is necessary for efficient transfer of nascent siRNAs and miRNAs from Dicer to AGO2. Here, using single-particle EM analysis, we show that human Dicer has an L-shaped structure. The RLC Dicer's N-terminal DExH/D domain, located in a short 'base branch', interacts with TRBP, whereas its C-terminal catalytic domains in the main body are proximal to AGO2. A model generated by docking the available atomic structures of Dicer and Argonaute homologs into the RLC reconstruction suggests a mechanism for siRNA transfer from Dicer to AGO2

A Novel Pathogenic Mechanism of Highly Pathogenic Avian Influenza H5N1 Viruses Involves Hemagglutinin Mediated Resistance to Serum Innate Inhibitors

In this study, the effect of innate serum inhibitors on influenza virus infection was addressed. Seasonal influenza A(H1N1) and A(H3N2), 2009 pandemic A(H1N1) (H1N1pdm) and highly pathogenic avian influenza (HPAI) A(H5N1) viruses were tested with guinea pig sera negative for antibodies against all of these viruses as evaluated by hemagglutination-inhibition and microneutralization assays. In the presence of serum inhibitors, the infection by each virus was inhibited differently as measured by the amount of viral nucleoprotein produced in Madin-Darby canine kidney cells. The serum inhibitors inhibited seasonal influenza A(H3N2) virus the most, while the effect was less in seasonal influenza A(H1N1) and H1N1pdm viruses. The suppression by serum inhibitors could be reduced by heat inactivation or treatment with receptor destroying enzyme. In contrast, all H5N1 strains tested were resistant to serum inhibitors. To determine which structure (hemagglutinin (HA) and/or neuraminidase (NA)) on the virus particles that provided the resistance, reverse genetics (rg) was applied to construct chimeric recombinant viruses from A/Puerto Rico/8/1934(H1N1) (PR8) plasmid vectors. rgPR8-H5 HA and rgPR8-H5 HANA were resistant to serum inhibitors while rgPR8-H5 NA and PR8 A(H1N1) parental viruses were sensitive, suggesting that HA of HPAI H5N1 viruses bestowed viral resistance to serum inhibition. These results suggested that the ability to resist serum inhibition might enable the viremic H5N1 viruses to disseminate to distal end organs. The present study also analyzed for correlation between susceptibility to serum inhibitors and number of glycosylation sites present on the globular heads of HA and NA. H3N2 viruses, the subtype with highest susceptibility to serum inhibitors, harbored the highest number of glycosylation sites on the HA globular head. However, this positive correlation cannot be drawn for the other influenza subtypes

Multi-color fluorescent reporter dengue viruses with improved stability for analysis of a multi-virus infection.

Reporter virus is a versatile tool to visualize and to analyze virus infections. However, for flaviviruses, it is difficult to maintain the inserted reporter genes on the viral genome, limiting its use in several studies that require homogeneous virus particles and several rounds of virus replication. Here, we showed that flanking inserted GFP genes on both sides with ribosome-skipping 2A sequences improved the stability and the consistency of their fluorescent signals for dengue-virus-serotype 2 (DENV2) reporter viruses. The reporter viruses can infect known susceptible mammalian cell lines and primary CD14+ human monocytes. This design can accommodate several fluorescent protein genes, enabling the generation of multi-color DENV2-16681 reporter viruses with comparable replication capabilities, as demonstrated by their abilities to maintain their fluorescent intensities during co-infections and to exclude superinfections regardless of the fluorescent tags. The reported design of multi-color DENV2 should be useful for high-throughput analyses, single-cell analysis, and characterizations of interference and superinfection in animal models

Influenza Neuraminidase Subtype N1: Immunobiological Properties and Functional Assays for Specific Antibody Response.

Influenza neuraminidase (NA) proteins expressed in TK- cells infected with recombinant vaccinia virus carrying NA gene of highly pathogenic avian influenza H5N1 virus or 2009 pandemic H1N1 (H1N1pdm) virus were characterized for their biological properties, i.e., cell localization, molecular weight (MW), glycosylation and sialidase activity. Immune sera collected from BALB/c mice immunized with these recombinant viruses were assayed for binding and functional activities of anti-NA antibodies. Recombinant NA proteins were found localized in cytoplasm and cytoplasmic membrane of the infected cells. H1N1pdm NA protein had MW at about 75 kDa while it was 55 kDa for H5N1 NA protein. Hyperglycosylation was more pronounced in H1N1pdm NA compared to H5N1 NA according to N-glycosidase F treatment. Three dimensional structures also predicted that H1N1 NA globular head contained 4 and that of H5N1 contained 2 potential glycosylation sites. H5N1 NA protein had higher sialidase activity than H1N1pdm NA protein as measured by both MUNANA-based assay and fetuin-based enzyme-linked lectin assay (ELLA). Plaque reduction assay demonstrated that anti-NA antibody could reduce number of plaques and plaque size through inhibiting virus release, not virus entry. Assay for neuraminidase-inhibition (NI) antibody by ELLA showed specific and cross reactivity between H5N1 NA and H1N1pdm NA protein derived from reverse genetic viruses or wild type viruses. In contrast, replication-inhibition assay in MDCK cells showed that anti-H1N1 NA antibody moderately inhibited viruses with homologous NA gene only, while anti-H5N1 NA antibody modestly inhibited the replication of viruses containing homologous NA gene and NA gene derived from H1N1pdm virus. Anti-H1N1 NA antibody showed higher titers of inhibiting virus replication than anti-H5N1 NA antibody, which are consistent with the results on reduction in plaque numbers and sizes as well as in inhibiting NA enzymatic activity. No assay showed cross reactivity with reassorted PR8 (H1N1) virus and H3N2 wild type viruses

Structural insights into RNA processing by the human RISC-loading complex.

Targeted gene silencing by RNA interference (RNAi) requires loading of a short guide RNA (small interfering RNA (siRNA) or microRNA (miRNA)) onto an Argonaute protein to form the functional center of an RNA-induced silencing complex (RISC). In humans, Argonaute2 (AGO2) assembles with the guide RNA-generating enzyme Dicer and the RNA-binding protein TRBP to form a RISC-loading complex (RLC), which is necessary for efficient transfer of nascent siRNAs and miRNAs from Dicer to AGO2. Here, using single-particle EM analysis, we show that human Dicer has an L-shaped structure. The RLC Dicer's N-terminal DExH/D domain, located in a short 'base branch', interacts with TRBP, whereas its C-terminal catalytic domains in the main body are proximal to AGO2. A model generated by docking the available atomic structures of Dicer and Argonaute homologs into the RLC reconstruction suggests a mechanism for siRNA transfer from Dicer to AGO2

Sequences of primers and protospacer sequence of gRNAs used in this work.

<p>Sequences of primers and protospacer sequence of gRNAs used in this work.</p

Improved stability of GFP reporter genes on DENV2 genome with 2x 2A design.

<p>A) Fluorescent microscopy of infected Vero cells during passaging. The left diagram outlines the passaging of a reporter virus. MOI of 0.01 was used to infect confluent Vero cells for each passage. Green-fluorescent images of infected Vero cells (right panel) were taken seven days post infection. P1, P2, P3, P4, P5, and P6 designate passage numbers. 1x 2A and 2x 2A denote the design of the reporter viruses. B) Flow-cytometry measurements of Vero cells infected with passaged DENV2-eGFP. Confluent Vero cells were infected with viruses from each passage (culture media collected at day 7) at MOI = 0.1. Infected cells were maintained for two days before whole-cell staining with 4G2 mouse antibody and Alexa-647-conjugated anti-mouse antibody. The percentage of each cell population is shown in each quardrant of the scatter plot. The top row shows the measurements of passaged DENV2-eGFP with the 1x 2A design while the bottom row show those of 2x 2A design. C) Quantification of the stability of GFP reporter genes based on flow-cytometry measurements. The stability was measured as the percentage of GFP-positive cells in the populations of infected cells (4G2-positive cells). % GFP/4G2 = (% cells with GFP)/(% cells with 4G2). D) RT-PCR of passaged DENV2-eGFP. Top diagram shows the locations of the primer binding sites (represented by arrows) on the viral genome of reporter DENV2-eGFP. Bottom is the image of agarose gel electrophoresis of the RT-PCR products of viral RNA extracted from passaged viruses.</p

A panel of multi-color fluorescent DENV2 with comparable replicative abilities.

<p>A) Multi-step replication kinetics of multi-color fluorescent DENV2. Confluent Vero cells were infected with one virus at MOI = 0.01. The culture media was sampled from the flask every 24 hours. Infectious virus titer in the media was assayed by foci-forming assay. B) Focus images of the wild-type (16681) and the multi-color DENV2 (top panel) and the bar graph comparing the sizes of their foci (bottom panel). Focus size was quantified by the number of pixels that it occupied on a digitized image taken on ELISpot reader. Each bar represents the average of the measured focus sizes and the error bars represent standard deviation. Between 143 and 713 foci were used for focus size quantification. C) Representative scatter plots of K562-CD209 infected with DENV2-eGFP and DENV2-mCherry. The first (with DENV2-eGFP) and the second (with DENV2-mCherry) infections were 24 hours apart. The infected cells were analyzed 3 days post infection. In the case of co-infection (right plot), the cells were infected by both viruses at the same time and analyzed 2 days post infection. D) Comparison of infection percentages of the second virus in K562-CD209 between the presence (diagonal-stripe bars) and the absence (empty-bar) of the first virus infections. The infection percentages in the absence of the first virus infections are normalized to 100% for comparing the extent of reduction (or exclusion) conferred by the first virus. E) Histograms of fluorescent intensities for each fluorescent reporter measured from populations of K562-CD209 infected with single virus, and co-infected with two, three, and four reporter DENV2 of different colors.</p

Inability of the reporter DENV2 to replicate in mosquito host.

<p>A) Fluorescent microscopy (top panel) and flow cytometry (bottom panel) of C6/36 cells infected with DENV2-16681 (WT) and DENV2-GFPs with 1x 2A and 2x 2A designs. C6/36 cells were infected with a DENV2 virus at MOI = 0.1 and cultured for 3 days before analyses. Infected cells were identified by immunostaining with 4G2 antibody. The number in each quadrant of the flow cytometry scatter plot represents the percentage of cells in the quadrant. B) Fluorescent microscopy of <i>Aedes aegypti</i> mosquitoes that received DENV2-16681 (WT), DENV2-Clover2-2x 2A, and DENV2-eGFP-2x 2A by intrathoracic injection. The mosquitoes were imaged seven days after injection. C) Propagation of viruses in the mosquitoes injected with DENV2-16681 (WT), DENV2-Clover2-2x 2A, and DENV2-eGFP-2x 2A. The infectious titer (FFU/mosquito) was quantified from the whole-body homogenate of a mosquito by immunostaining foci assay at 4, 7, and 10 days post intrathoracic injection (dpi). The table underneath the plot reports the descriptive statistics of the infectious-titer measurements that include the number of the mosquitoes (n), the minimum (Min), the maximum (Max), the median, the mean, and the standard deviation (SD) of the infectious titers for each condition.</p