bluuebunny/crossref_metadata_2025_split huggingface dataset:
Novel Inhibitors of Influenza Virus Fusion: Structure-Activity Relationship and Interaction with the Viral Hemagglutinin
Authors: Vanderlinden Evelien , Göktaş Fusun , Cesur Zafer , Froeyen Matheus , Reed Mark L. , Russell Charles J. , Cesur Nesrin , Naesens Lieve 
Published: February 2010

ABSTRACT

A new class of
N
-(1-thia-4-azaspiro[4.5]decan-4-yl)carboxamide inhibitors of influenza virus hemagglutinin (HA)-mediated membrane fusion that has a narrow and defined structure-activity relationship was identified. In Madin-Darby canine kidney (MDCK) cells infected with different strains of human influenza virus A/H3N2, the lead compound, 4c, displayed a 50% effective concentration of 3 to 23 μM and an antiviral selectivity index of 10. No activity was observed for A/H1N1, A/H5N1, A/H7N2, and B viruses. The activity of 4c was reduced considerably when added 30 min or later postinfection, indicating that 4c inhibits an early step in virus replication. 4c and its congeners inhibited influenza A/H3N2 virus-induced erythrocyte hemolysis at low pH. 4c-resistant virus mutants, selected in MDCK cells, contained either a single D112N change in the HA2 subunit of the viral HA or a combination of three substitutions, i.e., R220S (in HA1) and E57K (in HA2) and an A-T substitution at position 43 or 96 of HA2. The mutants showed efficiency for receptor binding and replication similar to that of wild-type virus yet displayed an increased pH of erythrocyte hemolysis. In polykaryon assays with cells expressing single-mutant HA proteins, the E57K, A96T, and D112N mutations resulted in 4c resistance, and the HA proteins containing R220S, A96T, and D112N mutations displayed an increased fusion pH. Molecular modeling identified a binding cavity for 4c involving arginine-54 and glutamic acid-57 in the HA2 subunit. Our studies with the new fusion inhibitor 4c confirm the importance of this HA region in the development of influenza virus fusion inhibitors.
Sumoylation of the Epstein-Barr Virus BZLF1 Protein Inhibits Its Transcriptional Activity and Is Regulated by the Virus-Encoded Protein Kinase
Authors: Hagemeier Stacy R. , Dickerson Sarah J. , Meng Qiao , Yu Xianming , Mertz Janet E. , Kenney Shannon C. 
Published: February 2010

ABSTRACT
The Epstein-Barr virus (EBV) immediate-early protein BZLF1 (Z) mediates the switch between latent and lytic EBV infection. Z not only activates early lytic viral gene transcription but also plays a direct role in lytic viral genome replication. Although a small fraction of Z is known to be sumoylated, the effects of this posttranslational modification on various different Z functions have not been well defined. In this report, we show that only the lysine at amino acid residue 12 is required for the sumoylation of Z, and that Z can be sumoylated by SUMO isoforms 1, 2, and 3. We also demonstrate that the sumo-defective Z mutants ZK12A and ZK12R have enhanced transcriptional activity. The sumoylated and nonsumoylated forms of Z were found to have a similar cellular location, both being localized primarily within the nuclear matrix. The Z sumo-defective mutants were, however, partially defective for disrupting promyelocytic leukemia (PML) bodies compared to the ability of wild-type Z. In addition, we show that lytic viral genome replication does not require the sumoylation of Z, although a Z mutant altered at both amino acids 12 and 13 is replication defective. Furthermore, we show that the sumoylation of Z is greatly increased (from less than 1 to about 11%) in lytically induced 293 cells infected with an EBV mutant virus deleted for the EBV-encoded protein kinase (EBV-PK) compared to that of 293 cells infected with wild-type EBV, and that the overexpression of EBV-PK leads to the reduced sumoylation of Z in EBV-negative cells. Our results suggest that the sumoylation of Z helps to promote viral latency, and that EBV-PK inhibits Z sumoylation during viral reactivation.
NCoR1 Mediates Papillomavirus E8^E2C Transcriptional Repression
Authors: Powell Maria L. C. , Smith Jennifer A. , Sowa Mathew E. , Harper J. Wade , Iftner Thomas , Stubenrauch Frank , Howley Peter M. 
Published: February 2010

ABSTRACT

The papillomavirus E2 open reading frame encodes the full-length E2 protein as well as an alternatively spliced product called E8^E2C. E8^E2C has been best studied for the high-risk human papillomaviruses, where it has been shown to regulate viral genome levels and, like the full-length E2 protein, to repress transcription from the viral promoter that directs the expression of the viral E6 and E7 oncogenes. The repression function of E8^E2C is dependent on the 12-amino-acid N-terminal sequence from the E8 open reading frame (ORF). In order to understand the mechanism by which E8^E2C mediates transcriptional repression, we performed an unbiased proteomic analysis from which we identified six

h

igh-confidence

c

andidate

i

nteracting

p

roteins (HCIPs) for E8^E2C; the top two are NCoR1 and TBLR1. We established an interaction of E8^E2C with an NCoR1/HDAC3 complex and demonstrated that this interaction requires the wild-type E8 open reading frame. Small interfering RNA (siRNA) knockdown studies demonstrated the involvement of NCoR1/HDAC3 in the E8^E2C-dependent repression of the viral long control region (LCR) promoter. Additional genetic work confirmed that the papillomavirus E2 and E8^E2C proteins repress transcription through distinct mechanisms.
Downregulation of Cdc2/CDK1 Kinase Activity Induces the Synthesis of Noninfectious Human Papillomavirus Type 31b Virions in Organotypic Tissues Exposed to Benzo[
a
]pyrene
Authors: Alam Samina , Bowser Brian S. , Conway Michael J. , Israr Mohd , Ryndock Eric J. , Xi Long Fu , Meyers Craig 
Published: February 2010

ABSTRACT

Epidemiological studies suggest that human papillomavirus (HPV)-infected women who smoke face an increased risk for developing cervical cancer. We have previously reported that exposure of HPV-positive organotypic cultures to benzo[
a
]pyrene (B
a
P), a major carcinogen in cigarette smoke, resulted in enhanced viral titers. Since B
a
P is known to deregulate multiple pathways of cellular proliferation, enhanced virion synthesis could result from carcinogen/host cell interaction. Here, we report that B
a
P-mediated upregulation of virus synthesis is correlated to an altered balance between cell cycle-specific cyclin-dependent kinase (CDK) activity profile compared with controls. Specifically, B
a
P treatment increased accumulation of hyperphosphorylated retinoblastoma protein (pRb) which coincided with increased cdc2/CDK1 kinase activity, but which further conflicted with the simultaneous upregulation of CDK inhibitors p16
INK4
and p27
KIP1
, which normally mediate pRb hypophosphorylation. In contrast, p21
WAF1
and p53 levels remained unchanged. Under these conditions, CDK6 and CDK2 kinase activities were decreased, whereas CDK4 kinase activity remained unchanged. The addition of purvalanol A, a specific inhibitor of CDK1 kinase, to B
a
P-treated cultures, resulted in the production of noninfectious HPV type 31b (HPV31b) particles. In contrast, infectivity of control virus was unaffected by purvalanol A treatment. B
a
P targeting of CDK1 occurred independently of HPV status, since B
a
P treatment also increased CDK1 activity in tissues derived from primary keratinocytes. Our data indicate that HPV31b virions synthesized in the presence of B
a
P were dependent on B
a
P-mediated alteration in CDK1 kinase activity for maintaining their infectivity.
The Epstein-Barr Virus (EBV)-Encoded Protein Kinase, EBV-PK, but Not the Thymidine Kinase (EBV-TK), Is Required for Ganciclovir and Acyclovir Inhibition of Lytic Viral Production
Authors: Meng Qiao , Hagemeier Stacy R. , Fingeroth Joyce D. , Gershburg Edward , Pagano Joseph S. , Kenney Shannon C. 
Published: February 2010

ABSTRACT

Ganciclovir (GCV) and acyclovir (ACV) are guanine nucleoside analogues that inhibit lytic herpesvirus replication. GCV and ACV must be monophosphorylated by virally encoded enzymes to be converted into nucleotides and incorporated into viral DNA. However, whether GCV and/or ACV phosphorylation in Epstein-Barr virus (EBV)-infected cells is mediated primarily by the EBV-encoded protein kinase (EBV-PK), the EBV-encoded thymidine kinase (EBV-TK), or both is controversial. To examine this question, we constructed EBV mutants containing stop codons in either the EBV-PK or EBV-TK open reading frame and selected for stable 293T clones latently infected with wild-type EBV or each of the mutant viruses. Cells were induced to the lytic form of viral replication with a BZLF1 expression vector in the presence and absence of various doses of GCV and ACV, and infectious viral titers were determined by a green Raji cell assay. As expected, virus production in wild-type EBV-infected 293T cells was inhibited by both GCV (50% inhibitory concentration [IC
50
] = 1.5 μM) and ACV (IC
50
= 4.1 μM). However, the EBV-PK mutant (which replicates as well as the wild-type (WT) virus in 293T cells) was resistant to both GCV (IC
50
= 19.6 μM) and ACV (IC
50
= 36.4 μM). Expression of the EBV-PK protein in
trans
restored GCV and ACV sensitivity in cells infected with the PK mutant virus. In contrast, in 293T cells infected with the TK mutant virus, viral replication remained sensitive to both GCV (IC
50
= 1.2 μM) and ACV (IC
50
= 2.8 μM), although susceptibility to the thymine nucleoside analogue, bromodeoxyuridine, was reduced. Thus, EBV-PK but not EBV-TK mediates ACV and GCV susceptibilities.
Antiretroviral Therapy in the Clinic
Authors: Tsibris Athe M. N. , Hirsch Martin S. 
Published: February 2010

ABSTRACT

Antiretroviral therapy in the developed world has resulted in substantial reductions in HIV-associated morbidity and mortality, changing an HIV diagnosis from a likely death sentence into a manageable chronic infection (F. J. Palella, Jr., K. M. Delaney, A. C. Moorman, M. O. Loveless, J. Fuhrer, G. A. Satten, D. J. Aschman, and S. D. Holmberg, N. Engl. J. Med.
338:
853-860, 1998). Several million years of life have been saved by effective anti-HIV treatment, although these successes should not obscure the magnitude of the ongoing worldwide HIV epidemic (R. P. Walensky, A. D. Paltiel, E. Losina, L. M. Mercincavage, B. R. Schackman, P. E. Sax, M. C. Weinstein, and K. A. Freedberg, J. Infect. Dis.
194:
11-19, 2006). Readers of the
Journal of Virology
are doubtless aware of the fundamental advances in retrovirology that have made possible the development of potent inhibitors of HIV replication. In this review, we focus on the issues surrounding how these drugs and drug regimens are actually used in clinical settings. Their proper use requires detailed knowledge of the natural history of HIV infection, the pharmacology of the individual drugs, the complexities of drug-drug interactions, and the use of sophisticated molecular tests for monitoring of viral load, immunologic response, and drug resistance.
Avian Reovirus μNS Protein Forms Homo-Oligomeric Inclusions in a Microtubule-Independent Fashion, Which Involves Specific Regions of Its C-Terminal Domain
Authors: Brandariz-Nuñez Alberto , Menaya-Vargas Rebeca , Benavente Javier , Martinez-Costas Jose 
Published: February 2010

ABSTRACT

Members of the genus
Orthoreovirus
replicate in cytoplasmic inclusions termed viral factories. Compelling evidence suggests that the nonstructural protein μNS forms the matrix of the factories and recruits specific viral proteins to these structures. In the first part of this study, we analyzed the properties of avian reovirus factories and μNS-derived inclusions and found that they are nonaggresome cytoplasmic globular structures not associated with the cytoskeleton which do not require an intact microtubule network for formation and maturation. We next investigated the capacity of avian reovirus μNS to form inclusions in transfected and baculovirus-infected cells. Our results showed that μNS is the main component of the inclusions formed by recombinant baculovirus expression. This, and the fact that μNS is able to self-associate inside the cell, suggests that μNS monomers contain all the interacting domains required for inclusion formation. Examination of the inclusion-forming capacities of truncated μNS versions allowed us to identify the region spanning residues 448 to 635 of μNS as the smallest that was inclusion competent, although residues within the region 140 to 380 seem to be involved in inclusion maturation. Finally, we investigated the roles that four different motifs present in μNS(448-635) play in inclusion formation, and the results suggest that the C-terminal tail domain is a key determinant in dictating the initial orientation of monomer-to-monomer contacts to form basal oligomers that control inclusion shape and inclusion-forming efficiency. Our results contribute to an understanding of the generation of structured protein aggregates that escape the cellular mechanisms of protein recycling.
T Cells Facilitate Recovery from Venezuelan Equine Encephalitis Virus-Induced Encephalomyelitis in the Absence of Antibody
Authors: Brooke Christopher B. , Deming Damon J. , Whitmore Alan C. , White Laura J. , Johnston Robert E. 
Published: February 2010

ABSTRACTVenezuelan equine encephalitis virus (VEEV) is a mosquito-borne RNA virus of the genusAlphavirusthat is responsible for a significant disease burden in Central and South America through sporadic outbreaks into human and equid populations. For humans, 2 to 4% of cases are associated with encephalitis, and there is an overall case mortality rate of approximately 1%. In mice, replication of the virus within neurons of the central nervous system (CNS) leads to paralyzing, invariably lethal encephalomyelitis. However, mice infected with certain attenuated mutants of the virus are able to control the infection within the CNS and recover. To better define what role T cell responses might be playing in this process, we infected B cell-deficient μMT mice with a VEEV mutant that induces mild, sublethal illness in immune competent mice. Infected μMT mice rapidly developed the clinical signs of severe paralyzing encephalomyelitis but were eventually able to control the infection and recover fully from clinical illness. Recovery in this system was T cell dependent and associated with a dramatic reduction in viral titers within the CNS, followed by viral persistence in the brain. Further comparison of the relative roles of T cell subpopulations within this system revealed that CD4+T cells were better producers of gamma interferon (IFN-γ) than CD8+T cells and were more effective at controlling VEEV within the CNS. Overall, these results suggest that T cells, especially CD4+T cells, can successfully control VEEV infection within the CNS and facilitate recovery from a severe viral encephalomyelitis.
In VivoTropism of Attenuated and Pathogenic Measles Virus Expressing Green Fluorescent Protein in Macaques
Authors: de Vries Rory D. , Lemon Ken , Ludlow Martin , McQuaid Stephen , Yüksel Selma , van Amerongen Geert , Rennick Linda J. , Rima Bert K. , Osterhaus Albert D. M. E. , de Swart Rik L. , Duprex W. Paul 
Published: February 2010

ABSTRACTThe global increase in measles vaccination has resulted in a significant reduction of measles mortality. The standard route of administration for the live-attenuated measles virus (MV) vaccine is subcutaneous injection, although alternative needle-free routes, including aerosol delivery, are under investigation.In vitro, attenuated MV has a much wider tropism than clinical isolates, as it can use both CD46 and CD150 as cellular receptors. To compare thein vivotropism of attenuated and pathogenic MV, we infected cynomolgus macaques with pathogenic or attenuated recombinant MV expressing enhanced green fluorescent protein (GFP) (strains IC323 and Edmonston, respectively) via the intratracheal or aerosol route. Surprisingly, viral loads and cellular tropism in the lungs were similar for the two viruses regardless of the route of administration, and CD11c-positive cells were identified as the major target population. However, only the pathogenic MV caused significant viremia, which resulted in massive virus replication in B and T lymphocytes in lymphoid tissues and viral dissemination to the skin and the submucosa of respiratory epithelia. Attenuated MV was rarely detected in lymphoid tissues, and when it was, only in isolated infected cells. Following aerosol inhalation, attenuated MV was detected at early time points in the upper respiratory tract, suggesting local virus replication. This contrasts with pathogenic MV, which invaded the upper respiratory tract only after the onset of viremia. This study shows that despitein vitrodifferences, attenuated and pathogenic MV show highly similarin vivotropism in the lungs. However, systemic spread of attenuated MV is restricted.
Potent Vesicular Stomatitis Virus-Based Avian Influenza Vaccines Provide Long-Term Sterilizing Immunity against Heterologous Challenge
Authors: Schwartz Jennifer A. , Buonocore Linda , Suguitan Amorsolo L. , Silaghi Alex , Kobasa Darwyn , Kobinger Gary , Feldmann Heinz , Subbarao Kanta , Rose John K. 
Published: February 2010

ABSTRACT
The emergence in 1997 and continuance today of a highly lethal H5N1 avian influenza virus (AIV) causing human disease has raised concern about an impending pandemic and the need for a vaccine to prepare for such an occurrence. We previously generated an efficacious vesicular stomatitis virus (VSV)-based AIV vaccine expressing H5 hemagglutinin (HA) from the fifth genomic position of VSV (J. A. Schwartz et al., Virology 366:166-173, 2007). Here we have generated and characterized VSV-based vaccines that express the A/Hong Kong/156/1997 (clade 0) H5 HA from the first position of the VSV genome. These vectors induce broadly cross-neutralizing antibodies against homologous and heterologous H5N1 viruses of different clades in mice. The vaccines provide complete protection against morbidity and mortality after heterologous challenge with clade 0 and clade 1 strains in animals even 1 year after vaccination. Postchallenge pulmonary virus loads show that these vectors provide sterilizing immunity. Therefore, VSV-based AIV vaccines are potent, broadly cross-protective pandemic vaccine candidates.
Structural Insights into the Molecular Mechanisms of
Cauliflower Mosaic Virus
Transmission by Its Insect Vector
Authors: Hoh François , Uzest Marilyne , Drucker Martin , Plisson-Chastang Célia , Bron Patrick , Blanc Stéphane , Dumas Christian 
Published: February 2010

ABSTRACT

Cauliflower mosaic virus
(CaMV) is transmitted from plant to plant through a seemingly simple interaction with insect vectors. This process involves an aphid receptor and two viral proteins, P2 and P3. P2 binds to both the aphid receptor and P3, itself tightly associated with the virus particle, with the ensemble forming a transmissible viral complex. Here, we describe the conformations of both unliganded CaMV P3 protein and its virion-associated form. X-ray crystallography revealed that the N-terminal domain of unliganded P3 is a tetrameric parallel coiled coil with a unique organization showing two successive four-stranded subdomains with opposite supercoiling handedness stabilized by a ring of interchain disulfide bridges. A structural model of virus-liganded P3 proteins, folding as an antiparallel coiled-coil network coating the virus surface, was derived from molecular modeling. Our results highlight the structural and biological versatility of this coiled-coil structure and provide new insights into the molecular mechanisms involved in CaMV acquisition and transmission by the insect vector.
Induction of Interferon and Interferon Signaling Pathways by Replication of Defective Interfering Particle RNA in Cells Constitutively Expressing Vesicular Stomatitis Virus Replication Proteins
Authors: Panda Debasis , Dinh Phat X. , Beura Lalit K. , Pattnaik Asit K. 
Published: February 2010

ABSTRACT
We show here that replication of defective interfering (DI) particle RNA in HEK293 cells stably expressing vesicular stomatitis virus (VSV) replication proteins potently activates interferon (IFN) and IFN signaling pathways through upregulation of IFN-β promoter, IFN-stimulated response element (ISRE) promoter, and NF-κB promoter activities. Replication of DI particle RNA, not mere expression of the viral replication proteins, was found to be critical for induction of IFN and IFN signaling. The stable cells supporting replication of DI RNA described in this report will be useful in further examining the innate immune signaling pathways and the host cell functions in viral genome replication.
PB1-F2 Expression by the 2009 Pandemic H1N1 Influenza Virus Has Minimal Impact on Virulence in Animal Models
Authors: Hai Rong , Schmolke Mirco , Varga Zsuzsanna T. , Manicassamy Balaji , Wang Taia T. , Belser Jessica A. , Pearce Melissa B. , García-Sastre Adolfo , Tumpey Terrence M. , Palese Peter 
Published: February 2010

ABSTRACT

Unlike previous pandemic viruses, the 2009 H1N1 pandemic influenza virus does not code for the virulence factor PB1-F2. The genome of the 2009 H1N1 virus contains three stop codons preventing PB1-F2 expression; however, PB1-F2 production could occur following genetic mutation or reassortment. Thus, it is of great interest to understand the impact that expression of the PB1-F2 protein might have in the context of the 2009 pandemic influenza virus, A/California/04/2009 (Cal/09). We have addressed this question by generating two Cal/09 viruses with productive PB1-F2 open reading frames containing either an asparagine at position 66 of PB1-F2 (66N) or a serine at position 66 (66S): this N66S change has previously been shown to be associated with increased virulence in mice. We used these viruses to investigate the effect on virulence conferred by expression of the 66N or the 66S PB1-F2 protein in both
in vitro
and
in vivo
systems. Our results show enhanced replication of the 66S virus in A549 cells, while studies of BALB/c and DBA/2 mice and ferrets revealed no significant differences in symptoms of infection with wild-type Cal/09 versus the 66N or 66S virus variant. Also, coinfection of mice with
Streptococcus pneumoniae
and the different viruses (recombinant wild-type [rWT] Cal/09 and the 66N and 66S viruses) did not result in significant differences in mortality. Mice infected with either PB1-F2-expressing virus did demonstrate altered protein levels of proinflammatory cytokines; differences were observed to be greater in infection caused by the 66S virus. In summary, our study demonstrates that PB1-F2 expression by the Cal/09 virus modulates the immune response to infection while having a minimal effect on virus virulence in two mammalian models.