bluuebunny/crossref_metadata_2025_split huggingface dataset:
Type I Interferon Production during Herpes Simplex Virus Infection Is Controlled by Cell-Type-Specific Viral Recognition through Toll-Like Receptor 9, the Mitochondrial Antiviral Signaling Protein Pathway, and Novel Recognition Systems
Authors: Rasmussen Simon B. , Sørensen Louise N. , Malmgaard Lene , Ank Nina , Baines Joel D. , Chen Zhijian J. , Paludan Søren R. 
Published: October 2007

ABSTRACT
Recognition of viruses by germ line-encoded pattern recognition receptors of the innate immune system is essential for rapid production of type I interferon (IFN) and early antiviral defense. We investigated the mechanisms of viral recognition governing production of type I IFN during herpes simplex virus (HSV) infection. We show that early production of IFN in vivo is mediated through Toll-like receptor 9 (TLR9) and plasmacytoid dendritic cells, whereas the subsequent alpha/beta IFN (IFN-α/β) response is derived from several cell types and induced independently of TLR9. In conventional DCs, the IFN response occurred independently of viral replication but was dependent on viral entry. Moreover, using a HSV-1 UL15 mutant, which fails to package viral DNA into the virion, we found that entry-dependent IFN induction also required the presence of viral genomic DNA. In macrophages and fibroblasts, where the virus was able to replicate, HSV-induced IFN-α/β production was dependent on both viral entry and replication, and ablated in cells unable to signal through the mitochondrial antiviral signaling protein pathway. Thus, during an HSV infection in vivo, multiple mechanisms of pathogen recognition are active, which operate in cell-type- and time-dependent manners to trigger expression of type I IFN and coordinate the antiviral response.
Two-Way Antigenic Cross-Reactivity between Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV) and Group 1 Animal CoVs Is Mediated through an Antigenic Site in the N-Terminal Region of the SARS-CoV Nucleoprotein
Authors: Vlasova Anastasia N. , Zhang Xinsheng , Hasoksuz Mustafa , Nagesha Hadya S. , Haynes Lia M. , Fang Ying , Lu Shan , Saif Linda J. 
Published: October 2007

ABSTRACTIn 2002, severe acute respiratory syndrome-associated coronavirus (SARS-CoV) emerged in humans, causing a global epidemic. By phylogenetic analysis, SARS-CoV is distinct from known CoVs and most closely related to group 2 CoVs. However, no antigenic cross-reactivity between SARS-CoV and known CoVs was conclusively and consistently demonstrated except for group 1 animal CoVs. We analyzed this cross-reactivity by an enzyme-linked immunosorbent assay (ELISA) and Western blot analysis using specific antisera to animal CoVs and SARS-CoV and SARS patient convalescent-phase or negative sera. Moderate two-way cross-reactivity between SARS-CoV and porcine CoVs (transmissible gastroenteritis CoV [TGEV] and porcine respiratory CoV [PRCV]) was mediated through the N but not the spike protein, whereas weaker cross-reactivity occurred with feline (feline infectious peritonitis virus) and canine CoVs. UsingEscherichia coli-expressed recombinant SARS-CoV N protein and fragments, the cross-reactive region was localized between amino acids (aa) 120 to 208. The N-protein fragments comprising aa 360 to 412 and aa 1 to 213 reacted specifically with SARS convalescent-phase sera but not with negative human sera in ELISA; the fragment comprising aa 1 to 213 cross-reacted with antisera to animal CoVs, whereas the fragment comprising aa 360 to 412 did not cross-react and could be a potential candidate for SARS diagnosis. Particularly noteworthy, a single substitution at aa 120 of PRCV N protein diminished the cross-reactivity. We also demonstrated that the cross-reactivity is not universal for all group 1 CoVs, because HCoV-NL63 did not cross-react with SARS-CoV. One-way cross-reactivity of HCoV-NL63 with group 1 CoVs was localized to aa 1 to 39 and at least one other antigenic site in the N-protein C terminus, differing from the cross-reactive region identified in SARS-CoV N protein. The observed cross-reactivity is not a consequence of a higher level of amino acid identity between SARS-CoV and porcine CoV nucleoproteins, because sequence comparisons indicated that SARS-CoV N protein has amino acid identity similar to that of infectious bronchitis virus N protein and shares a higher level of identity with bovine CoV N protein within the cross-reactive region. The TGEV and SARS-CoV N proteins are RNA chaperons with long disordered regions. We speculate that during natural infection, antibodies target similar short antigenic sites within the N proteins of SARS-CoV and porcine group 1 CoVs that are exposed to an immune response. Identification of the cross-reactive and non-cross-reactive N-protein regions allows development of SARS-CoV-specific antibody assays for screening animal and human sera.
Chaperones Activate Hepadnavirus Reverse Transcriptase by Transiently Exposing a C-Proximal Region in the Terminal Protein Domain That Contributes to ε RNA Binding
Authors: Stahl Michael , Beck Jürgen , Nassal Michael 
Published: October 2007

ABSTRACT
All hepatitis B viruses replicate by protein-primed reverse transcription, employing a specialized reverse transcriptase, P protein, that carries a unique terminal protein (TP) domain. To initiate reverse transcription, P protein must bind to a stem-loop, ε, on the pregenomic RNA template. TP then provides a Y residue for covalent attachment of the first nucleotide of an ε-templated DNA oligonucleotide (priming reaction) that serves to initiate full-length minus-strand DNA synthesis. ε binding requires the chaperone-dependent conversion of inactive P protein into an activated, metastable form designated P*. However, how P* differs structurally from P protein is not known. Here we used an in vitro reconstitution system for active duck hepatitis B virus P combined with limited proteolysis, site-specific antibodies, and defined P mutants to structurally compare nonactivated versus chaperone-activated versus primed P protein. The data show that Hsp70 action, under conditions identical to those required for functional activation, transiently exposes the C proximal TP region which is, probably directly, involved in ε RNA binding. Notably, after priming and ε RNA removal, a very similar new conformation appears stable without further chaperone activity; hence, the activation of P protein is triggered by energy-consuming chaperone action but may be completed by template RNA binding.
Interaction of Vesicular Stomatitis Virus P and N Proteins: Identification of Two Overlapping Domains at the N Terminus of P That Are Involved in N
0
-P Complex Formation and Encapsidation of Viral Genome RNA
Authors: Chen Mingzhou , Ogino Tomoaki , Banerjee Amiya K. 
Published: October 2007

ABSTRACT

The nucleocapsid (N) protein of nonsegmented negative-strand (NNS) RNA viruses, when expressed in eukaryotic cells, aggregates and forms nucleocapsid-like complexes with cellular RNAs. The phosphoprotein (P) has been shown to prevent such aggregation by forming a soluble complex with the N protein free from cellular RNAs (designated N
0
). The N
0
-P complex presumably mediates specific encapsidation of the viral genome RNA. The precise mechanism by which the P protein carries out this function remains unclear. Here, by using a series of deleted and truncated mutant forms of the P protein of vesicular stomatitis virus (VSV), Indiana serotype, we present evidence that the N-terminal 11 to 30 amino acids (aa) of the P protein are essential in keeping the N protein soluble. Furthermore, glutathione
S
-transferase fused to the N-terminal 40 aa by itself is able to form the N
0
-P complex. Interestingly, the N-terminal 40-aa stretch failed to interact with the viral genome N-RNA template whereas the C-terminal 72 aa of the P protein interacted specifically with the latter. With an in vivo VSV minigenome transcription system, we further show that a deletion mutant form of P (PΔ1-10) lacking the N-terminal 10 aa which is capable of forming the N
0
-P complex was unable to support VSV minigenome transcription, although it efficiently supported transcription in vitro in a transcription-reconstitution reaction when used as purified protein. However, the same mutant protein complemented minigenome transcription when expressed together with a transcription-defective P deletion mutant protein containing N-terminal aa 1 to 210 (PΔII+III). Since the minigenome RNA needs to be encapsidated before transcription ensues, it seems that the entire N-terminal 210 aa are required for efficient genome RNA encapsidation. Taking these results together, we conclude that the N-terminal 11 to 30 aa are required for N
0
-P complex formation but the N-terminal 210 aa are required for genome RNA encapsidation.
Human Herpesvirus 6A (HHV-6A) and HHV-6B Alter E2F1/Rb Pathways and E2F1 Localization and Cause Cell Cycle Arrest in Infected T Cells
Authors: Mlechkovich Guy , Frenkel Niza 
Published: October 2007

ABSTRACT
E2F transcription factors play pivotal roles in controlling the expression of genes involved in cell viability as well as genes involved in cell death. E2F1 is an important constituent of this protein family, which thus far contains eight members. The interaction of E2F1 with its major regulator, retinoblastoma protein (Rb), has been studied extensively in the past two decades, concentrating on the role of E2F1 in transcriptional regulation and the role of Rb in cell replication and cancer formation. Additionally, the effect of viral infections on E2F1/Rb interactions has been analyzed for different viruses, concentrating on cell division, which is essential for viral replication. In the present study, we monitored E2F1-Rb interactions during human herpesvirus 6A (HHV-6A) and HHV-6B infections of SupT1 T cells. The results have shown the following dramatic alterations in E2F1-Rb pathways compared to the pathways of parallel mock-infected control cultures. (i) The E2F1 levels were elevated during viral infections. (ii) The cellular localization of E2F1 was dramatically altered, and it was found to accumulate both in the cytoplasmic and nuclear fractions, as opposed to the strict nuclear localization seen in the mock-infected cells. (iii) Although E2F1 expression was elevated, two exemplary target genes, cyclin E and MCM5, were not upregulated. (iv) The Rb protein was dephosphorylated early postinfection, a trait that also occurred with UV-inactivated virus. (v) Infection was associated with significant reduction of E2F1/Rb complexing. (vi) HHV-6 infections were accompanied by cell cycle arrest. The altered E2F1-Rb interactions and functions might contribute to the observed cell cycle arrest.
Mapping Protease Inhibitor Resistance to Human Immunodeficiency Virus Type 1 Sequence Polymorphisms within Patients
Authors: Poon Art F. Y. , Kosakovsky Pond Sergei L. , Richman Douglas D. , Frost Simon D. W. 
Published: October 2007

ABSTRACTResistance genotyping provides an important resource for the clinical management of patients infected with human immunodeficiency virus type 1 (HIV-1). However, resistance to protease (PR) inhibitors (PIs) is a complex phenotype shaped by interactions among nearly half of the residues in HIV-1 PR. Previous studies of the genetic basis of PI resistance focused on fixed substitutions among populations of HIV-1, i.e., host-specific adaptations. Consequently, they are susceptible to a high false discovery rate due to founder effects. Here, we employ sequencing “mixtures” (i.e., ambiguous base calls) as a site-specific marker of genetic variation within patients that is independent of the phylogeny. We demonstrate that the transient response to selection by PIs is manifested as an excess of nonsynonymous mixtures. Using a sample of 5,651 PR sequences isolated from both PI-naive and -treated patients, we analyze the joint distribution of mixtures and eight PIs as a Bayesian network, which distinguishes residue-residue interactions from direct associations with PIs. We find that selection for resistance is associated with the emergence of nonsynonymous mixtures in two distinct groups of codon sites clustered along the substrate cleft and distal regions of PR, respectively. Within-patient evolution at several positions is independent of PIs, including those formerly postulated to be involved in resistance. These positions are under strong positive selection in the PI-naive patient population, implying that other factors can produce spurious associations with resistance, e.g., mutational escape from the immune response.
Analysis of Venezuelan Equine Encephalitis Virus Capsid Protein Function in the Inhibition of Cellular Transcription
Authors: Garmashova Natalia , Atasheva Svetlana , Kang Wenli , Weaver Scott C. , Frolova Elena , Frolov Ilya 
Published: October 2007

ABSTRACT

The encephalitogenic New World alphaviruses, including Venezuelan (VEEV), eastern (EEEV), and western equine encephalitis viruses, constitute a continuing public health threat in the United States. They circulate in Central, South, and North America and have the ability to cause fatal disease in humans and in horses and other domestic animals. We recently demonstrated that these viruses have developed the ability to interfere with cellular transcription and use it as a means of downregulating a cellular antiviral response. The results of the present study suggest that the N-terminal, ∼35-amino-acid-long peptide of VEEV and EEEV capsid proteins plays the most critical role in the downregulation of cellular transcription and development of a cytopathic effect. The identified VEEV-specific peptide C
VEE
33-68 includes two domains with distinct functions: the α-helix domain, helix I, which is critically involved in supporting the balance between the presence of the protein in the cytoplasm and nucleus, and the downstream peptide, which might contain a functional nuclear localization signal(s). The integrity of both domains not only determines the intracellular distribution of the VEEV capsid but is also essential for direct capsid protein functioning in the inhibition of transcription. Our results suggest that the VEEV capsid protein interacts with the nuclear pore complex, and this interaction correlates with the protein's ability to cause transcriptional shutoff and, ultimately, cell death. The replacement of the N-terminal fragment of the VEEV capsid by its Sindbis virus-specific counterpart in the VEEV TC-83 genome does not affect virus replication in vitro but reduces cytopathogenicity and results in attenuation in vivo. These findings can be used in designing a new generation of live, attenuated, recombinant vaccines against the New World alphaviruses.
Dynamics of T-Cell Responses and Memory T Cells during Primary Simian Immunodeficiency Virus Infection in Cynomolgus Macaques
Authors: Karlsson Ingrid , Malleret Benoît , Brochard Patricia , Delache Benoît , Calvo Julien , Le Grand Roger , Vaslin Bruno 
Published: October 2007

ABSTRACT

Cellular immune responses make an important contribution to both the control of human immunodeficiency virus (HIV) replication and disease progression. We used a pathogenic model of SIVmac251 infection of cynomolgus macaques to longitudinally evaluate cellular immune responses in association with various rates of disease progression. We found an inverse relationship between plasma viral load and the simian immunodeficiency virus (SIV)-specific T cells responses in peripheral blood and lymph nodes. SIV-specific T-cell responses in peripheral blood were transient during primary infection, with the highest responses detected around 3 months after infection. There was also a transient increase of central memory CD8
+
T cells in peripheral blood during primary infection, and effector memory T-cell counts in peripheral lymph nodes were increased. This study emphasizes the importance of the early virus-specific immune responses in the outcome of HIV/SIV disease and provides details about the changes of virus-specific immune responses over time.
Inhibition of Alpharetrovirus Replication by a Range of Human APOBEC3 Proteins
Authors: Wiegand Heather L. , Cullen Bryan R. 
Published: October 2007

ABSTRACT
The mammalian APOBEC3 family of cytidine deaminases includes members that can act as potent inhibitors of retroviral infectivity and retrotransposon mobility. Here, we have examined whether the alpharetrovirus Rous sarcoma virus (RSV) is susceptible to inhibition by a range of human APOBEC3 proteins. We report that RSV is highly susceptible to inhibition by human APOBEC3G, APOBEC3F, and APOBEC3B and moderately susceptible to inhibition by human APOBEC3C and APOBEC3A. For all five proteins, inhibition of RSV infectivity was associated with selective virion incorporation and with C-to-T editing of the proviral DNA minus strand. In the case of APOBEC3G, editing appeared to be critical for effective inhibition. These data represent the first report of inhibition of retroviral infectivity and induction of proviral DNA editing by human APOBEC3A and reveal that alpharetroviruses, which do not normally encounter APOBEC3 proteins in their avian hosts, are susceptible to inhibition by all human APOBEC3 proteins tested. These data further suggest that the resistance of mammalian retroviruses to inhibition by the APOBEC3 proteins expressed in their normal host species is likely to have evolved subsequent to the appearance of this family of mammalian antiretroviral proteins some 35 million years ago; i.e., the base state of a naïve retrovirus is susceptibility to inhibition.
ERK- and JNK-Dependent Signaling Pathways Contribute toBombyx moriNucleopolyhedrovirus Infection
Authors: Katsuma Susumu , Mita Kazuei , Shimada Toru 
Published: October 2007

ABSTRACTMitogen-activated protein kinases (MAPKs) often play important roles in virus infection. To explore intracellular signaling pathways induced by baculovirus infection, we examined the involvement of MAPKs inBombyx morinucleopolyhedrovirus (BmNPV) infection of BmN cells. We found that specific inhibitors of extracellular signal-regulated kinase (ERK) kinase and c-Jun NH2-terminal kinase (JNK) significantly reduced occlusion body (OB) formation and budded virus (BV) production. Next, we quantified OB and BV production after applying the inhibitors at different times postinfection (p.i.). The inhibitors significantly reduced OB and BV production to various extents when applied at 12 h p.i., indicating that the reduction of BmNPV infectivity by these inhibitors occurs at the late stage of infection. Also, we observed that these inhibitors markedly repressed or deregulated the expression of delayed early, late, and very late gene products. Western blot analysis using phospho-MAPK-specific antibodies showed that ERK and JNK were activated at the late stage of BmNPV infection. In addition, the magnitude and pattern of MAPK activation were dependent on the multiplicity of infection. To verify the effects of the inhibitors on BmNPV infection, we also attempted to knock down theB. morigenesBmErkandBmJnk, which encode ERK and JNK, respectively. Knockdown ofBmErkandBmJnkresulted in the reduced production of OBs and BVs, confirming that BmERK and BmJNK are involved in the BmNPV infection process. Taken together, these results indicate that the activation of MAPK signaling pathways is required for efficient infection by BmNPV.
Increased Immunogenicity of a DNA-Launched Venezuelan Equine Encephalitis Virus-Based Replicon DNA Vaccine
Authors: Ljungberg Karl , Whitmore Alan C. , Fluet Meagan E. , Moran Timothy P. , Shabman Reed S. , Collier Martha L. , Kraus Annette A. , Thompson Joseph M. , Montefiori David C. , Beard Clayton , Johnston Robert E. 
Published: October 2007

ABSTRACT
A novel genetic vaccine that is based on a Venezuelan equine encephalitis virus (VEE) replicon launched from plasmid DNA is described. The plasmid encodes a VEE replicon under the transcriptional control of the cytomegalovirus immediate-early promoter (VEE DNA). The VEE DNA consistently expressed 3- to 15-fold more green fluorescent protein in vitro than did a conventional DNA vaccine. Furthermore, transfection with the DNA-launched VEE replicon induced apoptosis and type I interferon production. Inoculation of mice with VEE DNA encoding human immunodeficiency virus type 1 gp160 significantly increased humoral responses by several orders of magnitude compared to an equal dose of a conventional DNA vaccine. These increases were also observed at 10- and 100-fold-lower doses of the VEE DNA. Cellular immune responses measured by gamma interferon and interleukin 2 enzyme-linked immunospot assay were significantly higher in mice immunized with the VEE DNA at decreased doses. The immune responses induced by the VEE DNA-encoded antigen, however, were independent of an intact type I interferon signaling pathway. Moreover, the DNA-launched VEE replicon induced an efficient prime to a VEE replicon particle (VRP) boost, increasing humoral and cellular immunity by at least 1 order of magnitude compared to VEE DNA only. Importantly, immunization with VEE DNA, as opposed to VRP, did not induce any anti-VRP neutralizing antibodies. Increased potency of DNA vaccines and reduced vector immunity may ultimately have an impact on the design of vaccination strategies in humans.
Inhibitory activity of cetuximab on epidermal growth factor receptor mutations in non–small cell lung cancers
Authors: Doody Jacqueline F. , Wang Ying , Patel Sheetal N. , Joynes Christopher , Lee Sui Ping , Gerlak Jason , Rolser Robin L. , Li Yanxia , Steiner Philipp , Bassi Rajiv , Hicklin Dan J. , Hadari Yaron R. 
Published: October 2007

AbstractMutations in the kinase domain of the epidermal growth factor receptor (EGFR) were identified in ∼15% of all patients with non–small cell lung cancer (NSCLC). These mutations have been established as an indicator of superior response to gefitinib and erlotinib, small molecule inhibitors of the EGFR kinase domain. Whether these mutations would also render patients more susceptible to treatment with cetuximab (Erbitux), an EGFR-neutralizing antibody, is yet to be determined. In this study, we attempted to evaluate the effect of cetuximab on several NSCLC lines harboring some of the more common EGFR mutations (L858R and delL747-T753insS), as well as the recently identified kinase inhibitor–resistant mutation, T790M. We could show that the kinase activity of the abovementioned EGFR mutants was hindered by cetuximab, as detected by both cell-based phosphorylation and proliferation assays. Interestingly, cetuximab also induced enhanced degradation of the EGFR mutants as compared with the wild-type receptor. Most importantly, cetuximab successfully inhibited the growth of NSCLC lines in xenograft models. These results indicate the promising potential of cetuximab as a regimen for patients with NSCLC bearing these mutations. [Mol Cancer Ther 2007;6(10):2642–51]