Population Genomics of GII.4 Noroviruses Reveal Complex Diversification and New Antigenic Sites Involved in the Emergence of Pandemic Strains

ABSTRACT GII.4 noroviruses are a major cause of acute gastroenteritis. Their dominance has been partially explained by the continuous emergence of antigenically distinct variants. To gain insights into the mechanisms of viral emergence and population dynamics of GII.4 noroviruses, we performed large-scale genomics, structural, and mutational analyses of the viral capsid protein (VP1). GII.4 noroviruses exhibited a periodic replacement of predominant variants with accumulation of amino acid substitutions. Genomic analyses revealed (i) a large proportion (87%) of conserved residues; (ii) variable residues that map on the previously determined antigenic sites; and (iii) variable residues that map outside the antigenic sites. Residues in the third pattern category formed motifs on the surface of VP1, which suggested extensions of previously predicted and new uncharacterized antigenic sites. The role of two motifs (C and G) in the antigenic makeup of the GII.4 capsid protein was confirmed with monoclonal antibodies and carbohydrate blocking assays. Amino acid profiles from antigenic sites (A, C, D, E, and G) correlated with the circulation patterns of GII.4 variants, with three of them (A, C, and G) containing residues (352, 357, 368, and 378) linked with the diversifying selective pressure on the emergence of new GII.4 variants. Notably, the emergence of each variant was followed by stochastic diversification with minimal changes that did not progress toward the next variant. This report provides a methodological framework for antigenic characterization of viruses and expands our understanding of the dynamics of GII.4 noroviruses and could facilitate the design of cross-reactive vaccines. IMPORTANCE Noroviruses are an important cause of viral gastroenteritis around the world. An obstacle delaying the development of norovirus vaccines is inadequate understanding of the role of norovirus diversity in immunity. Using a population genomics approach, we identified new residues on the viral capsid protein (VP1) from GII.4 noroviruses, the predominant genotype, that appear to be involved in the emergence and antigenic topology of GII.4 variants. Careful monitoring of the substitutions in those residues involved in the diversification and emergence of new viruses could help in the early detection of future novel variants with pandemic potential. Therefore, this novel information on the antigenic diversification could facilitate GII.4 norovirus vaccine design.

Population Genomics of GII.4 Noroviruses Reveal Complex Diversification and New Antigenic Sites Involved in the Emergence of Pandemic Strains

GII.4 noroviruses are a major cause of acute gastroenteritis. Their dominance has been partially explained by the continuous emergence of antigenically distinct variants. To gain insights on the mechanisms of viral emergence and population dynamics of GII.4 noroviruses, we performed large-scale genomics, structural, and mutational analyses of the viral capsid protein (VP1). GII.4 noroviruses exhibited a periodic replacement of predominant variants with accumulation of amino acid substitutions. Genomic analyses revealed (i) a large number (87%) of conserved residues; (ii) variable residues that map on the previously determined antigenic sites; and (iii) variable residues that map outside of the antigenic sites. Residues from the third pattern formed motifs on the surface of VP1, which suggested extensions of previously predicted and new uncharacterized antigenic sites. The role of two motifs (C and G) in the antigenic make-up of the GII.4 capsid protein was confirmed with monoclonal antibodies and carbohydrate blocking assays. Amino acid profiles from antigenic sites (A, C, D, E, and G) correlated with the circulation patterns of GII.4 variants, with two of them (C and G) containing residues (352, 357, 378) linked with the emergence of new GII.4 variants. Notably, the emergence of each variant was followed by a stochastic diversification with minimal changes at the antigenic sites that did not progress towards the next variant. This study provides a methodological framework for antigenic characterization of viruses, and expands our understanding of the dynamics of GII.4 noroviruses that could facilitate the design of cross-reactive vaccines.ImportanceNoroviruses are an important cause of viral gastroenteritis around the world. An obstacle delaying the development of norovirus vaccines is an inadequate understanding of the role of norovirus diversity in immunity. Using a population genomics approach, we identified new residues on the viral capsid protein (VP1) from GII.4 noroviruses, the predominant genotype, that appear to be involved in the emergence and antigenic topology of GII.4 variants. Careful monitoring of the substitutions in those residues involved in the diversification and emergence of new viruses could help in the early detection of future novel variants with pandemic potential. Therefore, this novel information on the antigenic diversification could facilitate GII.4 norovirus vaccine design.

The Cellular NMD Pathway Restricts Zika Virus Infection and Is Targeted by the Viral Capsid Protein

ABSTRACT Zika virus (ZIKV) infection of neural progenitor cells (NPCs) in utero is associated with neurological disorders, such as microcephaly, but a detailed molecular understanding of ZIKV-induced pathogenesis is lacking. Here we show that in vitro ZIKV infection of human cells, including NPCs, causes disruption of the nonsense-mediated mRNA decay (NMD) pathway. NMD is a cellular mRNA surveillance mechanism that is required for normal brain size in mice. Using affinity purification-mass spectrometry, we identified multiple cellular NMD factors that bind to the viral capsid protein, including the central NMD regulator up-frameshift protein 1 (UPF1). Endogenous UPF1 interacted with the ZIKV capsid protein in coimmunoprecipitation experiments, and capsid expression posttranscriptionally downregulated UPF1 protein levels, a process that we confirmed occurs during ZIKV infection. Cellular fractionation studies show that the ZIKV capsid protein specifically targets nuclear UPF1 for degradation via the proteasome. A further decrease in UPF1 levels by RNAi significantly enhanced ZIKV infection in NPC cultures, consistent with a model in which NMD restricts ZIKV infection in the fetal brain. We propose that ZIKV, via the capsid protein, has evolved a strategy to lower UPF1 levels and dampen antiviral activities of NMD, which in turn contributes to neuropathology in vivo. IMPORTANCE Zika virus (ZIKV) is a significant global health threat, as infection has been linked to serious neurological complications, including microcephaly. Using a human stem cell-derived neural progenitor model system, we find that a critical cellular quality control process called the nonsense-mediated mRNA decay (NMD) pathway is disrupted during ZIKV infection. Importantly, disruption of the NMD pathway is a known cause of microcephaly and other neurological disorders. We further identify an interaction between the capsid protein of ZIKV and up-frameshift protein 1 (UPF1), the master regulator of NMD, and show that ZIKV capsid targets UPF1 for degradation. Together, these results offer a new mechanism for how ZIKV infection can cause neuropathology in the developing brain.

The cellular NMD pathway restricts Zika virus infection and is targeted by the viral capsid protein

Zika virus (ZIKV) infection of neural progenitor cells (NPCs) in utero is associated with neurological disorders, such as microcephaly1, but a detailed molecular understanding of ZIKV-induced pathogenesis is lacking. Here we show that in vitro ZIKV infection of human cells, including NPCs, causes disruption of the nonsense-mediated mRNA decay (NMD) pathway. NMD is a cellular mRNA surveillance mechanism that is required for normal brain size in mice2–4. Using affinity purification-mass spectrometry, we identified multiple cellular NMD factors that bind to the viral capsid protein, including the central NMD regulator up-frameshift protein 1 (UPF1)5. Endogenous UPF1 interacted with the viral capsid protein in co-immunoprecipitation experiments and capsid expression post-transcriptionally downregulated UPF1, a process that we confirmed occurs during de novo ZIKV infection. A further decrease in UPF1 levels by RNAi significantly enhanced ZIKV infection in NPC cultures. We therefore propose that ZIKV, via the capsid protein, has evolved a strategy to dampen antiviral activities of NMD6,7, which subsequently contributes to neuropathology in vivo.