Nonstructural Proteins Are Preferential Positive Selection Targets in Zika Virus and Related Flaviviruses

<div><p>The <i>Flavivirus</i> genus comprises several human pathogens such as dengue virus (DENV), Japanese encephalitis virus (JEV), and Zika virus (ZIKV). Although ZIKV usually causes mild symptoms, growing evidence is linking it to congenital birth defects and to increased risk of Guillain-Barré syndrome. ZIKV encodes a polyprotein that is processed to produce three structural and seven nonstructural (NS) proteins. We investigated the evolution of the viral polyprotein in ZIKV and in related flaviviruses (DENV, Spondweni virus, and Kedougou virus). After accounting for saturation issues, alignment uncertainties, and recombination, we found evidence of episodic positive selection on the branch that separates DENV from the other flaviviruses. NS1 emerged as the major selection target, and selected sites were located in immune epitopes or in functionally important protein regions. Three of these sites are located in an NS1 region that interacts with structural proteins and is essential for virion biogenesis. Analysis of the more recent evolutionary history of ZIKV lineages indicated that positive selection acted on NS5 and NS4B, this latter representing the preferential target. All selected sites were located in the N-terminal portion of NS4B, which inhibits interferon response. One of the positively selected sites (26M/I/T/V) in ZIKV also represents a selection target in sylvatic DENV2 isolates, and a nearby residue evolves adaptively in JEV. Two additional positively selected sites are within a protein region that interacts with host (e.g. STING) and viral (i.e. NS1, NS4A) proteins. Notably, mutations in the NS4B region of other flaviviruses modulate neurovirulence and/or neuroinvasiveness. These results suggest that the positively selected sites we identified modulate viral replication and contribute to immune evasion. These sites should be prioritized in future experimental studies. However, analyses herein detected no selective events associated to the spread of the Asian/American ZIKV lineage.</p></div

Positively selected sites in ZIKV polyprotein.

<p>Positively selected sites in ZIKV polyprotein.</p

Ongoing positive selection in ZIKV isolates.

<p>(A) Maximum likelihood phylogeny for the nonstructural region (nucleotides 2371 to 8994). Amino acids at the five selected sites are shown for all ZIKV sequences. Viruses isolated from mosquitos and macaques are denoted with hash and asterisk symbols, respectively. Branch length is proportional to nucleotide substitutions per codon. Bootstrap values for internal branches >75% are shown. The phylogenetic tree is unrooted. (B) TMHMM prediction of transmembrane helices (TMH1-5) for the ZIKV NS4B protein and schematic representation of protein topology. Positively selected sites in the flavivirus phylogeny and in ZIKV strains are indicated by red and green triangles, respectively. Amino acid alignments of the regions surrounding selected sites are shown for 5 representative ZIKV, for DENV sequences belonging to the four serotypes, for JEV (NC_001437), and for WNV (strain NY-99, NC_001563). In the alignment, positively selected sites in ZIKV are shown in green; sites that are positively selected in other flaviviruses are marked in magenta. (C) Immune epitope mapping on NS5. Cyan bars indicate the number of epitopes overlapping each NS5 residue. Positively selected sites are colored as above. Mtase: methyltransferase domain; RdRp: RNA-dependent RNA polymerase domain.</p

Different selective pressures acting on flavivirus polyproteins.

<p>(A) Schematic representation of the ZIKV polyprotein. Proteins are colored in hues of blue depending on the percentage of negatively selected sites in ZIKV strains. The location of recombination breakpoints in flaviviruses and ZIKV is shown by striped rectangles. Positively selected sites in the flavivirus phylogeny and in ZIKV strains are colored in red and green, respectively. (B) Maximum likelihood unrooted tree for the flavivirus phylogeny. Branches analyzed in the branch-site tests are indicated with capital letters, with red indicating statistically significant evidence of positive selection. Branch length is proportional to nucleotide substitutions per codon. Bootstrap values for internal branches >75% are shown. See <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0004978#pntd.0004978.s002" target="_blank">S1 Table</a> for accession number and full names of analyzed viruses. (C) Immune epitope mapping and schematic representation of NS1 domains. Cyan bars indicate the number of epitopes overlapping each NS1 residue. Positively selected sites are also shown.</p

Table_1_EBV and multiple sclerosis: expression of LMP2A in MS patients.DOCX

Several evidences, including increased serum titers of Epstein–Barr virus (EBV)-specific antibodies and the presence of EBV DNA in brain of patients suggest a possible role of this virus in the pathogenesis of Multiple Sclerosis (MS), a chronic neurodegenerative disease with an unknown etiopathology. Aim of the present study is to verify if the expression of LMP2A and EBNA-1, two EBV genes, is altered in MS patients. EBV viral load, LMP2A and EBNA-1 gene expression and EBNA-1 antibodies titers were evaluated in blood of EBV-seropositive MS patients (n = 57; 31 relapsing remitting –RRMS- and 26 progressive -PMS-patients) and age- and sex-matched healthy controls (HC, n = 49). Results showed that EBNA-1 and VCA antibodies titers are significantly augmented in MS patients compared to HC (p < 0.05 for both antibodies); detection of EBV DNA was more frequent as well in MS patients compared to HC, although without reaching statistical significance. Regarding viral gene expression, LMP2A was significantly more frequently detected and more expressed in MS patients compared to HC (p < 0.005) whereas no differences were observed for EBNA-1. Considering patients alone, EBNA-1 was significantly more frequent in PMS compared to RRMS (p < 0.05), whereas no differences were observed for LMP2A. Increased expression of the LMP2A latency-associated gene in MS patients supports the hypothesis that EBV plays a role in disease etiopathology.</p

Branch-site analyses of the flavivirus polyprotein (nonstructural region).

<p>Branch-site analyses of the flavivirus polyprotein (nonstructural region).</p

Table_2_EBV and multiple sclerosis: expression of LMP2A in MS patients.DOCX

Several evidences, including increased serum titers of Epstein–Barr virus (EBV)-specific antibodies and the presence of EBV DNA in brain of patients suggest a possible role of this virus in the pathogenesis of Multiple Sclerosis (MS), a chronic neurodegenerative disease with an unknown etiopathology. Aim of the present study is to verify if the expression of LMP2A and EBNA-1, two EBV genes, is altered in MS patients. EBV viral load, LMP2A and EBNA-1 gene expression and EBNA-1 antibodies titers were evaluated in blood of EBV-seropositive MS patients (n = 57; 31 relapsing remitting –RRMS- and 26 progressive -PMS-patients) and age- and sex-matched healthy controls (HC, n = 49). Results showed that EBNA-1 and VCA antibodies titers are significantly augmented in MS patients compared to HC (p < 0.05 for both antibodies); detection of EBV DNA was more frequent as well in MS patients compared to HC, although without reaching statistical significance. Regarding viral gene expression, LMP2A was significantly more frequently detected and more expressed in MS patients compared to HC (p < 0.005) whereas no differences were observed for EBNA-1. Considering patients alone, EBNA-1 was significantly more frequent in PMS compared to RRMS (p < 0.05), whereas no differences were observed for LMP2A. Increased expression of the LMP2A latency-associated gene in MS patients supports the hypothesis that EBV plays a role in disease etiopathology.</p

Image_1_EBV and multiple sclerosis: expression of LMP2A in MS patients.pdf

Several evidences, including increased serum titers of Epstein–Barr virus (EBV)-specific antibodies and the presence of EBV DNA in brain of patients suggest a possible role of this virus in the pathogenesis of Multiple Sclerosis (MS), a chronic neurodegenerative disease with an unknown etiopathology. Aim of the present study is to verify if the expression of LMP2A and EBNA-1, two EBV genes, is altered in MS patients. EBV viral load, LMP2A and EBNA-1 gene expression and EBNA-1 antibodies titers were evaluated in blood of EBV-seropositive MS patients (n = 57; 31 relapsing remitting –RRMS- and 26 progressive -PMS-patients) and age- and sex-matched healthy controls (HC, n = 49). Results showed that EBNA-1 and VCA antibodies titers are significantly augmented in MS patients compared to HC (p < 0.05 for both antibodies); detection of EBV DNA was more frequent as well in MS patients compared to HC, although without reaching statistical significance. Regarding viral gene expression, LMP2A was significantly more frequently detected and more expressed in MS patients compared to HC (p < 0.005) whereas no differences were observed for EBNA-1. Considering patients alone, EBNA-1 was significantly more frequent in PMS compared to RRMS (p < 0.05), whereas no differences were observed for LMP2A. Increased expression of the LMP2A latency-associated gene in MS patients supports the hypothesis that EBV plays a role in disease etiopathology.</p

inter-species trees

Trees used in inter-species analyse

Sample collection and processing workflow for participants enrolled in the personalized medical protocol.

Sample collection and processing workflow for participants enrolled in the personalized medical protocol.</p