bluuebunny/crossref_metadata_2025_split huggingface dataset:
Potential Electrostatic Interactions in Multiple Regions Affect Human Metapneumovirus F-Mediated Membrane Fusion
Authors: Chang Andres , Hackett Brent A. , Winter Christine C. , Buchholz Ursula J. , Dutch Rebecca Ellis 
Published: July 2012

ABSTRACTThe recently identified human metapneumovirus (HMPV) is a worldwide respiratory virus affecting all age groups and causing pneumonia and bronchiolitis in severe cases. Despite its clinical significance, no specific antiviral agents have been approved for treatment of HMPV infection. Unlike the case for most paramyxoviruses, the fusion proteins (F) of a number of strains, including the clinical isolate CAN97-83, can be triggered by low pH. We recently reported that residue H435 in the HRB linker domain acts as a pH sensor for HMPV CAN97-83 F, likely through electrostatic repulsion forces between a protonated H435 and its surrounding basic residues, K295, R396, and K438, at low pH. Through site-directed mutagenesis, we demonstrated that a positive charge at position 435 is required but not sufficient for F-mediated membrane fusion. Arginine or lysine substitution at position 435 resulted in a hyperfusogenic F protein, while replacement with aspartate or glutamate abolished fusion activity. Studies with recombinant viruses carrying mutations in this region confirmed its importance. Furthermore, a second region within the F2domain identified as being rich in charged residues was found to modulate fusion activity of HMPV F. Loss of charge at residues E51, D54, and E56 altered local folding and overall stability of the F protein, with dramatic consequences for fusion activity. As a whole, these studies implicate charged residues and potential electrostatic interactions in function, pH sensing, and overall stability of HMPV F.
A Ribosome-Binding, 3′ Translational Enhancer Has a T-Shaped Structure and Engages in a Long-Distance RNA-RNA Interaction
Authors: Gao Feng , Kasprzak Wojciech , Stupina Vera A. , Shapiro Bruce A. , Simon Anne E. 
Published: July 2012

ABSTRACT

Many plant RNA viruses contain elements in their 3′ untranslated regions (3′ UTRs) that enhance translation. The PTE (
Panicum mosaic virus
-like translational enhancer) of
Pea enation mosaic virus
(PEMV) binds to eukaryotic initiation factor 4E (eIF4E), but how this affects translation from the 5′ end is unknown. We have discovered a three-way branched element just upstream of the PEMV PTE that engages in a long-distance kissing-loop interaction with a coding sequence hairpin that is critical for the translation of a reporter construct and the accumulation of the viral genome
in vivo
. Loss of the long-distance interaction was more detrimental than elimination of the adjacent PTE, indicating that the RNA-RNA interaction supports additional translation functions besides relocating the PTE to the 5′ end. The branched element is predicted by molecular modeling and molecular dynamics to form a T-shaped structure (TSS) similar to the ribosome-binding TSS of
Turnip crinkle virus
(TCV). The PEMV element binds to plant 80S ribosomes with a

K
d

(dissociation constant) of 0.52 μM and to 60S subunits with a

K
d

of 0.30 μM. Unlike the TCV TSS, the PEMV element also binds 40S subunits (

K
d

, 0.36 μM). Mutations in the element that suppressed translation reduced either ribosome binding or the RNA-RNA interaction, suggesting that ribosome binding is important for function. This novel, multifunctional element is designated a kl-TSS (kissing-loop T-shaped structure) to distinguish it from the TCV TSS. The kl-TSS has sequence and structural features conserved with the upper portion of most PTE-type elements, which, with the exception of the PEMV PTE, can engage in similar long-distance RNA-RNA interactions.
Stable Antigen Is Most Effective for Eliciting CD8+T-Cell Responses after DNA Vaccination and Infection with Recombinant Vaccinia VirusIn Vivo
Authors: Schliehe Christopher , Bitzer Annegret , van den Broek Maries , Groettrup Marcus 
Published: July 2012

ABSTRACTThe induction of strong CD8+T-cell responses against infectious diseases and cancer has remained a major challenge. Depending on the source of antigen and the infectious agent, priming of CD8+T cells requires direct and/or cross-presentation of antigenic peptides on major histocompatibility complex (MHC) class I molecules by professional antigen-presenting cells (APCs). However, both pathways show distinct preferences concerning antigen stability. Whereas direct presentation was shown to efficiently present peptides derived from rapidly degraded proteins, cross-presentation is dependent on long-lived antigen species. In this report, we analyzed the role of antigen stability on DNA vaccination and recombinant vaccinia virus (VV) infection using altered versions of the same antigen. The long-lived nucleoprotein (NP) of lymphocytic choriomeningitis virus (LCMV) can be targeted for degradation by N-terminal fusion to ubiquitin or, as we show here, to the ubiquitin-like modifier FAT10. Direct presentation by cells either transfected with NP-encoding plasmids or infected with recombinant VVin vitrowas enhanced in the presence of short-lived antigens.In vivo, however, the highest induction of NP-specific CD8+T-cell responses was achieved in the presence of long-lived NP. Our experiments provide evidence that targeting antigens for proteasomal degradation does not improve the immunogenicity of DNA vaccines and recombinant VVs. Rather, it is the long-lived antigen that is superior for the efficient activation of MHC class I-restricted immune responsesin vivo. Hence, our results suggest a dominant role for antigen cross-priming in DNA vaccination and recombinant VV infection.
Nectin4 Is an Epithelial Cell Receptor for Canine Distemper Virus and Involved in Neurovirulence
Authors: Pratakpiriya Watanyoo , Seki Fumio , Otsuki Noriyuki , Sakai Kouji , Fukuhara Hideo , Katamoto Hiromu , Hirai Takuya , Maenaka Katsumi , Techangamsuwan Somporn , Lan Nguyen Thi , Takeda Makoto , Yamaguchi Ryoji 
Published: July 2012

ABSTRACT

Canine distemper virus (CDV) uses signaling lymphocyte activation molecule (SLAM), expressed on immune cells, as a receptor. However, epithelial and neural cells are also affected by CDV
in vivo
. Wild-type CDV strains showed efficient replication with syncytia in Vero cells expressing dog nectin4, and the infection was blocked by an anti-nectin4 antibody. In dogs with distemper, CDV antigen was preferentially detected in nectin4-positive neurons and epithelial cells, suggesting that nectin4 is an epithelial cell receptor for CDV and also involved in its neurovirulence.
Peripheral Blood CD4 T-Cell and Plasmacytoid Dendritic Cell (pDC) Reactivity to Herpes Simplex Virus 2 and pDC Number Do Not Correlate with the Clinical or Virologic Severity of Recurrent Genital Herpes
Authors: Moss Nicholas J. , Magaret Amalia , Laing Kerry J. , Kask Angela Shaulov , Wang Minna , Mark Karen E. , Schiffer Joshua T. , Wald Anna , Koelle David M. 
Published: July 2012

ABSTRACTLeukocytes participate in the immune control of herpes simplex virus (HSV). Data from HIV coinfections, germ line mutations, and case reports suggest involvement of CD4 T cells and plasmacytoid dendritic cells (pDC). We investigated the relationships between these cells and recurrent genital herpes disease severity in the general population. Circulating CD4 T-cell responses to HSV-2 were measured in specimens from 67 immunocompetent individuals with measured genital lesion and HSV shedding rates. Similarly, pDC number and functional responses to HSV-2 were analyzed in 40 persons. CD4 responses and pDC concentrations and responses ranged as much as 100-fold between persons while displaying moderate within-person consistency over time. No correlations were observed between these immune response parameters and genital HSV-2 severity. Cytomegalovirus (CMV) coinfection was not correlated with differences in HSV-2-specific CD4 T-cell responses. The CD4 T-cell response to HSV-2 was much more polyfunctional than was the response to CMV. These data suggest that other immune cell subsets with alternate phenotypes or anatomical locations may be responsible for genital herpes control in chronically infected individuals.
Kaposi's Sarcoma-Associated Herpesvirus-Encoded LANA Recruits Topoisomerase IIβ for Latent DNA Replication of the Terminal Repeats
Authors: Purushothaman Pravinkumar , McDowell Maria E. , McGuinness James , Salas Ruth , Rumjahn Sharif M. , Verma Subhash C. 
Published: July 2012

ABSTRACT
The latency-associated nuclear antigen (LANA) encoded by Kaposi's sarcoma-associated herpesvirus (KSHV) plays a major role in maintaining latency and is critical for the perpetual segregation of viral episomes to the progeny nuclei of newly divided cells. LANA binds to KSHV terminal repeat (TR) DNA and tethers the viral episomes to host chromosomes through the association of chromatin-bound cellular proteins. TR elements serve as potential origin sites of KSHV replication and have been shown to play important roles in latent DNA replication and transcription of adjacent genes. Affinity chromatography and proteomics analysis using KSHV TR DNA and the LANA binding site as the affinity column identified topoisomerase IIβ (TopoIIβ) as a LANA-interacting protein. Here, we show that TopoIIβ forms complexes with LANA that colocalize as punctuate bodies in the nucleus of KSHV-infected cells. The specific TopoIIβ binding region of LANA has been identified to its N terminus and the first 32 amino acid residues containing the nucleosome-binding region crucial for binding. Moreover, this region could also act as a dominant negative to disrupt association of TopoIIβ with LANA. TopoIIβ plays an important role in LANA-dependent latent DNA replication, as addition of ellipticine, a selective inhibitor of TopoII, negatively regulated replication mediated by the TR. DNA break labeling and chromatin immunoprecipitation assay using biotin-16-dUTP and terminal deoxynucleotide transferase showed that TopoIIβ mediates a transient DNA break on viral DNA. These studies confirm that LANA recruits TopoIIβ at the origins of latent replication to unwind the DNA for replication.
Alanine Scanning of Poliovirus 2C
ATPase
Reveals New Genetic Evidence that Capsid Protein/2C
ATPase
Interactions Are Essential for Morphogenesis
Authors: Wang Chunling , Jiang Ping , Sand Claire , Paul Aniko V. , Wimmer Eckard 
Published: July 2012

ABSTRACT

Polypeptide 2C
ATPase
is one of the most thoroughly studied but least understood proteins in the life cycle of poliovirus. Within the protein, multiple functional domains important for uncoating, host cell membrane alterations, and RNA replication and encapsidation have previously been identified. In this study, charged to alanine-scanning mutagenesis was used to generate conditional-lethal mutations in hitherto-uncharacterized domains of the 2C
ATPase
polypeptide, particularly those involved in morphogenesis. Adjacent or clustered charged amino acids (2 to 4), scattered along the 2C
ATPase
coding sequence, were replaced with alanines. RNA transcripts of mutant poliovirus cDNA clones were transfected into HeLa cells. Subsequently, 10 lethal, 1 severely temperature-sensitive, 2 quasi-infectious, and 3 wild type-like mutants were identified. Using a luciferase-containing reporter virus, we demonstrated RNA replication defects in all lethal and quasi-infectious mutants. Temperature-sensitive mutants were defective in RNA replication only at the restricted temperatures. Furthermore, we characterized a quasi-infectious mutant (K
6
A/K
7
A) that produced a suppressor mutation (G
1
R) and a novel 2B∧2C
ATPase
cleavage site (Q∧R). Surprisingly, this cleavage site mutation did not interfere with normal processing of the polyprotein. These mutants have led to the identification of several new sites within the 2C
ATPase
polypeptide that are required for RNA replication. In addition, analysis of the suppressor mutants has revealed a new domain near the C terminus of 2C
ATPase
that is involved in encapsidation, possibly achieved through interaction with an amino acid sequence between NTP binding motifs A and B of 2C
ATPase
. Most importantly, the identification of suppressor mutations in both 2C
ATPase
and the capsid domains (VP1 and VP3) of poliovirus has confirmed that an interaction between 2C
ATPase
and capsid proteins is involved in viral morphogenesis.
Interferon Response Factors 3 and 7 Protect against Chikungunya Virus Hemorrhagic Fever and Shock
Authors: Rudd Penny A. , Wilson Jane , Gardner Joy , Larcher Thibaut , Babarit Candice , Le Thuy T. , Anraku Itaru , Kumagai Yutaro , Loo Yueh-Ming , Gale Michael , Akira Shizuo , Khromykh Alexander A. , Suhrbier Andreas 
Published: July 2012

ABSTRACT

Chikungunya virus (CHIKV) infections can produce severe disease and mortality. Here we show that CHIKV infection of adult mice deficient in interferon response factors 3 and 7 (IRF3/7
−/−
) is lethal. Mortality was associated with undetectable levels of alpha/beta interferon (IFN-α/β) in serum, ∼50- and ∼10-fold increases in levels of IFN-γ and tumor necrosis factor (TNF), respectively, increased virus replication, edema, vasculitis, hemorrhage, fever followed by hypothermia, oliguria, thrombocytopenia, and raised hematocrits. These features are consistent with hemorrhagic shock and were also evident in infected IFN-α/β receptor-deficient mice.
In situ
hybridization suggested CHIKV infection of endothelium, fibroblasts, skeletal muscle, mononuclear cells, chondrocytes, and keratinocytes in IRF3/7
−/−
mice; all but the latter two stained positive in wild-type mice. Vaccination protected IRF3/7
−/−
mice, suggesting that defective antibody responses were not responsible for mortality. IPS-1- and TRIF-dependent pathways were primarily responsible for IFN-α/β induction, with IRF7 being upregulated >100-fold in infected wild-type mice. These studies suggest that inadequate IFN-α/β responses following virus infection can be sufficient to induce hemorrhagic fever and shock, a finding with implications for understanding severe CHIKV disease and dengue hemorrhagic fever/dengue shock syndrome.
Rotavirus VP8*: Phylogeny, Host Range, and Interaction with Histo-Blood Group Antigens
Authors: Liu Yang , Huang Pengwei , Tan Ming , Liu Yiliu , Biesiada Jacek , Meller Jarek , Castello Alejandro A. , Jiang Baoming , Jiang Xi 
Published: July 2012

ABSTRACT
The distal portion of rotavirus (RV) VP4 spike protein (VP8*) is implicated in binding to cellular receptors, thereby facilitating viral attachment and entry. While VP8* of some animal RVs engage sialic acid, human RVs often attach to and enter cells in a sialic acid-independent manner. A recent study demonstrated that the major human RVs (P[4], P[6], and P[8]) recognize human histo-blood group antigens (HBGAs). In this study, we performed a phylogenetic analysis of RVs and showed further variations of RV interaction with HBGAs. On the basis of the VP8* sequences, RVs are grouped into five P genogroups (P[I] to P[V]), of which P[I], P[IV], and P[V] mainly infect animals, P[II] infects humans, and P[III] infects both animals and humans. The sialic acid-dependent RVs (P[1], P[2], P[3], and P[7]) form a subcluster within P[I], while all three major P genotypes of human RVs (P[4], P[6], and P[8]) are clustered in P[II]. We then characterized three human RVs (P[9], P[14], and P[25]) in P[III] and observed a new pattern of binding to the type A antigen which is distinct from that of the P[II] RVs. The binding was demonstrated by hemagglutination and saliva binding assay using recombinant VP8* and native RVs. Homology modeling and mutagenesis study showed that the locations of the carbohydrate binding interfaces are shared with the sialic acid-dependent RVs, although different amino acids are involved. The P[III] VP8* proteins also bind the A antigens of the porcine and bovine mucins, suggesting the A antigen as a possible factor for cross-species transmission of RVs. Our study suggests that HBGAs play an important role in RV infection and evolution.
Cyclophilins Facilitate Dissociation of the Human Papillomavirus Type 16 Capsid Protein L1 from the L2/DNA Complex following Virus Entry
Authors: Bienkowska-Haba Malgorzata , Williams Carlyn , Kim Seong Man , Garcea Robert L. , Sapp Martin 
Published: July 2012

ABSTRACT

Human papillomaviruses (HPV) are composed of the major and minor capsid proteins, L1 and L2, that encapsidate a chromatinized, circular double-stranded DNA genome. At the outset of infection, the interaction of HPV type 16 (HPV16) (pseudo)virions with heparan sulfate proteoglycans triggers a conformational change in L2 that is facilitated by the host cell chaperone cyclophilin B (CyPB). This conformational change results in exposure of the L2 N terminus, which is required for infectious internalization. Following internalization, L2 facilitates egress of the viral genome from acidified endosomes, and the L2/DNA complex accumulates at PML nuclear bodies. We recently described a mutant virus that bypasses the requirement for cell surface CyPB but remains sensitive to cyclosporine for infection, indicating an additional role for CyP following endocytic uptake of virions. We now report that the L1 protein dissociates from the L2/DNA complex following infectious internalization. Inhibition and small interfering RNA (siRNA)-mediated knockdown of CyPs blocked dissociation of L1 from the L2/DNA complex.
In vitro
, purified CyPs facilitated the dissociation of L1 pentamers from recombinant HPV11 L1/L2 complexes in a pH-dependent manner. Furthermore, CyPs released L1 capsomeres from partially disassembled HPV16 pseudovirions at slightly acidic pH. Taken together, these data suggest that CyPs mediate the dissociation of HPV L1 and L2 capsid proteins following acidification of endocytic vesicles.
Gag-Specific Cellular Immunity Determines
In Vitro
Viral Inhibition and
In Vivo
Virologic Control following Simian Immunodeficiency Virus Challenges of Vaccinated Rhesus Monkeys
Authors: Stephenson Kathryn E. , Li Hualin , Walker Bruce D. , Michael Nelson L. , Barouch Dan H. 
Published: July 2012

ABSTRACT

A comprehensive vaccine for human immunodeficiency virus type 1 (HIV-1) would block HIV-1 acquisition as well as durably control viral replication in breakthrough infections. Recent studies have demonstrated that Env is required for a vaccine to protect against acquisition of simian immunodeficiency virus (SIV) in vaccinated rhesus monkeys, but the antigen requirements for virologic control remain unclear. Here, we investigate whether CD8
+
T lymphocytes from vaccinated rhesus monkeys mediate viral inhibition
in vitro
and whether these responses predict virologic control following SIV challenge. We observed that CD8
+
lymphocytes from 23 vaccinated rhesus monkeys inhibited replication of SIV
in vitro
. Moreover, the magnitude of inhibition prior to challenge was inversely correlated with set point SIV plasma viral loads after challenge. In addition, CD8 cell-mediated viral inhibition in vaccinated rhesus monkeys correlated significantly with Gag-specific, but not Pol- or Env-specific, CD4
+
and CD8
+
T lymphocyte responses. These findings demonstrate that
in vitro
viral inhibition following vaccination largely reflects Gag-specific cellular immune responses and correlates with
in vivo
virologic control following infection. These data suggest the importance of including Gag in an HIV-1 vaccine in which virologic control is desired.
Interaction between the Human Cytomegalovirus Tegument Proteins UL94 and UL99 Is Essential for Virus Replication
Authors: Phillips Stacia L. , Cygnar Daniel , Thomas Alexandra , Bresnahan Wade A. 
Published: July 2012

ABSTRACT
Human cytomegalovirus (HCMV) virions are structurally complex, and the mechanisms by which they are assembled are poorly understood, especially with respect to the cytoplasmic phase of assembly, during which the majority of the tegument is acquired and final envelopment occurs. These processes occur at a unique cytoplasmic structure called the assembly complex, which is formed through a reorganization of the cellular secretory apparatus. The HCMV tegument protein UL99 (pp28) is essential for viral replication at the stage of secondary envelopment. We previously demonstrated that UL99 interacts with the essential tegument protein UL94 in infected cells as well as in the absence of other viral proteins. Here we show that UL94 and UL99 alter each other's localization and that UL99 stabilizes UL94 in a binding-dependent manner. We have mapped the interaction between UL94 and UL99 to identify the amino acids of each protein that are required for their interaction. Mutation of these amino acids in the context of the viral genome demonstrates that HCMV is completely defective for replication in the absence of the interaction between UL94 and UL99. Further, we demonstrate that in the absence of their interaction, both UL94 and UL99 exhibit aberrant localization and do not accumulate at the assembly complex during infection. Taken together, our data suggest that the interaction between UL94 and UL99 is essential for the proper localization of each protein to the assembly complex and thus for the production of infectious virus.
Dissecting Quasi-Equivalence in Nonenveloped Viruses: Membrane Disruption Is Promoted by Lytic Peptides Released from Subunit Pentamers, Not Hexamers
Authors: Domitrovic Tatiana , Matsui Tsutomu , Johnson John E. 
Published: July 2012

ABSTRACT

Nonenveloped viruses often invade membranes by exposing hydrophobic or amphipathic peptides generated by a proteolytic maturation step that leaves a lytic peptide noncovalently associated with the viral capsid. Since multiple copies of the same protein form many nonenveloped virus capsids, it is unclear if lytic peptides derived from subunits occupying different positions in a quasi-equivalent icosahedral capsid play different roles in host infection. We addressed this question with
Nudaurelia capensis omega virus
(NωV), an insect RNA virus with an icosahedral capsid formed by protein α, which undergoes autocleavage during maturation, producing the lytic γ peptide. NωV is a unique model because autocatalysis can be precisely initiated
in vitro
and is sufficiently slow to correlate lytic activity with γ peptide production. Using liposome-based assays, we observed that autocatalysis is essential for the potent membrane disruption caused by NωV. We observed that lytic activity is acquired rapidly during the maturation program, reaching 100% activity with less than 50% of the subunits cleaved. Previous time-resolved structural studies of partially mature NωV particles showed that, during this time frame, γ peptides derived from the pentamer subunits are produced and are organized in a vertical helical bundle that is projected toward the particle surface, while identical polypeptides in quasi-equivalent subunits are produced later or are in positions inappropriate for release. Our functional data provide experimental support for the hypothesis that pentamers containing a central helical bundle, observed in different nonenveloped virus families, are a specialized lytic motif.