Journal: BMC Infectious Diseases

Article Title: The PTAP sequence duplication in HIV-1 subtype C Gag p6 in drug-naive subjects of India and South Africa

doi: 10.1186/s12879-017-2184-4

Figure Lengend Snippet: a Schematic representation of HIV-1 Gag protein domains. The four major domains of Gag (MA, CA, p7, and p6) are depicted including the two linker sequences p1 and p2. HIV-1 Gag interacts with the ESCRT complex proteins Tsg101 and Alix to regulate viral budding. The sequence of subtype B NL4-3 gag p6 is presented and the sequence motifs PTAP and YPXnL, which serve as the binding motif for Tsg101 and Alix, respectively, are highlighted using the square boxes. Con_C represents the subtype C gag p6 consensus amino acid sequence. The dashes represent sequence identity and the dots sequence deletion. b A comparative analysis of the PTAP sequence duplication in subtypes B and C. In subtype B, a partial PTAP duplication consisting of three amino-acid residues (APP) is common. In contrast in subtype C, a sequence duplication of 14 amino acids is common. A 14 amino acid sequence duplication of subtype C derived from the primary clinical isolate T004 is presented. The amino acid sequences, the original and the duplicated sequences, in Gag and Gag-Pol are depicted. The arrows represent the length of sequence duplication and the direction of polymerization by the reverse transcriptase while synthesizing the cDNA from the viral RNA. The core PTAP motifs are highlighted using the square boxes. The sequences flanking the 3 or 14 aa residues are shown in gray

Article Snippet: RNA was extracted from 150 μl of plasma samples using a commercial Viral RNA isolation kit (NucleoSpin® RNA Virus, Ref. No. 740956.50, MACHEREY-NAGEL GmbH & Co. KG, Germany).

Techniques: Sequencing, Binding Assay, Derivative Assay, Reverse Transcription

Journal: BMC Infectious Diseases

Article Title: The PTAP sequence duplication in HIV-1 subtype C Gag p6 in drug-naive subjects of India and South Africa

doi: 10.1186/s12879-017-2184-4

Figure Lengend Snippet: Multiple sequence alignment of the PTAP sequences of eight primary viral isolates containing PTAP duplication. The sequences were derived from the plasma viral RNA collected at multiple longitudinal time-points and by sequencing the PCR fragments. In three subjects (T004, 2012, 2032), the PTAP motif duplication involved the duplication of 14 amino acids. In the other five subjects, the duplicated sequence length was shorter containing 12 (2018), 11(2020), 9 (2037) or 8 (T014, and 2006) residues. Of note, in all the sequences, the ‘PTAP’ core motif was intact in the original and the duplicated sequences. Note that in two subjects (2014 and 2018) a mixed infection of single- and double-PTAP viral strains could be seen by conventional sequencing. In subject T014, PTAP duplication was observed only at a single time-point M24. For subject 2006, the plasma sample was available only at the baseline. The vertical dashed line demarcates the original and the duplicated PTAP motifs. The arrows indicate the direction of the RT polymerization. Cons: The consensus subtype C gag sequence of 14 amino acids. The same 14 amino acid consensus sequence was used twice in the analysis for convenience. *For these five samples, only gag p6 , not the full-length gag , was amplified. These sequences have not been deposited in the Genbank and are not part of the phylogenetic tree presented in Fig. . D: the duplicated PTAP motif, O, the original PTAP motif

Article Snippet: RNA was extracted from 150 μl of plasma samples using a commercial Viral RNA isolation kit (NucleoSpin® RNA Virus, Ref. No. 740956.50, MACHEREY-NAGEL GmbH & Co. KG, Germany).

Techniques: Sequencing, Derivative Assay, Clinical Proteomics, Infection, Amplification

Journal: bioRxiv

Article Title: Zika virus noncoding RNA cooperates with the viral protein NS5 to inhibit STAT1 phosphorylation and facilitate viral pathogenesis

doi: 10.1101/2021.05.18.444753

Figure Lengend Snippet: (A) Detection of ZIKV sfRNA in Vero cells infected with WT, xrRNA1’ and xrRNA2’ viruses. Cells were infected at MOI=1, RNA was isolated at 72hpi and used for Northern blotting. Blot was hybridised with a probe complementary to the most terminal 31nt of viral genome. Bottom panel shows rRNA visualised by Et-Br staining as a loading control. The blot is a representative image from three independent experiments that showed similar results. (B) Quantification of viral RNA in samples used for northern blotting in (A). RNA copy numbers were determined by qRT-PCR with normalisation to input RNA amount. The values are the means from three biological replicates ±SD, statistical analysis is by one-way ANOVA. (C) ZIKV plaque morphology on a monolayer of Vero cells at 72hpi. Bottom panel demonstrates virus replication foci visualised by immunostaining of Vero cells inoculated with the same virus samples. (D) Cytotoxicity of WT and sfRNA-deficient ZIKV mutants determined using Viral ToxGlo Assay. Vero cells were infected at the indicated viral dose per well and CPE was measured at 72hpi. %CPE is calculated with the reference to uninfected cells grown for the same period of time. Individual values from two independent experiments with technical triplicates in each are displayed. (E) ZIKV titres in culture fluids of human and mouse cells infected with WT and sfRNA deficient ZIKV. Cells were infected at MOI=0.1. Values are the means from three independent experiments + SD. Statistical analysis is by two-way ANOVA with Dunnett correction. (F) Replication of WT and sfRNA- deficient ZIKV in AG129 mice. Mice were inoculated with 10 4 FFU of the viruses by subcutaneous injection and were monitored for disease symptoms twice a day for 15 days and blood was collected daily via tail bleeding. Mean values were used to build the curves in viremia analysis and all individual values are shown on the plot. The results of the statistical analysis for viremia are summarised in Supplementary table 1, survival rates were compared using Gehan-Breslow-Wilcoxon test. Viral titres were determined by foci-forming immunoassay with cross-reactive mouse anti-E protein monoclonal antibodies (4G2) as presented in (E) and human anti-E protein (Z67) as presented in (F).

Article Snippet: Viral RNA was isolated from cell culture fluids using the NucleoSpin RNA Virus Kit (Macherey-Nagel, Germany).

Techniques: Infection, Isolation, Northern Blot, Staining, Control, Quantitative RT-PCR, Virus, Immunostaining, Injection, Bioprocessing

Journal: bioRxiv

Article Title: Zika virus noncoding RNA cooperates with the viral protein NS5 to inhibit STAT1 phosphorylation and facilitate viral pathogenesis

doi: 10.1101/2021.05.18.444753

Figure Lengend Snippet: (A) Differential gene expression in BeWo cells infected with WT and xrRNA2’ ZIKV. Points indicating the genes that responded differently (FDR-adjusted P-value < 0.05) to infection with WT and mutant viruses are shown in red. (B) Pathways and biological processes affected by production of ZIKV sfRNA. The sfRNA-affected genes identified in (A) were subjected to GO and KEGG enrichment analysis. The data on enrichment significance was then combined with expression values for each gene and z-scores were calculated. Z-scores show directionality in expression changes of the genes associated with each sfRNA-affected process – negative indicate for inhibition and positive indicate for activation. (C) Expression of the individual genes associated with biological processes affected by production of ZIKV sfRNA in BeWo cells. Values are Z-scores with green indicating for negative and purple for positive and are the means from three biological replicates. (D) Expression of ISGs and IFNs in BeWo cells infected with WT virus (cyan bars) and xrRNA2’ mutant virus (magenta bars) ZIKV. Cells were infected at MOI=1, total RNA was isolated at the indicated time points and used for qRT-PCR. Relative mRNA quantity was determined using ΔΔ Ct method and is relative to Mock with normalisation to GAPDH . The values are the means of three biological replicates ±SD. Statistical analysis was performed using multiple t-tests; * p<0.05 , ** p<0.01 , *** p<0.001 , **** p<0.0001 . (E) Network of interactions between the sfRNA-affected genes involved in antiviral response and apoptosis. Two individual networks were reconstructed form the genes identified in (C) using Genemania CytoScape plug-in and then merged. The resulted network was subjected to CytoScape network analysis to calculate the Betweennes Centrality values as a measure for the weight of each nod in the combined network. Size of the nods indicates for Betweenness Centrality, colour of the nods indicates the difference in gene expression between the cells infected with WT and xrRNA2’ mutant ZIKV. Positions of the nods are relative to their connectivity within and between the two subnetworks.

Article Snippet: Viral RNA was isolated from cell culture fluids using the NucleoSpin RNA Virus Kit (Macherey-Nagel, Germany).

Techniques: Gene Expression, Infection, Mutagenesis, Expressing, Inhibition, Activation Assay, Virus, Isolation, Quantitative RT-PCR

Journal: bioRxiv

Article Title: Zika virus noncoding RNA cooperates with the viral protein NS5 to inhibit STAT1 phosphorylation and facilitate viral pathogenesis

doi: 10.1101/2021.05.18.444753

Figure Lengend Snippet: (A) The sfRNA-binding proteins identified in lysates of A549 cells infected with sfRNA-deficient ZIKV mutant using RNA affinity pull-down. Top 10 most significant sfRNA- interacting proteins are highlighted in red circles and named. The values are the means from three independent experiments. (B) Schematics of the reporter constructs generated for production of ZIKV sfRNA and NS5 alone and in combination. The C22G mutations that abolish XRN-1 resistance in each of ZIKV xrRNA elements and disrupt production of sfRNA are highlighted. (C) Northern blotting demonstrating production of sfRNA in HEK293T cells transfected with expression constructs shown in (B). RNA was isolated at 48h post transfection and 5ug was loaded per lane. Bottom panel shows 5.8S rRNA on an Et-Br-stained gel as a loading control. (D) Western-blot detection of phosphorylated STAT1 (pSTAT1), total STAT1 and ZIKV NS5 in HEK293T cells transfected with expression constructs shown in (B) and treated with IFN α . IFN-treatment was performed at 48hpt and cells were lysed for Western blotting after 15 min of treatment. GAPDH is shown as a loading control. (E) Quantification of pSTAT1 levels in (D) by densitometry. Normalised levels represent the ratios of pSTAT1 band density to GAPDH band density and expressed as a percentage relative to the values observed in the samples transfected with pcDNA-GFP-3’UTR-Mut plasmid. (F) Quantification of NS5 levels in (D) by densitometry. (G) Western-blot detection of structural (E-protein) and non-structural (NS5, NS3, NS1) proteins in the lysates of Vero cells infected with WT and sfRNA-deficient (xrRNA2’) ZIKV. Cells were infected at MOI=5 and assays were performed at 48hpi. (H) Quantification of the viral proteins levels in (G) by densitometry. Normalised levels represent the ratios of each protein band density to GAPDH band density and expressed as a percentage of the protein level in xrRNA2’-infected cells to WT ZIKV-infected cells. The blots in (C, D, G) are representative from three independent experiments that showed similar results. The values in (E, F, H) are the means from three independent experiments ±SD. Statistical analysis was performed by one-way ANOVA with Dunnett correction (E) or Student’s t-test (F, H).

Article Snippet: Viral RNA was isolated from cell culture fluids using the NucleoSpin RNA Virus Kit (Macherey-Nagel, Germany).

Techniques: Binding Assay, Infection, Mutagenesis, Construct, Generated, Northern Blot, Transfection, Expressing, Isolation, Staining, Control, Western Blot, Plasmid Preparation