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ABSTRACT Viral noncoding RNAs have acquired increasing prominence as important regulators of infection and mediators of pathogenesis. Circular RNAs (circRNAs) generated by backsplicing events have been identified in several oncogenic human DNA viruses. Here, we show that Merkel cell polyomavirus (MCV), the etiologic cause of ∼80% of Merkel cell carcinomas (MCCs), also expresses circular RNAs. By RNase R-resistant RNA sequencing, four putative circRNA backsplice junctions (BSJs) were identified from the MCV early region (ER). The most abundantly expressed MCV circRNA, designated circMCV-T, is generated through backsplicing of all of ER exon II to form a 762-nucleotide (nt) circular RNA molecule. Curiously, circMCV-T, as well as two other less abundantly expressed putative MCV circRNAs, overlaps in a complementary fashion with the MCV microRNA (miRNA) locus that encodes MCV-miR-M1. circMCV-T is consistently detected in concert with linear T antigen transcripts throughout infection, suggesting a crucial role for this RNA molecule in the regulatory functions of the early region, known to be vital for viral replication. Knocking out the hairpin structure of MCV-miR-M1 in genomic early region expression constructs and using a new high-efficiency, recombinase-mediated, recircularized MCV molecular clone demonstrates that circMCV-T levels decrease in the presence of MCV-miR-M1, underscoring the interplay between MCV circRNA and miRNA. Furthermore, circMCV-T partially reverses the known inhibitory effect of MCV-miR-M1 on early gene expression. RNase R-resistant RNA sequencing of lytic rat polyomavirus 2 (RatPyV2) identified an analogously located circRNA, stipulating a crucial, conserved regulatory function of this class of RNA molecules in the family of polyomaviruses. IMPORTANCE Covalently closed circular RNAs were recently described in the human DNA tumor viruses Epstein-Barr virus (EBV), Kaposi’s sarcoma-associated herpesvirus (KSHV), and human papillomavirus (HPV). Here, we show that MCV, another DNA tumor virus, generates circRNAs from its early regulatory region in concert with T antigen linear transcripts. MCV circMCV-T interacts with another MCV noncoding RNA, miR-M1, to functionally modulate early region transcript expression important for viral replication and long-term episomal persistence. This work describes a dynamic regulatory network integrating circRNA/miRNA/mRNA biomolecules and underscores the intricate functional modulation between several classes of polyomavirus-encoded RNAs in the control of viral replication.

ABSTRACT Streptococcus pyogenes (group A Streptococcus [GAS]) is an important human pathogen causing a broad spectrum of diseases and associated with significant global morbidity and mortality. Almost all GAS isolates express a surface hyaluronic acid capsule, a virulence determinant that facilitates host colonization and impedes phagocyte killing. However, recent epidemiologic surveillance has reported a sustained increase in both mucosal and invasive infections caused by nonencapsulated GAS, which questions the indispensable role of hyaluronic acid capsule in GAS pathogenesis. In this study, we found that pilus of M4 GAS not only significantly promotes biofilm formation, adherence, and cytotoxicity to human upper respiratory tract epithelial cells and keratinocytes, but also promotes survival in human whole blood and increased virulence in murine models of invasive infection. T4 antigen, the pilus backbone protein of M4 GAS, binds haptoglobin, an abundant human acute-phase protein upregulated upon infection and inflammation, on the bacterial surface. Haptoglobin sequestration reduces the susceptibility of nonencapsulated M4 GAS to antimicrobial peptides released from activated neutrophils and platelets. Our results reveal a previously unappreciated virulence-promoting role of M4 GAS pili, in part mediated by co-opting the biology of haptoglobin to mitigate host antimicrobial defenses. IMPORTANCE Group A Streptococcus (GAS) is a strict human pathogen causing more than 700 million infections globally each year. The majority of the disease-causing GAS are encapsulated, which greatly guarantees survival and dissemination in the host. Emergence of the capsule-negative GAS, such as M4 GAS, in recent epidemiologic surveillance alarms the necessity to elucidate the virulence determinants of these pathogens. Here, we found that M4 pili play an important role in promoting M4 GAS adherence and cytotoxicity to human pharyngeal epithelial cells and keratinocytes. The same molecule also significantly enhanced M4 GAS survival and replication in human whole blood and experimental murine infection. T4 antigen, which composes the backbone of M4 pili, was able to sequester the very abundant serum protein haptoglobin to further confer M4 GAS resistance to antibacterial substances released by neutrophils and platelets.

JC polyomavirus (JCPyV) infects 50 to 80% of the population and is the causative agent of a fatal demyelinating disease of the central nervous system (CNS). JCPyV presents initially as a persistent infection in the kidneys of healthy people, but during immunosuppression, the virus can reactivate and cause progressive multifocal leukoencephalopathy (PML). Within the CNS, JCPyV predominately targets two cell types, oligodendrocytes and astrocytes. Until recently, the role of astrocytes has been masked by the pathology in the myelin-producing oligodendrocytes, which are lytically destroyed by the virus. To better understand how astrocytes are impacted during JCPyV infection, the temporal regulation and infectious cycle of JCPyV were analyzed in primary normal human astrocytes (NHAs). Previous research to define the molecular mechanisms underlying JCPyV infection has mostly relied on the use of cell culture models, such as SVG-A cells (SVGAs), an immortalized, mixed population of glial cells transformed with simian virus 40 (SV40) T antigen. However, SVGAs present several limitations due to their immortalized characteristics, and NHAs represent an innovative approach to study JCPyV infection in vitro. Using infectivity assays, quantitative PCR, and immunofluorescence assay approaches, we have further characterized JCPyV infectivity in NHAs. The JCPyV infectious cycle is significantly delayed in NHAs, and the expression of SV40 T antigen alters the cellular environment, which impacts viral infection in immortalized cells. This research establishes a foundation for the use of primary NHAs in future studies and will help unravel the role of astrocytes in PML pathogenesis. [ABSTRACT FROM AUTHOR]

The human JC polyomavirus (JCPyV) infects the majority of the population worldwide and presents as an asymptomatic, persistent infection in the kidneys. In individuals who are immunocompromised, JCPyV can become reactivated and cause a lytic infection in the central nervous system resulting in the fatal, demyelinating disease progressive multifocal leukoencephalopathy (PML). Infection is initiated by interactions between the capsid protein viral protein 1 (VP1) and the α2,6-linked sialic acid on lactoseries tetrasaccharide c (LSTc), while JCPyV internalization is facilitated by 5-hydroxytryptamine 2 receptors (5-HT2Rs). The mechanisms by which the serotonin receptors mediate virus entry and the signaling cascades required to drive viral infection remain poorly understood. JCPyV was previously shown to induce phosphorylation of extracellular signal-regulated kinase (ERK), a downstream target of the mitogen-activated protein kinase (MAPK) pathway, upon virus entry. However, it remained unclear whether ERK activation was required for JCPyV infection. Both ERK-specific small interfering RNA (siRNA) and ERK inhibitor treatments resulted in significantly diminished JCPyV infection in both kidney and glial cells yet had no effect on the infectivity of the polyomavirus simian virus 40 (SV40). Experiments characterizing the role of ERK during steps in the viral life cycle indicate that ERK activation is required for viral transcription, as demonstrated by a significant reduction in production of large T antigen (TAg), a key viral protein associated with the initiation of viral transcription and viral replication. These findings delineate the role of the MAPK-ERK signaling pathway in JCPyV infection, elucidating how the virus reprograms the host cell to promote viral pathogenesis. IMPORTANCE Viral infection is dependent upon host cell factors, including the activation of cellular signaling pathways. These interactions between viruses and host cells are necessary for infection and play an important role in viral disease outcomes. The focus of this study was to determine how the human JC polyomavirus (JCPyV), a virus that resides in the kidney of the majority of the population and can cause the fatal, demyelinating disease progressive multifocal leukoencephalopathy (PML) in the brains of immunosuppressed individuals, usurps a cellular signaling pathway to promote its own infectious life cycle. We demonstrated that the activation of extracellular signal-regulated kinase (ERK), a component of the mitogenactivated protein kinase (MAPK) pathway, promotes JCPyV transcription, which is required for viral infection. Our findings demonstrate that the MAPK-ERK signaling pathway is a key determinant of JCPyV infection, elucidating new information regarding the signal reprogramming of host cells by a pathogenic virus. [ABSTRACT FROM AUTHOR]

Human polyomavirus (HPyV) DNA genomes contain three regions denoted the early viral gene region (EVGR), encoding the regulatory T-antigens and one microRNA, the late viral gene region (LVGR), encoding the structural Vp capsid proteins, and the noncoding control region (NCCR). The NCCR harbors the origin of viral genome replication and bidirectional promoter/enhancer functions governing EVGR and LVGR expression on opposite DNA strands. Despite principal similarities, HPyV NCCRs differ in length, sequence, and architecture. To functionally compare HPyV NCCRs, sequences from human isolates were inserted into a bidirectional reporter vector using dsRed2 for EVGR expression and green fluorescent protein (GFP) for LVGR expression. Transfecting HPyV NCCR reporter vectors into human embryonic kidney 293 (HEK293) cells and flow cytometry normalized to archetype BKPyV NCCR revealed a hierarchy of EVGR expression levels with MCPyV, HPyV12, and STLPyV NCCRs conferring stronger levels and HPyV6, HPyV9, and HPyV10 NCCRs weaker levels, while LVGR expression was less variable and showed comparable activity levels. Transfection of HEK293T cells expressing simian virus 40 (SV40) large T antigen (LTag) increased EVGR expression for most HPyV NCCRs, which correlated with the number of LTag-binding sites (Spearman's r, 0.625; P < 0.05) and decreased following SV40 LTag small interfering RNA (siRNA) knockdown. LTagdependent activation was specifically confirmed for two different MCPyV NCCRs in 293MCT cells expressing the cognate MCPyV LTag. HPyV NCCR expression in different cell lines derived from skin (A375), cervix (HeLaNT), lung (A549), brain (Hs683), and colon (SW480) demonstrated that host cell properties significantly modulate the baseline HPyV NCCR activity, which partly synergized with SV40 LTag expression. Clinically occurring NCCR sequence rearrangements of HPyV7 PITT-1 and -2 and HPyV9 UF1 were found to increase EVGR expression compared to the respective HPyV archetype, but this was partly host cell type specific. IMPORTANCE HPyV NCCRs integrate essential viral functions with respect to host cell specificity, persistence, viral replication, and disease. Here, we show that HPyV NCCRs not only differ in sequence length, number, and position of LTag- and common transcription factor-binding sites but also confer differences in bidirectional viral gene expression. Importantly, EVGR reporter expression was significantly modulated by LTag expression and by host cell properties. Clinical sequence variants of HPyV7 and HPyV9 NCCRs containing deletions and insertions were associated with increased EVGR expression, similar to BKPyV and JCPyV rearrangements, emphasizing that HPyV NCCR sequences are major determinants not only of host cell tropism but also of pathogenicity. These results will help to define secondary HPyV cell tropism beyond HPyV surface receptors, to identify key viral and host factors shaping the viral life cycle, and to develop preclinical models of HPyV persistence and replication and suitable antiviral targets. [ABSTRACT FROM AUTHOR]

Viral noncoding RNAs have acquired increasing prominence as important regulators of infection and mediators of pathogenesis. Circular RNAs (circRNAs) generated by backsplicing events have been identified in several oncogenic human DNA viruses. Here, we show that Merkel cell polyomavirus (MCV), the etiologic cause of ∼80% of Merkel cell carcinomas (MCCs), also expresses circular RNAs. By RNase R-resistant RNA sequencing, four putative circRNA backsplice junctions (BSJs) were identified from the MCV early region (ER). The most abundantly expressed MCV circRNA, designated circMCV-T, is generated through backsplicing of all of ER exon II to form a 762-nucleotide (nt) circular RNA molecule. Curiously, circMCV-T, as well as two other less abundantly expressed putative MCV circRNAs, overlaps in a complementary fashion with the MCV microRNA (miRNA) locus that encodes MCV-miR-M1. circMCV-T is consistently detected in concert with linear T antigen transcripts throughout infection, suggesting a crucial role for this RNA molecule in the regulatory functions of the early region, known to be vital for viral replication. Knocking out the hairpin structure of MCV-miR-M1 in genomic early region expression constructs and using a new high-efficiency, recombinase-mediated, recircularized MCV molecular clone demonstrates that circMCV-T levels decrease in the presence of MCV-miR-M1, underscoring the interplay between MCV circRNA and miRNA. Furthermore, circMCV-T partially reverses the known inhibitory effect of MCV-miR-M1 on early gene expression. RNase R-resistant RNA sequencing of lytic rat polyomavirus 2 (RatPyV2) identified an analogously located circRNA, stipulating a crucial, conserved regulatory function of this class of RNA molecules in the family of polyomaviruses. IMPORTANCE Covalently closed circular RNAs were recently described in the human DNA tumor viruses Epstein-Barr virus (EBV), Kaposi's sarcoma-associated herpesvirus (KSHV), and human papillomavirus (HPV). Here, we show that MCV, another DNA tumor virus, generates circRNAs from its early regulatory region in concert with T antigen linear transcripts. MCV circMCV-T interacts with another MCV noncoding RNA, miR-M1, to functionally modulate early region transcript expression important for viral replication and long-term episomal persistence. This work describes a dynamic regulatory network integrating circRNA/miRNA/mRNA biomolecules and underscores the intricate functional modulation between several classes of polyomavirus-encoded RNAs in the control of viral replication., (Copyright © 2020 Abere et al.)

Streptococcus pyogenes (group A Streptococcus [GAS]) is an important human pathogen causing a broad spectrum of diseases and associated with significant global morbidity and mortality. Almost all GAS isolates express a surface hyaluronic acid capsule, a virulence determinant that facilitates host colonization and impedes phagocyte killing. However, recent epidemiologic surveillance has reported a sustained increase in both mucosal and invasive infections caused by nonencapsulated GAS, which questions the indispensable role of hyaluronic acid capsule in GAS pathogenesis. In this study, we found that pilus of M4 GAS not only significantly promotes biofilm formation, adherence, and cytotoxicity to human upper respiratory tract epithelial cells and keratinocytes, but also promotes survival in human whole blood and increased virulence in murine models of invasive infection. T4 antigen, the pilus backbone protein of M4 GAS, binds haptoglobin, an abundant human acute-phase protein upregulated upon infection and inflammation, on the bacterial surface. Haptoglobin sequestration reduces the susceptibility of nonencapsulated M4 GAS to antimicrobial peptides released from activated neutrophils and platelets. Our results reveal a previously unappreciated virulence-promoting role of M4 GAS pili, in part mediated by co-opting the biology of haptoglobin to mitigate host antimicrobial defenses. IMPORTANCE Group A Streptococcus (GAS) is a strict human pathogen causing more than 700 million infections globally each year. The majority of the disease-causing GAS are encapsulated, which greatly guarantees survival and dissemination in the host. Emergence of the capsule-negative GAS, such as M4 GAS, in recent epidemiologic surveillance alarms the necessity to elucidate the virulence determinants of these pathogens. Here, we found that M4 pili play an important role in promoting M4 GAS adherence and cytotoxicity to human pharyngeal epithelial cells and keratinocytes. The same molecule also significantly enhanced M4 GAS survival and replication in human whole blood and experimental murine infection. T4 antigen, which composes the backbone of M4 pili, was able to sequester the very abundant serum protein haptoglobin to further confer M4 GAS resistance to antibacterial substances released by neutrophils and platelets., (Copyright © 2020 Chen et al.)

Group A Streptococcus (GAS) ( Streptococcus pyogenes ) is an important human pathogen associated with significant global morbidity and mortality for which there is no safe and efficacious vaccine. The T antigen, a protein that polymerizes to form the backbone of the GAS pilus structure, is a potential vaccine candidate. Previous surveys of the tee gene, which encodes the T antigen, have identified 21 different tee types and subtypes such that any T antigen-based vaccine must be multivalent and carefully designed to provide broad strain coverage. In this study, the crystal structures of three two-domain T antigens (T3.2, T13, and T18.1) were determined and found to have remarkable structural similarity to the previously reported T1 antigen, despite moderate overall sequence similarity. This has enabled reliable modeling of all major two-domain T antigens to reveal that T antigen sequence variation is distributed along the full length of the protein and shields a highly conserved core. Immunoassays performed with sera from immunized animals and commercial T-typing sera identified a significant cross-reactive antibody response between T18.1, T18.2, T3.2, and T13. The existence of shared epitopes between T antigens, combined with the remarkably conserved structure and high level of surface sequence divergence, has important implications for the design of multivalent T antigen-based vaccines., (Copyright © 2019 American Society for Microbiology.)

Quantitative PCR is a diagnostic mainstay of clinical virology, and accurate quantitation of viral load among labs requires the use of international standards. However, the use of multiple passages of viral isolates to obtain sufficient material for international standards may result in genomic changes that complicate their use as quantitative standards. We performed next-generation sequencing to obtain single-nucleotide resolution and relative copy number of JC virus (JCV) clinical standards. Strikingly, the WHO international standard and the Exact v1/v2 prototype standards for JCV showed 8-fold and 4-fold variation in genomic coverage between different loci in the viral genome, respectively, due to large deletions in the large T antigen region. Intriguingly, several of the JCV standards sequenced in this study with large T antigen deletions were cultured in cell lines immortalized using simian virus 40 (SV40) T antigen, suggesting the possibility of transcomplementation in cell culture. Using a cutoff 5% allele fraction for junctional reads, 7 different rearrangements were present in the JC virus sequences present in the WHO standard across multiple library preparations and sequencing runs. Neither the copy number differences nor the rearrangements were observed in a clinical sample with a high copy number of JCV or a plasmid control. These results were also confirmed by the quantitative real-time PCR (qPCR), droplet digital PCR (ddPCR), and Sanger sequencing of multiple rearrangements. In summary, targeting different regions of the same international standard can result in up to an 8-fold difference in quantitation. We recommend the use of next-generation sequencing to validate standards in clinical virology., (Copyright © 2017 Greninger et al.)