bluuebunny/crossref_metadata_2025 huggingface dataset:
ORF66 Protein Kinase Function Is Required for T-Cell Tropism of Varicella-Zoster Virus In Vivo
Authors: Schaap-Nutt Anne , Sommer Marvin , Che Xibing , Zerboni Leigh , Arvin Ann M.
Published: November 2006

Several functions have been attributed to the serine/threonine protein kinase encoded by open reading frame 66 (ORF66) of varicella-zoster virus (VZV), including modulation of the apoptosis and interferon pathways, down-regulation of major histocompatibility complex class I cell surface expression, and regulation of IE62 localization. The amino acid sequence of the ORF66 protein contains a recognizable conserved kinase domain. Point mutations were introduced into conserved protein kinase motifs to evaluate their importance to ORF66 protein functions. Two substitution mutants were generated, including a G102A substitution, which blocked autophosphorylation and altered IE62 localization, and an S250P substitution, which had no effect on either autophosphorylation or IE62 localization. Both kinase domain mutants grew to titers equivalent to recombinant parent Oka (pOka) in vitro. pOka66G102A had slightly reduced growth in skin, which was comparable to the reduction observed when ORF66 translation was prevented by stop codon insertions in pOka66S. In contrast, infection of T-cell xenografts with pOka66G102A was associated with a significant decrease in infectious virus production equivalent to the impaired T-cell tropism found with pOka66S infection of T-cell xenografts in vivo. Disrupting kinase activity with the G102A mutation did not alter IE62 cytoplasmic localization in VZV-infected T cells, suggesting that decreased T-cell tropism is due to other ORF66 protein functions. The G102A mutation reduced the antiapoptotic effects of VZV infection of T cells. These experiments indicate that the T-cell tropism of VZV depends upon intact ORF66 protein kinase function.
The Hepatitis C Virus RNA 3′-Untranslated Region Strongly Enhances Translation Directed by the Internal Ribosome Entry Site
Authors: Song Yutong , Friebe Peter , Tzima Eleni , Jünemann Christiane , Bartenschlager Ralf , Niepmann Michael
Published: November 2006

The positive-strand RNA genome of the hepatitis C virus (HCV) is flanked by 5′- and 3′-untranslated regions (UTRs). Translation of the viral RNA is directed by the internal ribosome entry site (IRES) in the 5′-UTR, and subsequent viral RNA replication requires sequences in the 3′-UTR and in the 5′-UTR. Addressing previous conflicting reports on a possible function of the 3′-UTR for RNA translation in this study, we found that reporter construct design is an important parameter in experiments testing 3′-UTR function. A translation enhancer function of the HCV 3′-UTR was detected only after transfection of monocistronic reporter RNAs or complete RNA genomes having a 3′-UTR with a precise 3′ terminus. The 3′-UTR strongly stimulates HCV IRES-dependent translation in human hepatoma cell lines but only weakly in nonliver cell lines. The variable region, the poly(U · C) tract, and the most 3′ terminal stem-loop 1 of the highly conserved 3′ X region contribute significantly to translation enhancement, whereas stem-loops 2 and 3 of the 3′ X region are involved only to a minor extent. Thus, the signals for translation enhancement and for the initiation of RNA minus-strand synthesis in the HCV 3′-UTR partially overlap, supporting the idea that these sequences along with viral and possibly also cellular factors may be involved in an RNA 3′-5′ end interaction and a switch between translation and RNA replication.
Overexpression of the Lens Epithelium-Derived Growth Factor/p75 Integrase Binding Domain Inhibits Human Immunodeficiency Virus Replication
Authors: De Rijck Jan , Vandekerckhove Linos , Gijsbers Rik , Hombrouck Anneleen , Hendrix Jelle , Vercammen Jo , Engelborghs Yves , Christ Frauke , Debyser Zeger
Published: November 2006

We initially identified lens epithelium-derived growth factor/p75 (LEDGF/p75) as a binding partner of human immunodeficiency virus type 1 (HIV-1) integrase. To investigate the role of LEDGF/p75 in HIV replication and its potential as a new antiviral target, we stably overexpressed two different fragments containing the integrase binding domain (IBD) of LEDGF/p75 fused to enhanced green fluorescent protein (eGFP). HIV-1 replication was severely inhibited by overexpression of the eGFP-IBD fusion proteins, while no inhibition was observed in cell lines overexpressing the interaction-deficient D366A mutant. Quantitative PCR pinpointed the block to the integration step, whereas nuclear import was not affected. Competition of the IBD fusion proteins with endogenous LEDGF/p75 for binding to integrase led to a potent defect in HIV-1 replication in both HeLaP4- and MT-4-derived cell lines. A previously described diketo acid-resistant HIV-1 strain remained fully susceptible to inhibition, suggesting that this strategy will also work in patients who harbor strains resistant to the current experimental integrase inhibitors. These data support LEDGF/p75 as an important cofactor for HIV replication and provide proof of concept for the LEDGF/p75-integrase interaction as a novel target for treating HIV-1 infection.
Novel Fiber-Dependent Entry Mechanism for Adenovirus Serotype 5 in LacrimalAcini
Authors: Xie Jiansong , Chiang Lilian , Contreras Janette , Wu Kaijin , Garner Judy A. , Medina-Kauwe Lali , Hamm-Alvarez Sarah F.
Published: November 2006

The established mechanism for infection of most cells with adenovirus serotype 5 (Ad5) involves fiber capsid protein binding to
coxsackievirus-adenovirus receptor (CAR) at the cell surface, followed by penton base capsid protein binding to α
v
integrins, which triggers clathrin-mediated endocytosis of the virus. Here we determined the identity of the capsid proteins responsible for
mediating Ad5 entry into the acinar epithelial cells of the lacrimal gland. Ad5 transduction of primary rabbit lacrimal acinar cells was
inhibited by excess Ad5 fiber or knob (terminal region of the fiber) but not excess penton base. Investigation of the interactions of
recombinant Ad5 penton base, fiber, and knob with lacrimal acini revealed that the penton base capsid protein remained surface
associated, while the knob domain of the fiber capsid protein was rapidly internalized. Introduction of rabbit CAR-specific small
interfering RNA (siRNA) into lacrimal acini under conditions that reduced intracellular CAR mRNA significantly inhibited Ad5
transduction, in contrast to a control (nonspecific) siRNA. Preincubation of Ad5 with excess heparin or pretreatment of acini with
a heparinase cocktail each inhibited Ad5 transduction by a separate and apparently additive mechanism. Functional and imaging studies revealed
that Ad5, fiber, and knob, but not penton base, stimulated macropinocytosis in acini and that inhibition of macropinocytosis
significantly reduced Ad5 transduction of acini. However, inhibition of macropinocytosis did not reduce Ad5 uptake. We propose that
internalization of Ad5 into lacrimal acini is through a novel fiber-dependent mechanism that includes CAR and heparan sulfate
glycosaminoglycans and that the subsequent intracellular trafficking of Ad5 is enhanced by fiber-induced macropinocytosis.
Flavivirus Infection Activates the XBP1 Pathway of the Unfolded Protein Response To Cope with Endoplasmic Reticulum Stress
Authors: Yu Chia-Yi , Hsu Yun-Wei , Liao Ching-Len , Lin Yi-Ling
Published: November 2006

The unfolded protein response (UPR) is a coordinated change in gene expression triggered by perturbations in functions of the endoplasmic reticulum (ER). XBP1, a key transcription factor of the UPR, is activated by an IRE1-mediated splicing event, which results in a frameshift and encodes a protein with transcriptional activity. Here, we report that XBP1 was activated during flaviviral infection, as evidenced by
XBP1
mRNA splicing and protein expression, as well as induction of the downstream genes
ERdj4
,
EDEM1
, and
p58(IPK)
in Japanese encephalitis virus (JEV)- and dengue virus serotype 2 (DEN-2)-infected cells. Reporter systems based on IRE1-mediated
XBP1
splicing were established, and several flaviviral proteins associated with the ER, including glycoproteins and small hydrophobic membrane-anchored proteins, were found to trigger the splicing event. Notably, nonstructural protein NS2B-3 of DEN-2, but not of JEV, was a potent inducer of
XBP1
splicing through an unclear mechanism(s). Reduction of XBP1 by a small interfering RNA had no effect on cells' susceptibility to the two viruses but exacerbated the flavivirus-induced cytopathic effects. Overall, flaviviruses trigger the XBP1 signaling pathway and take advantage of this cellular response to alleviate virus-induced cytotoxicity.
Inhibition of Coxsackievirus B3 in Cell Cultures and in Mice by Peptide-Conjugated Morpholino Oligomers Targeting the Internal Ribosome Entry Site
Authors: Yuan Ji , Stein David A. , Lim Travis , Qiu Dexin , Coughlin Shaun , Liu Zhen , Wang Yinjing , Blouch Robert , Moulton Hong M. , Iversen Patrick L. , Yang Decheng
Published: November 2006

Coxsackievirus B3 (CVB3) is a primary cause of viral myocarditis, yet no effective therapeutic against CVB3 is available. Nucleic acid-based interventional strategies against various viruses, including CVB3, have shown promise experimentally, but limited stability and inefficient delivery in vivo remain as obstacles to their potential as therapeutics. We employed phosphorodiamidate morpholino oligomers (PMO) conjugated to a cell-penetrating arginine-rich peptide, P007 (to form PPMO), to address these issues. Eight CVB3-specific PPMO were evaluated with HeLa cells and HL-1 cardiomyocytes in culture and in a murine infection model. One of the PPMO (PPMO-6), designed to target a sequence in the 3′ portion of the CVB3 internal ribosomal entry site, was found to be especially potent against CVB3. Treatment of cells with PPMO-6 prior to CVB3 infection produced an approximately 3-log
10
decrease in viral titer and largely protected cells from a virus-induced cytopathic effect. A similar antiviral effect was observed when PPMO-6 treatment began shortly after the virus infection period. A/J mice receiving intravenous administration of PPMO-6 once prior to and once after CVB3 infection showed an ∼2-log
10
-decreased viral titer in the myocardium at 7 days postinfection and a significantly decreased level of cardiac tissue damage, compared to the controls. Thus, PPMO-6 provided potent inhibition of CVB3 amplification both in cell cultures and in vivo and appears worthy of further evaluation as a candidate for clinical development.
Entire Genome of a Strain of Human Immunodeficiency Virus Type 1 with a Deletion of
nef
That Was Recovered 20 Years after Primary Infection: Large Pool of Proviruses with Deletions of
env
Authors: Calugi Graziella , Montella Francesco , Favalli Cartesio , Benedetto Arrigo
Published: November 2006

We report the complete sequence analysis of the provirus harbored in a long-term nonprogressor (patient SG1) 20 years after the first infection with a human immunodeficiency virus type 1 strain lacking
nef
. The sequencing showed large deletions in the
nef-nef
and
nef-
U3 regions. Except for
vpu
, all of the other accessory genes were intact. The
gag
and
pol
genes did not show significant alterations. We found large deletions in
env
, spanning the V1, V2, V3, V4, and V5 regions. We believe that, when down-regulation of the class 1 major histocompatibility complex molecules is inhibited by the lack of
nef
function, the cells containing Env-defective molecules evade cytotoxic T lymphocyte killing and accumulate progressively.
Human Cytomegalovirus (HCMV) Infection of Endothelial Cells Promotes Naïve Monocyte Extravasation and Transfer of Productive Virus To Enhance Hematogenous Dissemination of HCMV
Authors: Bentz Gretchen L. , Jarquin-Pardo Marta , Chan Gary , Smith M. Shane , Sinzger Christian , Yurochko Andrew D.
Published: November 2006

Human cytomegalovirus (HCMV) pathogenesis is dependent on the hematogenous spread of the virus to host tissue. While data suggest that infected monocytes are required for viral dissemination from the blood to the host organs, infected endothelial cells are also thought to contribute to this key step in viral pathogenesis. We show here that HCMV infection of endothelial cells increased the recruitment and transendothelial migration of monocytes. Infection of endothelial cells promoted the increased surface expression of cell adhesion molecules (intercellular cell adhesion molecule 1, vascular cell adhesion molecule 1, E-selectin, and platelet endothelial cell adhesion molecule 1), which were necessary for the recruitment of naïve monocytes to the apical surface of the endothelium and for the migration of these monocytes through the endothelial cell layer. As a mechanism to account for the increased monocyte migration, we showed that HCMV infection of endothelial cells increased the permeability of the endothelium. The cellular changes contributing to the increased permeability and increased naïve monocyte transendothelial migration include the disruption of actin stress fiber formation and the decreased expression of lateral junction proteins (occludin and vascular endothelial cadherin). Finally, we showed that the migrating monocytes were productively infected with the virus, documenting that the virus was transferred to the migrating monocyte during passage through the lateral junctions. Together, our results provide evidence for an active role of the infected endothelium in HCMV dissemination and pathogenesis.
Inhibition of \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{tRNA}_{3}^{\mathrm{Lys}}\) \end{document}-Primed Reverse Transcription by Human APOBEC3G during Human Immunodeficiency Virus Type 1 Replication
Authors: Guo Fei , Cen Shan , Niu Meijuan , Saadatmand Jenan , Kleiman Lawrence
Published: November 2006

Cells are categorized as being permissive or nonpermissive according to their ability to produce infectious human immunodeficiency virus type 1 (HIV-1) lacking the viral protein Vif. Nonpermissive cells express the human cytidine deaminase APOBEC3G (hA3G), and Vif has been shown to bind to APOBEC3G and facilitate its degradation. Vif-negative HIV-1 virions produced in nonpermissive cells incorporate hA3G and have a severely reduced ability to produce viral DNA in newly infected cells. While it has been proposed that the reduction in DNA production is due to hA3G-facilitated deamination of cytidine, followed by DNA degradation, we provide evidence here that a decrease in the synthesis of the DNA by reverse transcriptase may account for a significant part of this reduction. During the infection of cells with Vif-negative HIV-1 produced from 293T cells transiently expressing hA3G, much of the inhibition of early (≥50% reduction) and late (≥95% reduction) viral DNA production, and of viral infectivity (≥95% reduction), can occur independently of DNA deamination. The inhibition of the production of early minus-sense strong stop DNA is also correlated with a similar inability of tRNA
3
Lys
to prime reverse transcription. A similar reduction in tRNA
3
Lys
priming and viral infectivity is also seen in the naturally nonpermissive cell H9, albeit at significantly lower levels of hA3G expression.
Recombination Patterns in Aphthoviruses Mirror Those Found in Other Picornaviruses
Authors: Heath Livio , van der Walt Eric , Varsani Arvind , Martin Darren P.
Published: November 2006

Foot-and-mouth disease virus
(FMDV) is thought to evolve largely through genetic drift driven by the inherently error-prone nature of its RNA polymerase. There is, however, increasing evidence that recombination is an important mechanism in the evolution of these and other related picornoviruses. Here, we use an extensive set of recombination detection methods to identify 86 unique potential recombination events among 125 publicly available FMDV complete genome sequences. The large number of events detected between members of different serotypes suggests that horizontal flow of sequences among the serotypes is relatively common and does not incur severe fitness costs. Interestingly, the distribution of recombination breakpoints was found to be largely nonrandom. Whereas there are clear breakpoint cold spots within the structural genes, two statistically significant hot spots precisely separate these from the nonstructural genes. Very similar breakpoint distributions were found for other picornovirus species in the genera
Enterovirus
and
Teschovirus
. Our results suggest that genome regions encoding the structural proteins of both FMDV and other picornaviruses are functionally interchangeable modules, supporting recent proposals that the structural and nonstructural coding regions of the picornaviruses are evolving largely independently of one another.
The NPro Product of Bovine Viral Diarrhea Virus Inhibits DNA Binding by Interferon Regulatory Factor 3 and Targets It for Proteasomal Degradation
Authors: Hilton Louise , Moganeradj Kartykayan , Zhang Gang , Chen Yun-Hsiang , Randall Richard E. , McCauley John W. , Goodbourn Stephen
Published: November 2006

Bovine viral diarrhea virus
(BVDV) is a pestivirus that can establish a persistent infection in the developing fetus and has the ability to disable the production of type I interferon. In this report, we extend our previous observations that BVDV encodes a protein able to specifically block the activity of interferon regulatory factor 3 (IRF-3), a transcription factor essential for interferon promoter activation, by demonstrating that this is a property of the N-terminal protease fragment (NPro) of the BVDV polyprotein. Although BVDV infections cause relocalization of cellular IRF-3 from the cytoplasm to the nucleus early in infection, NPro blocks IRF-3 from binding to DNA. NPro has the additional property of targeting IRF-3 for polyubiquitination and subsequent destruction by cellular multicatalytic proteasomes. The autoprotease activity of NPro is not required for the inhibition of type I interferon induction or the targeting of IRF-3 for degradation.
Autographa californica
Multiple Nucleopolyhedrovirus Nucleocapsid Assembly Is Interrupted upon Deletion of the
38K
Gene
Authors: Wu Wenbi , Lin Tiehao , Pan Lijing , Yu Mei , Li Zhaofei , Pang Yi , Yang Kai
Published: November 2006

38K
(
ac98
) of
Autographa californica
multiple nucleopolyhedrovirus (Ac
M
NPV) is a highly conserved baculovirus gene whose function is unknown. To determine the role of
38K
in the baculovirus life cycle, a
38K
knockout bacmid containing the Ac
M
NPV genome was generated through homologous recombination in
Escherichia coli
. Furthermore, a
38K
repair bacmid was constructed by transposing the
38K
open reading frame with its native promoter region into the
polyhedrin
locus of the
38K
knockout bacmid. After transfection of these viruses into
Spodoptera frugiperda
cells, the
38K
knockout bacmid led to a defect in production of infectious budded virus, while the
38K
repair bacmid rescued this defect, allowing budded-virus titers to reach wild-type levels. Slot blot analysis indicated that
38K
deletion did not affect the levels of viral DNA replication. Subsequent immunoelectron-microscopic analysis revealed that masses of electron-lucent tubular structures containing the capsid protein VP39 were present in cells transfected with
38K
knockout bacmids, suggesting that nucleocapsid assembly was interrupted. In contrast, the production of normal nucleocapsids was restored when the
38K
knockout bacmid was rescued with a copy of
38K
. Recombinant virus that expresses 38K fused to green fluorescent protein as a visual marker was constructed to monitor protein transport and localization within the nucleus during infection. Fluorescence was first detected along the cytoplasmic periphery of the nucleus and subsequently localized to the center of the nucleus. These results demonstrate that 38K plays a role in nucleocapsid assembly and is essential for viral replication in the Ac
M
NPV life cycle.
Human Cytomegalovirus Blocks Tumor Necrosis Factor Alpha- and Interleukin-1β-Mediated NF-κB Signaling
Authors: Montag Christina , Wagner Jutta , Gruska Iris , Hagemeier Christian
Published: November 2006

NF-κB plays an important role in the early cellular response to pathogens by activating genes involved in inflammation, immune response, and cell proliferation and survival. NF-κB is also utilized by many viral pathogens, like human cytomegalovirus (HCMV), to activate their own gene expression programs, reflecting intricate roles for NF-κB in both antiviral defense mechanisms and viral physiology. Here we show that the NF-κB signaling pathway stimulated by proinflammatory cytokines tumor necrosis factor alpha (TNF-α) and interleukin-1β (IL-1β) becomes inhibited in HCMV-infected cells. The block to NF-κB signaling is first noticeable during the early phase of infection but is fully established only at later times. Biochemical and genetic evidence demonstrates that the viral inhibition of proinflammatory signaling by distinct cytokines occurs upstream of the convergence point of NF-κB-activating pathways, i.e., the IκB kinase complex, and that it is mediated via different mechanisms. Consistent with this, we further show that an HCMV variant that has lost the ability to downregulate TNF-α-induced NF-κB signaling also fails to downregulate surface expression of TNF receptor 1, thereby mechanistically linking the inhibition of TNF-α-induced NF-κB signaling by HCMV to TNF receptor targeting. Our data support a model whereby HCMV inhibits cytokine-induced NF-κB signaling at later times during infection, and we suggest that this contributes to the inhibition of the cell's antiviral defense program.
Effects of Picornavirus 3A Proteins on Protein Transport and GBF1-Dependent COP-I Recruitment
Authors: Wessels Els , Duijsings Daniël , Lanke Kjerstin H. W. , van Dooren Sander H. J. , Jackson Catherine L. , Melchers Willem J. G. , van Kuppeveld Frank J. M.
Published: November 2006

The 3A protein of the coxsackievirus B3 (CVB3), an enterovirus that belongs to the family of the picornaviruses, inhibits endoplasmic reticulum-to-Golgi transport. Recently, we elucidated the underlying mechanism by showing that CVB3 3A interferes with ADP-ribosylation factor 1 (Arf1)-dependent COP-I recruitment to membranes by binding and inhibiting the function of GBF1, a guanine nucleotide exchange factor that is required for the activation of Arf1 (E. Wessels et al., Dev. Cell 11:191-201, 2006). Here, we show that the 3A protein of poliovirus, another enterovirus, is also able to interfere with COP-I recruitment through the same mechanism. No interference with protein transport or COP-I recruitment was observed for the 3A proteins of any of the other picornaviruses tested here (human rhinovirus [HRV], encephalomyocarditis virus, foot-and-mouth disease virus, and hepatitis A virus). We show that the 3A proteins of HRV, which are the most closely related to the enteroviruses, are unable to inhibit COP-I recruitment, due to a reduced ability to bind GBF1. When the N-terminal residues of the HRV 3A proteins are replaced by those of CVB3 3A, chimeric proteins are produced that have gained the ability to bind GBF1 and, by consequence, to inhibit protein transport. These results show that the N terminus of the CVB3 3A protein is important for binding of GBF1 and its transport-inhibiting function. Taken together, our data demonstrate that the activity of the enterovirus 3A protein to inhibit GBF1-dependent COP-I recruitment is unique among the picornaviruses.
RhoA-GTPase Facilitates Entry of Kaposi's Sarcoma-Associated Herpesvirus into Adherent Target Cells in a Src-Dependent Manner
Authors: Veettil Mohanan Valiya , Sharma-Walia Neelam , Sadagopan Sathish , Raghu Hari , Sivakumar Ramu , Naranatt Pramod P. , Chandran Bala
Published: November 2006

Kaposi's sarcoma-associated herpesvirus (KSHV) (human herpesvirus 8) binds to adherent target cell surface heparan sulfate molecules via its envelope glycoproteins gB and gpK8.1A, to integrins via gB, to the transporter CD98/xCT complex, and possibly to another molecule(s). This is followed by virus entry overlapping with the induction of preexisting host cell signal pathways, such as focal adhesion kinase, Src, phosphatidylinositol 3-kinase (PI3-K), Rho-GTPases, protein kinase C-ζ, and extracellular signal-regulated kinase 1/2. Here, using hemagglutinin-tagged plasmids expressing wild-type, dominant-positive, and dominant-negative forms of RhoA in HEK (human embryonic kidney) 293 cells, we investigated the role of RhoA-GTPase in virus entry. The dominant-negative form of RhoA GTPase and treatment of target cells with
Clostridium difficile
toxin B (CdTxB), a specific inactivator of Rho-GTPases, significantly blocked KSHV entry. KSHV infection induced closely similar levels of FAK and PI3-K in all three cell types. In contrast, very strong Src activation was observed in KSHV-infected dominant-positive RhoA cells compared to wild-type cells, and only moderate Src activation was seen in dominant-negative cells. Inhibition of Src activation by CdTxB and reduction of RhoA activation by Src inhibitors suggest that KSHV-induced Src is involved in RhoA activation, which in turn is involved in a feedback-sustained activation of Src. Since the decreased entry in RhoA dominant-negative cells may be due to inefficient signaling downstream of RhoA, we examined the induction of RhoA-activated Dia-2, which is also known to induce Src. Dia-2 coimmunoprecipitated with activated Src, which was inhibited by Src inhibitors, in the infected cells. Together with the reduced virus entry in RhoA dominant-negative cells, these results suggest that activated RhoA-dependent Dia-2 probably functions as a link between RhoA and Src in KSHV-infected cells, mediating the sustained Src activation, and that KSHV-induced Src and RhoA play roles in facilitating entry into adherent target cells.
In Vitro and In Vivo Fitness of Respiratory Syncytial Virus Monoclonal Antibody Escape Mutants
Authors: Zhao Xiaodong , Liu Enmei , Chen Fu-Ping , Sullender Wayne M.
Published: November 2006

Respiratory syncytial virus (RSV) is the only infectious disease for which a monoclonal antibody (MAb) is used in humans. Palivizumab (PZ) is a humanized murine MAb to the F protein of RSV. PZ-resistant viruses appear after in vitro and in vivo growth of RSV in the presence of PZ. Fitness for replication could be a determinant of the likelihood of dissemination of resistant viruses. We assessed the fitness of two PZ-resistant viruses (F212 and MP4). F212 grew less well in cell culture than the parent A2 virus and was predicted to be less fit than A2. Equal amounts of F212 and A2 were mixed and passaged in cell culture. F212 disappeared from the viral population, indicating it was less fit than the A2 virus. The MP4 virus grew as well as A2 in culture and in cotton rats. A2/MP4 virus input ratios of 1:1, 10:1, 100:1, and 1,000:1 were compared in competitive replication. For all input ratios except 1,000:1, the MP4 virus became dominant, supplanting the A2 virus. The MP4 virus also dominated the A2 virus during growth in cotton rats. Thus, the mutant MP4 virus was more fit than A2 virus in both in vitro and in vivo competitive replication. Whether this fitness difference was due to the identified nucleotide substitutions in the F gene or to mutations elsewhere in the genome is unknown. Understanding the mechanisms by which mutant virus fitness increased or decreased could prove useful for consideration in attenuated vaccine design efforts.
Comparison of Avian and Human Influenza A Viruses Reveals a Mutational Bias on the Viral Genomes
Authors: Rabadan Raul , Levine Arnold J. , Robins Harlan
Published: November 2006

In the last few years, the genomic sequence data for thousands of influenza A virus strains, including the 1918 pandemic strain, and hundreds of isolates of the avian influenza virus H5N1, which is causing an increasing number of human fatalities, have become publicly available. This large quantity of sequence data allows us to do comparative genomics with the human and avian versions of the virus. We find that the nucleotide compositions of influenza A viruses infecting the two hosts are sufficiently different that we can determine the host at almost 100% accuracy. This assignment works at the segment level, which allows us to construct the reassortment history of individual segments within each strain. We suggest that the different nucleotide compositions can be explained by a host-dependent mutation bias. To support this idea, we estimate the fixation rates for the different polymerase segments and the ratios of synonymous to nonsynonymous changes. Additionally, we provide evidence supporting the hypothesis that the H1N1 influenza virus entered the human population just prior to the 1918 outbreak, with an earliest bound of 1910.
Cellular Immune Responses in Children and Adults Receiving Inactivated or Live Attenuated Influenza Vaccines
Authors: He Xiao-Song , Holmes Tyson H. , Zhang Caiqiu , Mahmood Kutubuddin , Kemble George W. , Lewis David B. , Dekker Cornelia L. , Greenberg Harry B. , Arvin Ann M.
Published: November 2006

The patterns of cellular immune responses induced by live attenuated influenza vaccine (LAIV) versus those of the trivalent inactivated influenza vaccine (TIV) have not been studied extensively, especially in children. The goals of this study were to evaluate the effects of TIV and LAIV immunization on cellular immunity to live influenza A virus in children and adults and to explore factors associated with variations in responses to influenza vaccines among individuals. A gamma interferon (IFN-γ) flow cytometry assay was used to measure IFN-γ-producing (IFN-γ+) NK and T cells in peripheral blood mononuclear cell cultures stimulated with a live influenza A virus strain before and after LAIV or TIV immunization of children and adults. The mean percentages of influenza A virus-specific IFN-γ+CD4 and CD8 T cells increased significantly after LAIV, but not TIV, immunization in children aged 5 to 9 years. No increases in the mean levels of influenza A virus-reactive IFN-γ+T cells and NK cells were observed in adults given LAIV or TIV. TIV induced a significant increase in influenza A virus-reactive T cells in 6-month- to 4-year-old children; LAIV was not evaluated in this age group. The postvaccination changes (n-fold) in the percentages of influenza A virus-reactive IFN-γ+T and NK cells in adults were highly variable and correlated inversely with the prevaccination percentages, in particular with that of the CD56dimNK cell subset. In conclusion, our findings identify age, type of vaccine, and prevaccination levels of immune reactivity to influenza A virus as factors significantly associated with the magnitude of cellular immune responses to influenza vaccines.
Generation of Filamentous Instead of Icosahedral Particles by Repression of African Swine Fever Virus Structural Protein pB438L
Authors: Epifano Carolina , Krijnse-Locker Jacomine , Salas María L. , Salas José , Rodríguez Javier M.
Published: November 2006

The mechanisms involved in the construction of the icosahedral capsid of the African swine fever virus (ASFV) particle are not well understood at present. Capsid formation requires protein p72, the major capsid component, but other viral proteins are likely to play also a role in this process. We have examined the function of the ASFV structural protein pB438L, encoded by gene B438L, in virus morphogenesis. We show that protein pB438L associates with membranes during the infection, behaving as an integral membrane protein. Using a recombinant ASFV that inducibly expresses protein pB438L, we have determined that this structural protein is essential for the formation of infectious virus particles. In the absence of the protein, the virus assembly sites contain, instead of icosahedral particles, large aberrant tubular structures of viral origin as well as bilobulate forms that present morphological similarities with the tubules. The filamentous particles, which possess an aberrant core shell domain and an inner envelope, are covered by a capsid-like layer that, although containing the major capsid protein p72, does not acquire icosahedral morphology. This capsid, however, is to some extent functional, as the filamentous particles can move from the virus assembly sites to the plasma membrane and exit the cell by budding. The finding that, in the absence of protein pB438L, the viral particles formed have a tubular structure in which the icosahedral symmetry is lost supports a role for this protein in the construction or stabilization of the icosahedral vertices of the virus particle.
The Epstein-Barr Virus-Encoded LMP-1 Oncoprotein Negatively Affects Tyk2 Phosphorylation and Interferon Signaling in Human B Cells
Authors: Geiger Timothy R. , Martin Jennifer M.
Published: November 2006

Epstein-Barr virus (EBV) establishes a persistent infection in the human host and is associated with a variety of human cancers. Persistent infection results from a balance between the host immune response and viral immune evasion mechanisms. EBV infection is controlled initially by the innate immune response and later by T-cell-mediated adaptive immunity. EBV has evolved mechanisms to evade the host immune response so that it can persist for the lifetime of the host. Latent membrane protein 1 (LMP-1) is the EBV oncoprotein essential for B-cell immortalization by EBV. We show here that LMP-1 interacts with Tyk2, a signaling intermediate in the alpha interferon (IFN-α) signaling pathway, via a previously uncharacterized LMP-1 signaling domain. LMP-1 prevents Tyk2 phosphorylation and inhibits IFN-α-stimulated STAT2 nuclear translocation and interferon-stimulated response element transcriptional activity. Long-term culture of EBV+lymphoblastoid cells in IFN-α is associated with outgrowth of a population expressing elevated LMP-1 protein levels, suggesting that cells expressing higher levels of LMP-1 survive the antiproliferative selective pressure imposed by IFN-α. These results show that LMP-1 can protect EBV+cells from the IFN-α-stimulated antiviral/antiproliferative response and suggest that chronic IFN-α treatment may encourage the outgrowth of cells expressing elevated, and therefore potentially oncogenic, LMP-1 levels in EBV+individuals.
Genomic Sequence of
Spodoptera frugiperda Ascovirus 1a
, an Enveloped, Double-Stranded DNA Insect Virus That Manipulates Apoptosis for Viral Reproduction
Authors: Bideshi Dennis K. , Demattei Marie-Véronique , Rouleux-Bonnin Florence , Stasiak Karine , Tan Yeping , Bigot Sylvie , Bigot Yves , Federici Brian A.
Published: November 2006

Ascoviruses (family
Ascoviridae
) are double-stranded DNA viruses with circular genomes that attack lepidopterans, where they produce large, enveloped virions, 150 by 400 nm, and cause a chronic, fatal disease with a cytopathology resembling that of apoptosis. After infection, host cell DNA is degraded, the nucleus fragments, and the cell then cleaves into large virion-containing vesicles. These vesicles and virions circulate in the hemolymph, where they are acquired by parasitic wasps during oviposition and subsequently transmitted to new hosts. To develop a better understanding of ascovirus biology, we sequenced the genome of the type species
Spodoptera frugiperda ascovirus 1a
(SfAV-1a). The genome consisted of 156,922 bp, with a G+C ratio of 49.2%, and contained 123 putative open reading frames coding for a variety of enzymes and virion structural proteins, of which tentative functions were assigned to 44. Among the most interesting enzymes, due to their potential role in apoptosis and viral vesicle formation, were a caspase, a cathepsin B, several kinases, E3 ubiquitin ligases, and especially several enzymes involved in lipid metabolism, including a fatty acid elongase, a sphingomyelinase, a phosphate acyltransferase, and a patatin-like phospholipase. Comparison of SfAV-1a proteins with those of other viruses showed that 10% were orthologs of
Chilo
iridescent virus proteins, the highest correspondence with any virus, providing further evidence that ascoviruses evolved from a lepidopteran iridovirus. The SfAV-1a genome sequence will facilitate the determination of how ascoviruses manipulate apoptosis to generate the novel virion-containing vesicles characteristic of these viruses and enable study of their origin and evolution.
Common and Specific Properties of Herpesvirus UL34/UL31 Protein Family Members Revealed by Protein ComplementationAssay
Authors: Schnee Margit , Ruzsics Zsolt , Bubeck Anja , Koszinowski Ulrich H.
Published: November 2006

The proteins encoded by the UL34 and UL31 genes of herpes simplex virus are conserved among herpesviruses. They form a complex that is essential for the egress of the herpesvirus nucleocapsids from the nucleus. In previous work on the homologous protein complex in murine cytomegalovirus (MCMV), we defined their mutual binding domains. Here, we started to map binding domains within the UL34/UL31 proteins of alpha-, beta-, and gammaherpesviruses and to locate other functional properties. A protein complementation assay (PCA) using the TEM-1 β-lactamase fragments fused to UL31 and UL34 protein homologues was used to study protein-protein interactions in cells. Wild-type MCMV M50 and M53 provided a strong reaction in the PCA, whereas mutants unable to form a complex did not. The homologous pairs of herpes simplex virus type 1, pseudorabies virus, human cytomegalovirus (HCMV), Epstein-Barr virus (EBV), and murine herpes virus 68 proteins also reacted, with the exception of the EBV proteins. Cross-complementation was found to be positive only within the same herpesvirus subfamily. Moreover, the HCMV homologues rescued replication-defective MCMV genomes lacking one or the other gene. We identified the binding site of M53 for M50 in the first conserved region (CR1) (M. Loetzerich, Z. Ruzsics, and U. H. Koszinowski, J. Virol. 80:73-84). Here we show that the CR1 of all tested UL31 proteins contains the UL34 binding site, and chimeric proteins carrying the subfamily-specific CR1 rescued the ability to cross-complement in the PCA.
Evidence that the Immediate-Early Gene Product ICP4 Is Necessary for the Genome of the Herpes Simplex Virus Type 1 ICP4 Deletion Mutant Strain
d
120 To Circularize in Infected Cells
Authors: Su Ying-Hsiu , Zhang Xianchao , Wang Xiaohe , Fraser Nigel W. , Block Timothy M.
Published: November 2006

Following infection, the physical state of linear herpes simplex virus (HSV) genomes may change into an “endless” or circular form. In this study, using Southern blot analysis of the HSV genome, we provide evidence that immediate-early protein ICP4 is involved in the process of converting the linear HSV-1 ICP4-deleted mutant strain
d
120 genome into its endless form. Under conditions where de novo viral DNA synthesis was inhibited, the genome of the ICP4 deletion mutant
d
120 failed to assume an endless conformation following infection of Vero cells (compared with the ability of wild-type strain KOS). This defect was reversed in the Vero-derived cell line E5, which produces the ICP4 protein, suggesting that ICP4 is necessary and sufficient to complement the
d
120 defect. When ICP4 protein was provided by the replication-defective DNA polymerase mutant HP66, the genomes of mutant
d
120 could assume an endless conformation in Vero cells. Western blot analysis using antibody specific to the ICP4 protein showed that although the
d
120 virions contained ICP4 protein, the majority of that ICP4 protein was in a 40-kDa truncated form, with only a small fraction present as a full-length 175-kDa protein. When expression of ICP4 protein from E5 cells was inhibited by cycloheximide, the
d
120 virion-associated ICP4 protein was unable to mediate endless formation after infection of E5 cells. Collectively, these data suggest that ICP4 protein has an important role in mediating the endless formation of the HSV-1 genome upon infection and that this function can be provided in
trans
.
The Leading Edge of Stem Cell Therapeutics
Authors: Singec Ilyas , Jandial Rahul , Crain Andrew , Nikkhah Guido , Snyder Evan Y.
Published: November 2006

Stem cells, by virtue of their defining property of self-renewal, represent an unlimited source of potentially functional human cells for basic research and regenerative medicine. Having validated the feasibility of cell-based therapeutic strategies over the past decade, mostly through the use of rodent cells, the stem cell field has now embarked upon a detailed characterization of human cells. Recent progress has included improved cell culture conditions, long-term propagation, directed differentiation, and transplantation of both human embryonic and somatic stem cells. Continued progress in understanding basic human stem cell biology, combined with a better handle on the fundamental pathophysiology of human diseases one wishes to target (including the use of human stem cells in primate and other large animal models of human disease), should help to move this technology closer to clinical application.
Bringing the Courts Back In: Interbranch Perspectives on the Role of Courts in American Politics and Policy Making
Authors: Barnes Jeb
Published: November 2006

▪ Abstract  Understanding the role of law and courts in American politics and policy making is inherently complex. The dominant response to this problem has been to specialize narrowly in Supreme Court decision making. The problem is that American politics and policy making are inherently interactive and thus cannot be easily parsed into component parts. Consistent with this observation, a growing literature assumes that courts must be studied from an interbranch perspective, which holds that American politics and policy making emanate from interaction among overlapping and diversely representative forums. The result is studies that analyze how law and courts fit into broader political and policy-making processes and reveal the political significance of seemingly technical legal matters. By redescribing courts and judicial decision making in political terms, interbranch analysis not only enriches the study of judicial behavior but also promises to bring law and courts back into the mainstream of the study of American politics and public administration—where they belong.

[N]o skill in the science of government has yet been able to discriminate and define, with sufficient certainty, its three great provinces—the legislative, executive, and judiciary. … Questions daily occur in the course of practice which prove the obscurity which reigns in these subjects, and which puzzle the greatest adepts in political science.

James Madison, Federalist Paper No. 37 (1788 [1987], p. 244)
Prevention of angiotensin II-induced cardiac remodeling by angiotensin-(1–7)
Authors: Grobe Justin L. , Mecca Adam P. , Lingis Melissa , Shenoy Vinayak , Bolton Tonya A. , Machado Juline M. , Speth Robert C. , Raizada Mohan K. , Katovich Michael J.
Published: November 2006

Cardiac remodeling, which typically results from chronic hypertension or following an acute myocardial infarction, is a major risk factor for the development of heart failure and, ultimately, death. The renin-angiotensin system (RAS) has previously been established to play an important role in the progression of cardiac remodeling, and inhibition of a hyperactive RAS provides protection from cardiac remodeling and subsequent heart failure. Our previous studies have demonstrated that overexpression of angiotensin-converting enzyme 2 (ACE2) prevents cardiac remodeling and hypertrophy during chronic infusion of angiotensin II (ANG II). This, coupled with the knowledge that ACE2 is a key enzyme in the formation of ANG-(1–7), led us to hypothesize that chronic infusion of ANG-(1–7) would prevent cardiac remodeling induced by chronic infusion of ANG II. Infusion of ANG II into adult Sprague-Dawley rats resulted in significantly increased blood pressure, myocyte hypertrophy, and midmyocardial interstitial fibrosis. Coinfusion of ANG-(1–7) resulted in significant attenuations of myocyte hypertrophy and interstitial fibrosis, without significant effects on blood pressure. In a subgroup of animals also administered [d-Ala7]-ANG-(1–7) (A779), an antagonist to the reported receptor for ANG-(1–7), there was a tendency to attenuate the antiremodeling effects of ANG-(1–7). Chronic infusion of ANG II, with or without coinfusion of ANG-(1–7), had no effect on ANG II type 1 or type 2 receptor binding in cardiac tissue. Together, these findings indicate an antiremodeling role for ANG-(1–7) in cardiac tissue, which is not mediated through modulation of blood pressure or altered cardiac angiotensin receptor populations and may be at least partially mediated through an ANG-(1–7) receptor.
Smoking, Snuff Dipping and the Risk of Amyotrophic Lateral Sclerosis – A Prospective Cohort Study
Authors: Fang Fang , Bellocco Rino , Hernán Miguel A. , Ye Weimin
Published: November 2006

Background: Little is known about the etiology of amyotrophic lateral sclerosis (ALS). The association between cigarette smoking, but not other types of smoking and snuff dipping, and the risk of ALS has been evaluated in several epidemiologic studies. The findings were inconclusive. Methods: We studied the association of smoking and snuff dipping with the risk of ALS in the Swedish Construction Workers Cohort, which includes 280,558 male construction workers enrolled between 1978 and 1993 with detailed information on tobacco use. Incident cases of ALS were identified through cross-linkage to the Swedish Inpatient Register. Relative risks and their corresponding 95% confidence intervals (CIs) were estimated using the Cox proportional hazards regression model. Results: After a mean follow-up duration of 19.6 years, we identified 160 incident cases of ALS through 2004. Compared with non-tobacco use, the relative risk of ALS was 0.8 (95% CI 0.6–1.1) for tobacco smoking and 0.6 (95% CI 0.3–1.5) for snuff dipping, respectively. For tobacco smoking, further stratified analyses of smoking status or types of tobacco smoking did not reveal any excess risks in any strata. Conclusions: Our study provides no evidence that smoking or snuff dipping is associated with an increased ALS risk among men.
Glucocorticoid Receptor Expression in the Cortex of the Neonatal Rat Brain with and without Focal Cerebral Ischemia
Authors: Lee Ben H. , Wen Tong-Chun , Rogido Marta , Sola Augusto
Published: November 2006

Background/Aims: Glucocorticoid receptors (GR) mediate cellular processes which may be neuroprotective and/or neurotoxic to the neonatal rat brain. Our aim was to describe GR ontogeny in the developing rat brain cortex and changes in GR expression after permanent neonatal focal cerebral ischemia (FCI). Methods: GR Western blots and immunohistochemical stains were performed on neonatal rat cortices on P1, P3, P7, P10, P15, and P30 and on P7 at 1 h, 3 h, 6 h, 12 h, 24 h, and 72 h after FCI or sham-operation (S-O), 8 per group. Nissl staining was performed on FCI or S-O P7 cortical samples. Results: Cortical GR expression was increased by 65.2% at P7, 110.1% at P15, and 87.0% at P30, compared to P1. On P7, GR expression decreased in the ischemic cortex after 6 h and in the non-ischemic cortex after 24 h of FCI (p < 0.05). Cortical GR expression was not altered in S-O P7 rats. Immunohistochemistry supported Western blot findings. Nissl staining revealed no gross decrease in neuronal number in non-ischemic cortices after 24 h of FCI, compared to baseline. Conclusions: Neonatal rat cortical GR expression increases during P1 to P30, peaking at P15. At P7, cortical GR expression appears downregulated in the ischemic cortex after 6 h and in the non-ischemic cortex after 24 h of FCI. Thus, cortical GR may play important roles in normal brain development and neonatal brain injury responses.