bluuebunny/crossref_metadata_2025 huggingface dataset:
Structural Characterization of the UL25 DNA-Packaging Protein from Herpes Simplex Virus Type 1
Authors: Bowman Brian R. , Welschhans Robert L. , Jayaram Hariharan , Stow Nigel D. , Preston Valerie G. , Quiocho Florante A.
Published: February 2006

Herpesviruses replicate their double stranded DNA genomes as high-molecular-weight concatemers which are subsequently cleaved into unit-length genomes by a complex mechanism that is tightly coupled to DNA insertion into a preformed capsid structure, the procapsid. The herpes simplex virus type 1 UL25 protein is incorporated into the capsid during DNA packaging, and previous studies of a null mutant have demonstrated that its function is essential at the late stages of the head-filling process, either to allow packaging to proceed to completion or for retention of the viral genome within the capsid. We have expressed and purified an N-terminally truncated form of the 580-residue UL25 protein and have determined the crystallographic structure of the region corresponding to amino acids 134 to 580 at 2.1-Å resolution. This structure, the first for any herpesvirus protein involved in processing and packaging of viral DNA, reveals a novel fold, a distinctive electrostatic distribution, and a unique “flexible” architecture in which numerous flexible loops emanate from a stable core. Evolutionary trace analysis of UL25 and its homologues in other herpesviruses was used to locate potentially important amino acids on the surface of the protein, leading to the identification of four putative docking regions for protein partners.
Hierarchy among Viral RNA (vRNA) Segments in Their Role in vRNA Incorporation into Influenza A Virions
Authors: Muramoto Yukiko , Takada Ayato , Fujii Ken , Noda Takeshi , Iwatsuki-Horimoto Kiyoko , Watanabe Shinji , Horimoto Taisuke , Kida Hiroshi , Kawaoka Yoshihiro
Published: February 2006

The genome of influenza A viruses comprises eight negative-strand RNA segments. Although all eight segments must be present in cells for efficient viral replication, the mechanism(s) by which these viral RNA (vRNA) segments are incorporated into virions is not fully understood. We recently found that sequences at both ends of the coding regions of the HA, NA, and NS vRNA segments of A/WSN/33 play important roles in the incorporation of these vRNAs into virions. In order to similarly identify the regions of the PB2, PB1, and PA vRNAs of this strain that are critical for their incorporation, we generated a series of mutant vRNAs that possessed the green fluorescent protein gene flanked by portions of the coding and noncoding regions of the respective segments. For all three polymerase segments, deletions at the ends of their coding regions decreased their virion incorporation efficiencies. More importantly, these regions not only affected the incorporation of the segment in which they reside, but were also important for the incorporation of other segments. This effect was most prominent with the PB2 vRNA. These findings suggest a hierarchy among vRNA segments for virion incorporation and may imply intersegment association of vRNAs during virus assembly.
Glycosylation of the Severe Acute Respiratory Syndrome Coronavirus Triple-Spanning Membrane Proteins 3a and M
Authors: Oostra M. , de Haan C. A. M. , de Groot R. J. , Rottier P. J. M.
Published: February 2006

The severe acute respiratory syndrome coronavirus (SARS-CoV) open reading frame 3a protein has recently been shown to be a structural protein. The protein is encoded by one of the so-called group-specific genes and has no sequence homology with any of the known structural or group-specific proteins of coronaviruses. It does, however, have several similarities to the coronavirus M proteins; (i) they are triple membrane spanning with the same topology, (ii) they have similar intracellular localizations (predominantly Golgi), (iii) both are viral structural proteins, and (iv) they appear to interact with the E and S proteins, as well as with each other. The M protein plays a crucial role in coronavirus assembly and is glycosylated in all coronaviruses, either by N-linked or by O-linked oligosaccharides. The conserved glycosylation of the coronavirus M proteins and the resemblance of the 3a protein to them led us to investigate the glycosylation of these two SARS-CoV membrane proteins. The proteins were expressed separately using the vaccinia virus T7 expression system, followed by metabolic labeling. Pulse-chase analysis showed that both proteins were modified, although in different ways. While the M protein acquired cotranslationally oligosaccharides that could be removed by PNGaseF, the 3a protein acquired its modifications posttranslationally, and they were not sensitive to the
N
-glycosidase enzyme. The SARS-CoV 3a protein, however, was demonstrated to contain sialic acids, indicating the presence of oligosaccharides. O-glycosylation of the 3a protein was indeed confirmed using an in situ O-glycosylation assay of endoplasmic reticulum-retained mutants. In addition, we showed that substitution of serine and threonine residues in the ectodomain of the 3a protein abolished the addition of the O-linked sugars. Thus, the SARS-CoV 3a protein is an O-glycosylated glycoprotein, like the group 2 coronavirus M proteins but unlike the SARS-CoV M protein, which is N glycosylated.
Different De Novo Initiation Strategies Are Used by Influenza Virus RNA Polymerase on Its cRNA and Viral RNA Promoters during Viral RNA Replication
Authors: Deng Tao , Vreede Frank T. , Brownlee George G.
Published: February 2006

Various mechanisms are used by single-stranded RNA viruses to initiate and control their replication via the synthesis of replicative intermediates. In general, the same virus-encoded polymerase is responsible for both genome and antigenome strand synthesis from two different, although related promoters. Here we aimed to elucidate the mechanism of initiation of replication by influenza virus RNA polymerase and establish whether initiation of cRNA and viral RNA (vRNA) differed. To do this, two in vitro replication assays, which generated transcripts that had 5′ triphosphate end groups characteristic of authentic replication products, were developed. Surprisingly, mutagenesis screening suggested that the polymerase initiated pppApG synthesis internally on the model cRNA promoter, whereas it initiated pppApG synthesis terminally on the model vRNA promoter. The internally synthesized pppApG could subsequently be used as a primer to realign, by base pairing, to the terminal residues of both the model cRNA and vRNA promoters. In vivo evidence, based on the correction of a mutated or deleted residue 1 of a cRNA chloramphenicol acetyltransferase reporter construct, supported this internal initiation and realignment model. Thus, influenza virus RNA polymerase uses different initiation strategies on its cRNA and vRNA promoters. To our knowledge, this is novel and has not previously been described for any viral RNA-dependent RNA polymerase. Such a mechanism may have evolved to maintain genome integrity and to control the level of replicative intermediates in infected cells.
Distinct Viral Populations Differentiate and Evolve Independently in a Single Perennial Host Plant
Authors: Jridi Chiraz , Martin Jean-François , Marie-Jeanne Véronique , Labonne Gérard , Blanc Stéphane
Published: February 2006

The complex structure of virus populations has been the object of intensive study in bacteria, animals, and plants for over a decade. While it is clear that tremendous genetic diversity is rapidly generated during viral replication, the distribution of this diversity within a single host remains an obscure area in this field of science. Among animal viruses, only
Human immunodeficiency virus
and
Hepatitis C virus
populations have recently been thoroughly investigated at an intrahost level, where they are structured as metapopulations, demonstrating that the host cannot be considered simply as a “bag” containing a homogeneous or unstructured swarm of mutant viral genomes. In plants, a few reports suggested a possible heterogeneous distribution of virus variants at different locations within the host but provided no clues as to how this heterogeneity is structured. Here, we report the most exhaustive study of the structure and evolution of a virus population ever reported at the intrahost level through the analysis of a
Prunus
tree infected by
Plum pox virus
for over 13 years following a single inoculation event and by using analysis of molecular variance at different hierarchical levels combined with nested clade analysis. We demonstrate that, following systemic invasion of the host, the virus population differentiates into several distinct populations that are isolated in different branches, where they evolve independently through contiguous range expansion while colonizing newly formed organs. Moreover, we present and discuss evidence that the tree harbors a huge “bank” of viral clones, each isolated in one of the myriad leaves.
A Chromatin Insulator-Like Element in the Herpes Simplex Virus Type 1 Latency-Associated Transcript Region Binds CCCTC-Binding Factor and Displays Enhancer-Blocking and Silencing Activities
Authors: Amelio Antonio L. , McAnany Peterjon K. , Bloom David C.
Published: February 2006

A previous study demonstrated that the latency-associated transcript (LAT) promoter and the LAT enhancer/reactivation critical region (
rcr
) are enriched in acetyl histone H3 (K9, K14) during herpes simplex virus type 1 (HSV-1) latency, whereas all lytic genes analyzed (ICP0, UL54, ICP4, and DNA polymerase) are not (N. J. Kubat, R. K. Tran, P. McAnany, and D. C. Bloom, J. Virol.
78:
1139-1149, 2004). This suggests that the HSV-1 latent genome is organized into histone H3 (K9, K14) hyperacetylated and hypoacetylated regions corresponding to transcriptionally permissive and transcriptionally repressed chromatin domains, respectively. Such an organization implies that chromatin insulators, similar to those of cellular chromosomes, may separate distinct transcriptional domains of the HSV-1 latent genome. In the present study, we sought to identify
cis
elements that could partition the HSV-1 genome into distinct chromatin domains. Sequence analysis coupled with chromatin immunoprecipitation and luciferase reporter assays revealed that (i) the long and short repeats and the unique-short region of the HSV-1 genome contain clustered CTCF (CCCTC-binding factor) motifs, (ii) CTCF motif clusters similar to those in HSV-1 are conserved in other alphaherpesviruses, (iii) CTCF binds to these motifs on latent HSV-1 genomes in vivo, and (iv) a 1.5-kb region containing the CTCF motif cluster in the LAT region possesses insulator activities, specifically, enhancer blocking and silencing. The finding that CTCF, a cellular protein associated with chromatin insulators, binds to motifs on the latent genome and insulates the LAT enhancer suggests that CTCF may facilitate the formation of distinct chromatin boundaries during herpesvirus latency.
Selective Lymphocyte Depletion during the Early Stage of the Immune Response to Foot-and-Mouth Disease Virus Infection in Swine
Authors: Díaz-San Segundo Fayna , Salguero Francisco J. , de Avila Ana , Fernández de Marco M. Mar , Sánchez-Martín Miguel A. , Sevilla Noemí
Published: February 2006

Foot-and-mouth disease virus
(FMDV) is the causative agent of a highly contagious vesicular disease of cloven-hoofed animals. In the present study we use FMDV serotype C infection of swine to determine, by analytical techniques, the direct ex vivo visualization of virus-infected immune cells during the first 17 days of infection. We report, for the first time, that FMDV C-S8c1 can infect T and B cells at short periods of time postinoculation, corresponding with the peak of the viremia. There is a significant lymphopenia that involves CD3
+
CD4
−
CD8
+/−
, CD3
+
CD4
−
CD8
+
Tc, and CD3
+
CD4
+
CD8
+
memory Th but not CD3
+
CD4
+
CD8
−
naïve Th lymphocytes. In addition, a profound depletion of the vast majority of peripheral T cells in lymph nodes and spleen is observed. This selective depletion of T cells is not due mainly to in situ death via apoptosis as visualized by the terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL) technique. Thus, early infection of T cells by FMDV may be the main cause of the observed T-cell depletion. Importantly, this lack of T cells is reflected in a reduced response to mitogen activation, which in many cases is totally eliminated. These data suggest a mechanism by which the virus causes a transient immunosuppression, subvert the immune systems, and spreads. These results have important implications for our understanding of early events in the development of a robust immune response against FMDV.
Modeling and Estimation of Replication Fitness of Human Immunodeficiency Virus Type 1 In Vitro Experiments by Using a Growth Competition Assay
Authors: Wu Hulin , Huang Yangxin , Dykes Carrie , Liu Dacheng , Ma Jingming , Perelson Alan S. , Demeter Lisa M.
Published: February 2006

Growth competition assays have been developed to quantify the relative fitnesses of human immunodeficiency virus (HIV-1) mutants. In this article we develop mathematical models to describe viral/cellular dynamic interactions in the assay experiment, from which new competitive fitness indices or parameters are defined. These indices include the log fitness ratio (LFR), the log relative fitness (LRF), and the production rate ratio (PRR). From the population genetics perspective, we clarify the confusion and correct the inconsistency in the definition of relative fitness in the literature of HIV-1 viral fitness. The LFR and LRF are easier to estimate from the experimental data than the PRR, which was misleadingly defined as the relative fitness in recent HIV-1 research literature. Calculation and estimation methods based on two data points and multiple data points were proposed and were carefully studied. In particular, we suggest using both standard linear regression (method of least squares) and a measurement error model approach for more-accurate estimates of competitive fitness parameters from multiple data points. The developed methodologies are generally applicable to any growth competition assays. A user-friendly computational tool also has been developed and is publicly available on the World Wide Web at
http://www.urmc.rochester.edu/bstools/vfitness/virusfitness.htm
.
Expression of
Autographa californica
Multiple Nucleopolyhedrovirus Genes in Mammalian Cells and Upregulation of the Host
β-Actin
Gene
Authors: Fujita Ryosuke , Matsuyama Takahiro , Yamagishi Junya , Sahara Ken , Asano Shinichiro , Bando Hisanori
Published: February 2006

The gene expression of
Autographa californica
multiple nucleopolyhedrovirus (AcMNPV) was examined in two types of mammalian cells, human HeLa14 and hamster BHK cells. DNA microarray analysis followed by reverse transcription-PCR identified at least 12 viral genes transcribed in both HeLa14 cells and BHK cells inoculated with AcMNPV. 5′ rapid amplification of cDNA ends was carried out to examine the transcriptional fidelity of these genes in HeLa14 cells. The transcription of
ie-1
,
ie-0
and
gp64
was initiated at a baculovirus early gene motif, CAGT, accompanied by a TATA motif. In addition, the same splicing observed for
ie-0
mRNA in Sf9 cells occurred in HeLa14 cells. While the transcription initiation sites for
pe38
and
p6.9
were not located in the CAGT motif, most of them were in a typical eukaryotic RNA polymerase II promoter structure (a conventional TATA motif and/or an initiator). Interestingly, the expression of
β-actin
was upregulated in the mammalian cells inoculated with AcMNPV. Subsequent experiments using UV-inactivated virus confirmed the upregulation, suggesting that de novo synthesis of viral products is not required for the event. These results indicated that the AcMNPV genome acts as a template for transcription in mammalian cells through the usual infection pathway, though there is no evidence for the functional expression of viral genes at present.
A Mutation in the Human Immunodeficiency Virus Type 1 Gag Protein Destabilizes the Interaction of the Envelope Protein Subunits gp120 and gp41
Authors: Davis Melody R. , Jiang Jiyang , Zhou Jing , Freed Eric O. , Aiken Christopher
Published: February 2006

The Gag protein of human immunodeficiency virus type 1 (HIV-1) associates with the envelope protein complex during virus assembly. The available evidence indicates that this interaction involves recognition of the gp41 cytoplasmic tail (CT) by the matrix protein (MA) region of Pr55Gag. Here we show that substitution of Asp for Leu at position 49 (L49D) in MA results in a specific reduction in particle-associated gp120 without affecting the levels of gp41. Mutant virions were markedly reduced in single-cycle infectivity despite a relatively modest defect in fusion with target cells. Studies with HIV-1 particles containing decreased levels of envelope proteins suggested that the L49D mutation also inhibits a postentry step in infection. Truncation of the gp41 tail, or pseudotyping by vesicular stomatitis virus glycoprotein, restored both the fusion and infectivity of L49D mutant virions to wild-type levels. Truncation of gp41 also resulted in equivalent levels of gp120 on particles with and without the MA mutation and enhanced the replication of the L49D mutant virus in T cells. The impaired fusion and infectivity of L49D mutant particles were also complemented by a single point mutation in the gp41 CT that disrupted the tyrosine-containing endocytic motif. Our results suggest that an altered interaction between the MA domain of Gag and the gp41 cytoplasmic tail leads to dissociation of gp120 from gp41 during HIV-1 particle assembly, thus resulting in impaired fusion and infectivity.
Association between Hepatitis C Virus and Very-Low-Density Lipoprotein (VLDL)/LDL Analyzed in Iodixanol Density Gradients
Authors: Nielsen Søren U. , Bassendine Margaret F. , Burt Alastair D. , Martin Caroline , Pumeechockchai Wanna , Toms Geoffrey L.
Published: February 2006

Hepatitis C virus (HCV) RNA circulates in the blood of persistently infected patients in lipoviroparticles (LVPs), which are heterogeneous in density and associated with host lipoproteins and antibodies. The variability and lability of these virus-host complexes on fractionation has hindered our understanding of the structure of LVP and determination of the physicochemical properties of the HCV virion. In this study, HCV from an antibody-negative immunodeficient patient was analyzed using three fractionation techniques, NaBr gradients, isotonic iodixanol, and sucrose gradient centrifugation. Iodixanol gradients were shown to best preserve host lipoprotein-virus complexes, and all HCV RNA was found at densities below 1.13 g/ml, with the majority at low density, ≤1.08 g/ml. Immunoprecipitation with polyclonal antibodies against human ApoB and ApoE precipitated 91.8% and 95.0% of HCV with low density, respectively, suggesting that host lipoprotein is closely associated with HCV in a particle resembling VLDL. Immunoprecipitation with antibodies against glycoprotein E2 precipitated 25% of HCV with low density, providing evidence for the presence of E2 in LVPs. Treatment of serum with 0.5% deoxycholic acid in the absence of salt produced HCV with a density of 1.12 g/ml and a sedimentation coefficient of 215S. The diameters of these particles were calculated as 54 nm. Treatment of serum with 0.18% NP-40 produced HCV with a density of 1.18 g/ml, a sedimentation coefficient of 180S, and a diameter of 42 nm. Immunoprecipitation analysis showed that ApoB remained associated with HCV after treatment of serum with deoxycholic acid or NP-40, whereas ApoE was removed from HCV with these detergents.
MicroRNA-Guided Processing Impairs
Plum Pox Virus
Replication, but the Virus Readily Evolves To Escape This Silencing Mechanism
Authors: Simón-Mateo Carmen , García Juan Antonio
Published: February 2006

Since the discovery of microRNA (miRNA)-guided processing, a new type of RNA silencing, the possibility that such a mechanism could play a role in virus defense has been proposed. In this work, we have analyzed whether
Plum pox virus
(PPV) chimeras bearing miRNA target sequences (miR171, miR167, and miR159), which have been reported to be functional in
Arabidopsis
, were affected by miRNA function in three different host plants. Some of these PPV chimeras had clearly impaired infectivity compared with those carrying nonfunctional miRNA target sequences. The behaviors of PPV chimeras were similar but not identical in all the plants tested, and the deleterious effect on virus infectivity depended on the miRNA sequence cloned and on the site of insertion in the viral genome. The effect of the miRNA target sequence was drastically alleviated in transgenic plants expressing the silencing suppressor P1/HCPro. Furthermore, we show that virus chimeras readily escape RNA silencing interference through mutations within the miRNA target sequence, which mainly affected nucleotides matching the 5′-terminal region of the miRNA.
Potassium Ion Channels of Chlorella Viruses Cause Rapid Depolarization of Host Cells during Infection
Authors: Frohns Florian , Käsmann Anja , Kramer Detlef , Schäfer Britta , Mehmel Mario , Kang Ming , Van Etten James L. , Gazzarrini Sabrina , Moroni Anna , Thiel Gerhard
Published: February 2006

Previous studies have established that chlorella viruses encode K
+
channels with different structural and functional properties. In the current study, we exploit the different sensitivities of these channels to Cs
+
to determine if the membrane depolarization observed during virus infection is caused by the activities of these channels. Infection of
Chlorella
NC64A with four viruses caused rapid membrane depolarization of similar amplitudes, but with different kinetics. Depolarization was fastest after infection with virus SC-1A (half time [
t
1/2
], about 9 min) and slowest with virus NY-2A (
t
1/2
, about 12 min). Cs
+
inhibited membrane depolarization only in viruses that encode a Cs
+
-sensitive K
+
channel. Collectively, the results indicate that membrane depolarization is an early event in chlorella virus-host interactions and that it is correlated with viral-channel activity. This suggestion was supported by investigations of thin sections of
Chlorella
cells, which show that channel blockers inhibit virus DNA release into the host cell. Together, the data indicate that the channel is probably packaged in the virion, presumably in its internal membrane. We hypothesize that fusion of the virus internal membrane with the host plasma membrane results in an increase in K
+
conductance and membrane depolarization; this depolarization lowers the energy barrier for DNA release into the host.
Ataxia-Telangiectasia-Mutated (ATM) Protein Can Enhance Human Immunodeficiency Virus Type 1 Replication by Stimulating Rev Function
Authors: Ariumi Yasuo , Trono Didier
Published: February 2006

The ataxia-telangiectasia-mutated (ATM) kinase plays a central role in responses to various forms of DNA damage and has been suggested to facilitate human immunodeficiency virus type 1 (HIV-1) integration. Here, we describe a series of experiences that indicate that ATM can enhance HIV-1 replication by stimulating the action of the Rev viral posttranscriptional regulator. The Rev-dependent stimulation of viral late gene expression was observed with ATM-overexpressing cells, a result confirmed with a Rev-dependent reporter construct. Both parameters were also enhanced upon treatment of HeLa cells with caffeine, a xanthine that, in this cellular context, stimulates ATM activity. As well, decreased levels of virions with reduced infectivity were released by ATM knockdown cells. Notably, ATM overexpression did not stimulate the HIV-1 late gene expression within the context of Rev-independent constructs or the Rex-dependent production of capsid from human T-cell leukemia virus type 1 proviral constructs. Altogether, these results indicate that ATM can positively influence HIV-1 Rev function.
Genetic Association of the Antiviral Restriction Factor TRIM5α with Human Immunodeficiency Virus Type 1 Infection
Authors: Speelmon Emily C. , Livingston-Rosanoff Devon , Li Shuying Sue , Vu Quyen , Bui John , Geraghty Daniel E. , Zhao Lue Ping , McElrath M. Juliana
Published: February 2006

The innate antiviral factor TRIM5α restricts the replication of some retroviruses through its interaction with the viral capsid protein, leading to abortive infection. While overexpression of human TRIM5α results in modest restriction of human immunodeficiency virus type 1 (HIV-1), this inhibition is insufficient to block productive infection of human cells. We hypothesized that polymorphisms withinTRIM5may result in increased restriction of HIV-1 infection. We sequenced theTRIM5gene (excluding exon 5) and the 4.8-kb 5′ putative regulatory region in genomic DNA from 110 HIV-1-infected subjects and 96 exposed seronegative persons, along with targeted gene sequencing in a further 30 HIV-1-infected individuals. Forty-eight single nucleotide polymorphisms (SNPs), including 20 with allele frequencies of >1.0%, were identified. Among these were two synonymous and eight nonsynonymous coding polymorphisms. We observed no association betweenTRIM5polymorphism in HIV-1-infected subjects and their set-point viral load after acute infection, although oneTRIM5haplotype was weakly associated with more rapid CD4+T-cell loss. Importantly, aTRIM5haplotype containing the nonsynonymous SNP R136Q showed increased frequency among HIV-1-infected subjects relative to exposed seronegative persons, with an odds ratio of 5.49 (95% confidence interval = 1.83 to 16.45;P= 0.002). Nonetheless, we observed no effect of individual TRIM5α nonsynonymous mutations on the in vitro HIV-1 susceptibility of CD4+T cells. Therefore, any effect of TRIM5α polymorphism on HIV-1 infection in primary lymphocytes may depend on combinations of SNPs or on DNA sequences in linkage disequilibrium with the TRIM5α coding sequence.
Pausing during Reverse Transcription Increases the Rate of Retroviral Recombination
Authors: Lanciault Christian , Champoux James J.
Published: February 2006

Retroviruses package two copies of genomic RNA into viral particles. During the minus-sense DNA synthesis step of reverse transcription, the nascent DNA can transfer multiple times between the two copies of the genome, resulting in recombination. The mechanism for this process is similar to the process of obligate strand transfers mediated by the repeat and primer binding site sequences. The location at which the DNA 3′ terminus completely transfers to the second RNA strand defines the point of crossover. Previous work in vitro demonstrated that reverse transcriptase pausing has a significant impact on the location of the crossover, with a proportion of complete transfer events occurring very close to pause sites. The role of pausing in vivo, however, is not clearly understood. By employing a murine leukemia virus-based single-cycle infection assay, strong pausing was shown to increase the probability of recombination, as reflected in the reconstitution of green fluorescent protein expression. The infection assay results were directly correlated with the presence of strong pause sites in reverse transcriptase primer extension assays in vitro. Conversely, when pausing was diminished in vitro, without changing the sequence of the RNA template involved in recombination, there was a significant reduction in recombination in vivo. Together, these data demonstrate that reverse transcriptase pausing, as observed in vitro, directly correlates with recombination during minus-sense DNA synthesis in vivo.
A Novel Alternative Splicing Isoform of Human T-Cell Leukemia Virus Type 1 bZIP Factor (HBZ-SI) Targets Distinct Subnuclear Localization
Authors: Murata Ken , Hayashibara Toshihisa , Sugahara Kazuyuki , Uemura Akiko , Yamaguchi Taku , Harasawa Hitomi , Hasegawa Hiroo , Tsuruda Kazuto , Okazaki Toshiro , Koji Takehiko , Miyanishi Takayuki , Yamada Yasuaki , Kamihira Shimeru
Published: February 2006

Adult T-cell leukemia (ATL) is associated with prior infection with human T-cell leukemia virus type 1 (HTLV-1); however, the mechanism by which HTLV-1 causes adult T-cell leukemia has not been fully elucidated. Recently, a functional basic leucine zipper (bZIP) protein coded in the minus strand of HTLV-1 genome (HBZ) was identified. We report here a novel isoform of the HTLV-1 bZIP factor (HBZ), HBZ-SI, identified by means of reverse transcription-PCR (RT-PCR) in conjunction with 5′ and 3′ rapid amplification of cDNA ends (RACE). HBZ-SI is a 206-amino-acid-long protein and is generated by alternative splicing between part of the HBZ gene and a novel exon located in the 3′ long terminal repeat of the HTLV-1 genome. Consequently, these isoforms share >95% amino acid sequence identity, and differ only at their N termini, indicating that HBZ-SI is also a functional protein. Duplex RT-PCR and real-time quantitative RT-PCR analyses showed that the mRNAs of these isoforms were expressed at equivalent levels in all ATL cell samples examined. Nonetheless, we found by Western blotting that the HBZ-SI protein was preferentially expressed in some ATL cell lines examined. A key finding was obtained from the subcellular localization analyses of these isoforms. Despite their high sequence similarity, each isoform was targeted to distinguishable subnuclear structures. These data show the presence of a novel isoform of HBZ in ATL cells, and in addition, shed new light on the possibility that each isoform may play a unique role in distinct regions in the cell nucleus.
Preferential Infection of Mature Dendritic Cells by Mouse Hepatitis Virus Strain JHM
Authors: Zhou Haixia , Perlman Stanley
Published: February 2006

Mouse hepatitis virus strain JHM (MHV-JHM) causes acute encephalitis and acute and chronic demyelinating diseases in mice. Dendritic cells (DCs) are key cells in the initiation of innate and adaptive immune responses, and infection of these cells could potentially contribute to a dysregulated immune response; consistent with this, recent results suggest that DCs are readily infected by another strain of mouse hepatitis virus, the A59 strain (MHV-A59). Herein, we show that the JHM strain also productively infected DCs. Moreover, mature DCs were at least 10 times more susceptible than immature DCs to infection with MHV-JHM. DC function was impaired after MHV-JHM infection, resulting in decreased stimulation of CD8 T cells in vitro. Preferential infection of mature DCs was not due to differential expression of the MHV-JHM receptor CEACAM-1a on mature or immature cells or to differences in apoptosis. Although we could not detect infected DCs in vivo, both CD8
+
and CD11b
+
splenic DCs were susceptible to infection with MHV-JHM directly ex vivo. This preferential infection of mature DCs may inhibit the development of an efficient immune response to the virus.
Nature of Nonfunctional Envelope Proteins on the Surface of Human Immunodeficiency Virus Type 1
Authors: Moore Penny L. , Crooks Emma T. , Porter Lauren , Zhu Ping , Cayanan Charmagne S. , Grise Henry , Corcoran Paul , Zwick Michael B. , Franti Michael , Morris Lynn , Roux Kenneth H. , Burton Dennis R. , Binley James M.
Published: February 2006

Human immunodeficiency virus type 1 (HIV-1) neutralizing antibodies are thought be distinguished from nonneutralizing antibodies by their ability to recognize functional gp120/gp41 envelope glycoprotein (Env) trimers. The antibody responses induced by natural HIV-1 infection or by vaccine candidates tested to date consist largely of nonneutralizing antibodies. One might have expected a more vigorous neutralizing response, particularly against virus particles that bear functional trimers. The recent surprising observation that nonneutralizing antibodies can specifically capture HIV-1 may provide a clue relating to this paradox. Specifically, it was suggested that forms of Env, to which nonneutralizing antibodies can bind, exist on virus surfaces. Here, we present evidence that HIV-1 particles bear nonfunctional gp120/gp41 monomers and gp120-depleted gp41 stumps. Using a native electrophoresis band shift assay, we show that antibody-trimer binding predicts neutralization and that the nonfunctional forms of Env may account for virus capture by nonneutralizing antibodies. We hypothesize that these nonfunctional forms of Env on particle surfaces serve to divert the antibody response, helping the virus to evade neutralization.
Natural Killer Cell Function Is Well Preserved in Asymptomatic Human Immunodeficiency Virus Type 2 (HIV-2) Infection but Similar to That of HIV-1 Infection When CD4 T-Cell Counts Fall
Authors: Nuvor Samuel Victor , van der Sande Marianne , Rowland-Jones Sarah , Whittle Hilton , Jaye Assan
Published: February 2006

Natural killer (NK) cells are potent effectors of natural immunity and their activity prevents human immunodeficiency virus type 1 (HIV-1) viral entry and viral replication. We sought to determine whether NK immune responses are associated with different clinical course of HIV-1 and HIV-2 infections. A cross-sectional analysis of NK cell responses was undertaken in 30 HIV-1 and 30 HIV-2 subjects in each of three categories of CD4+-T-cell counts (>500, 200 to 500, and <200 cells/μl) and in 50 HIV-uninfected control subjects. Lytic activity and gamma interferon (IFN-γ) secretion were measured by chromium release and enzyme-linked immunospot assays, respectively. Flow cytometry was used to assess intracellular cytokines and chemokines. Levels of NK cytotoxicity were significantly higher in HIV-2 than in HIV-1 infections in subjects with high CD4+-T-cell counts and were similar to that of the healthy controls. In these HIV-2 subjects, cytolytic activity was positively correlated to NK cell count and inversely related to plasma viremia. Levels of intracellular MIP-1β, RANTES, tumor necrosis factor alpha, and IFN-γ produced by NK CD56brightcells were significantly higher in HIV-2- than HIV-1-infected subjects with high CD4+-T-cell counts but fell to similar levels as CD4 counts dropped. The data suggest efficient cytolytic and chemokine-suppressive activity of NK cells early in HIV-2 infection, which is associated with high CD4+T-cell counts. Enhancement of these functions may be important in immune-based therapy to control HIV disease.
Effect of Epitope Position on Neutralization by Anti-Human Immunodeficiency Virus Monoclonal Antibody 2F5
Authors: Ou Wu , Lu Ning , Yu Sloane S. , Silver Jonathan
Published: February 2006

The membrane-proximal region of the human immunodeficiency virus type 1 (HIV-1) transmembrane protein (TM) is critical for envelope (Env)-mediated membrane fusion and contains the target for broadly reactive neutralizing antibody 2F5. It has been proposed that 2F5 neutralization might involve interaction of its long, hydrophobic, complementarity-determining region (CDR) H3, with adjacent viral membrane. Using Moloney murine leukemia virus (MLV) as a tool, we examined the effect of epitope position on 2F5 neutralization. When the 2F5 epitope was inserted in the proline-rich region of MLV Env surface protein (SU), 2F5 blocked cell fusion and virus infection, whereas MLV with a hemagglutinin (HA) epitope at the same position was not neutralized by anti-HA, even though the antibodies bound their respective Envs on the surface of infected cells and viruses equally well. When the 2F5 epitope was inserted in the MLV Env TM at a position comparable to its natural position in HIV-1 TM, 2F5 antibody blocked Env-mediated cell fusion. Epitope position had subtle effects on neutralization by 2F5: the antibody concentration for 50% inhibition of cell fusion was more than 10-fold lower when the 2F5 epitope was in SU than in TM, and inhibition was less complete at high concentrations of antibody; we discuss possible explanations for these effects of epitope position. Since membrane proximity was not required for neutralization by 2F5 antibody, we speculate that the CDR H3 of 2F5 contributes to neutralization by destabilizing an adjacent protein rather than by inserting into an adjacent membrane.
Virus and Cell RNAs Expressed during Epstein-Barr Virus Replication
Authors: Yuan Jing , Cahir-McFarland Ellen , Zhao Bo , Kieff Elliott
Published: February 2006

Changes in Epstein-Barr virus (EBV) and cell RNA levels were assayed following immunoglobulin G (IgG) cross-linking-induced replication in latency 1-infected Akata Burkitt B lymphoblasts. EBV replication as assayed by membrane gp350 expression was ∼5% before IgG cross-linking and increased to more than 50% 48 h after induction. Seventy-two hours after IgG cross-linking, gp350-positive cells excluded propidium iodide as well as gp350-negative cells. EBV RNA levels changed temporally in parallel with previously defined sensitivity to inhibitors of protein or viral DNA synthesis. BZLF1 immediate-early RNA levels doubled by 2 h and reached a peak at 4 h, whereas BMLF1 doubled by 4 h with a peak at 8 h, and BRLF1 doubled by 8 h with peak at 12 h. Early RNAs peaked at 8 to 12 h, and late RNAs peaked at 24 h. Hybridization to intergenic sequences resulted in evidence for new EBV RNAs. Surprisingly, latency III (LTIII) RNAs for LMP1, LMP2, EBNALP, EBNA2, EBNA3A, EBNA3C, and BARTs were detected at 8 to 12 h and reached maxima at 24 to 48 h. EBNA2 and LMP1 were at full LTIII levels by 48 h and localized to gp350-positive cells. Thus, LTIII expression is a characteristic of late EBV replication in both B lymphoblasts and epithelial cells in immune-comprised people (J. Webster-Cyriaque, J. Middeldorp, and N. Raab-Traub, J. Virol.74:7610-7618, 2000). EBV replication significantly altered levels of 401 Akata cell RNAs, of which 122 RNAs changed twofold or more relative to uninfected Akata cells. Mitogen-activated protein kinase levels were significantly affected. Late expression of LTIII was associated with induction of NF-κB responsive genes including IκBα and A20. The exclusion of propidium, expression of EBV LTIII RNAs and proteins, and up-regulation of specific cell RNAs are indicative of vital cell function late in EBV replication.
Crucial Role of the 5′ Conserved Structure ofBamboo Mosaic VirusSatellite RNA in Downregulation of Helper Viral RNA Replication
Authors: Hsu Yau-Heiu , Chen Hsin-Chuan , Cheng Jeannie , Annamali Padmanaban , Lin Bin-Yen , Wu Chiang-Tai , Yeh Wen-Bin , Lin Na-Sheng
Published: February 2006

Satellite RNA ofBamboo mosaic virus(satBaMV), a single-stranded mRNA type satellite encoding a protein of 20 kDa (P20), depends on the helper BaMV for replication and encapsidation. Two satBaMV isolates, BSF4 and BSL6, exhibit distinctly differential phenotypes inNicotiana benthamianaplants when coinoculated with BaMV RNA. BSL6 significantly reduces BaMV RNA replication and suppresses the BaMV-induced symptoms, whereas BSF4 does not. By studies with chimeric satBaMVs generated by exchanging the components between BSF4 and BSL6, the genetic determinants responsible for the downregulation of BaMV replication and symptom expression were mapped at the 5′ untranslated region (UTR) of BSL6. The 5′ UTR of BSL6 alone is sufficient to diminish BaMV RNA replication when the 5′ UTR is inserted incisinto the BaMV expression vector or when coinoculation with mutants that block the synthesis of P20 protein takes place. Further, the 5′ UTR of natural satBaMV isolates contains one hypervariable (HV) region which folds into a conserved apical hairpin stem-loop (AHSL) structure (W. B. Yeh, Y. H. Hsu, H. C. Chen, and N. S. Lin, Virology 330:105-115, 2004). Interchanges of AHSL segment of HV regions between BSF4 and BSL6 led to the ability of chimeric satBaMV to interfere with BaMV replication and symptom expression. The conserved secondary structure within the HV region is a potent determinant of the downregulation of helper virus replication.
Human Herpesvirus 1
UL24
Gene Encodes a Potential PD-(D/E)XK Endonuclease
Authors: Kniżewski Łukasz , Kinch Lisa , Grishin Nick V. , Rychlewski Leszek , Ginalski Krzysztof
Published: February 2006

Using Meta-BASIC, a highly sensitive method for detection of distant similarity between proteins, we have identified another potential PD-(D/E)XK endonuclease in human herpesvirus 1 (HHV-1) encoded by the
UL24
gene. The universal presence of
UL24
in completed herpesviral genomes of three major subfamilies,
Alphaherpesvirinae
,
Betaherpesvirinae
, and
Gammaherpesvirinae
, suggests a fundamental role for this predicted PD-(D/E)XK endonuclease activity in the viral life cycle.
The Level of Reverse Transcriptase (RT) in Human Immunodeficiency Virus Type 1 Particles Affects Susceptibility to Nonnucleoside RT Inhibitors but Not to Lamivudine
Authors: Ambrose Zandrea , Julias John G. , Boyer Paul L. , KewalRamani Vineet N. , Hughes Stephen H.
Published: February 2006

We investigated the relationship between the level of reverse transcriptase (RT) in human immunodeficiency virus type 1 (HIV-1) particles and susceptibility to nonnucleoside reverse transcriptase inhibitors (NNRTIs). HIV-1 virions containing different active levels of RT were generated. Susceptibility to the NNRTIs efavirenz and nevirapine was inversely proportional to the level of enzymatically active RT. However, the sensitivity of HIV-1 to the nucleoside analog 3TC was not affected by the level of RT per particle. These data indicate that the susceptibility of HIV-1 to NNRTIs is influenced by RT activity.
Herpes Simplex Virus Tegument Protein VP16 Is a Component of Primary Enveloped Virions
Authors: Naldinho-Souto Raquel , Browne Helena , Minson Tony
Published: February 2006

Immunogold electron microscopy was used to determine whether the tegument proteins VP13/14, VP22, and VP16 of herpes simplex virus type 1 (HSV1) are components of primary enveloped virions. Whereas VP13/14 and VP22 were not detected in virus particles in the perinuclear space and were present in only mature extracellular virions, VP16 was acquired prior to primary envelopment of the virus at the inner nuclear membrane. This finding highlights potential similarities and differences between HSV1 and the related alphaherpesvirus, pseudorabies virus, in which the homologues of all three of these tegument proteins are not incorporated into the virion until secondary envelopment.
EpCAM and gpA33 are markers of Barrett’s metaplasia
Authors: Wong N A C S , Warren B F , Piris J , Maynard N , Marshall R , Bodmer W F
Published: February 2006

Aims:To characterise a specific and sensitive marker of Barrett’s metaplasia (BM).

Methods:Cases of normal oesophageal squamous mucosa (11 fresh endoscopic biopsies and 10 formalin fixed, paraffin embedded tissue blocks), BM (11 biopsies and 11 tissue blocks), and normal gastric body mucosa (five biopsies and five tissue blocks) were analysed using reverse transcriptase PCR, Western blotting, and immunohistochemistry for EpCAM, and reverse transcriptase PCR for gpA33.

Results:Strong EpCAM mRNA expression was detected in all the BM cases, in contrast to weak expression in all the normal gastric mucosal samples and no expression in any of the normal oesophageal mucosal samples tested. Strong gpA33 mRNA expression was detected in all the BM cases, in contrast to weak expression in a quarter of the normal gastric mucosal samples and no expression in any of the normal oesophageal mucosal samples tested. Western blotting showed EpCAM protein expression in all the BM cases and in none of the normal gastric or oesophageal mucosal samples tested. Immunohistochemistry showed strong EpCAM protein expression in BM and complete absence of expression in normal oesophageal squamous epithelium. Scattered EpCAM expressing cells were found in the gland bases of normal gastric body mucosa.

Conclusions:EpCAM protein and gpA33 mRNA expressions are specific and sensitive markers of BM.
Carriage of group B streptococcus in pregnant women from Oxford, UK
Authors: Jones N , Oliver K , Jones Y , Haines A , Crook D
Published: February 2006

Objective:To investigate asymptomatic vagino-rectal carriage of group B streptococcus (GBS) in pregnant women.

Methods:Women in the final trimester of pregnancy were recruited. A single vagino-rectal swab was taken, with consent, for culture of GBS. Two microbiological methods for isolation of GBS from vagino-ractal swabs were compared. The distribution of capsular serotypes of the GBS identified was determined. Epidemiological data for a subset (n = 167) of the pregnant women participating were examined.

Results:21.3% were colonised vagino-rectally with GBS. Risk factors for neonatal GBS disease (maternal fever, prolonged rupture of membranes, and preterm delivery) were present in 34 of 167 women (20.4%), and the presence of these factors correlated poorly with GBS carriage. Capsular serotypes III (26.4%), IA (25.8%), V (18.9%), and IB (15.7%) were prevalent in the GBS isolates. Selective broth culture of vagino-rectal swabs was superior to selective plate culture, but the combination of both methods was associated with increased detection of GBS (7.5%). An algorithm for the identification of GBS from vagino-rectal swabs was developed.

Conclusions:GBS carriage is prevalent in pregnant women in Oxfordshire, UK. The poor correlation between risk factors and GBS carriage requires further investigation in larger groups, given that the identification of these surrogate markers is recommended to guide administration of intrapartum antibiotic prophylaxis by the Royal College of Obstetricians of the UK. A selective broth culture detected more GBS carriers than a selective plate culture.
Relationship of p53, Bcl-2, Ki-67 index and E-cadherin expression in early invasive breast cancers with comedonecrosis as an accelerated apoptosis
Authors: Hosaka N , Ryu T , Cui W , Li Q , Nishida A , Miyake T , Takaki T , Inaba M , Ikehara S
Published: February 2006

Aims:To study the relationship between comedonecrosis formation and morphology, apoptosis, and p53, Bcl-2, Ki-67 index and E-cadherin expression in early invasive breast cancer.

Experimental design:Early invasive breast cancers were first divided into two groups according to the presence (CN+ tumours) or absence (CN− tumours) of comedonecrosis. The histological grade, apoptosis, and expression of E-cadherin, Ki-67, p53 and Bcl-2 in the cancer-affected area, and in normal ducts from the specimen, were then examined.

Results:Less tubule and gland formation was seen in CN+ tumours than in CN− tumours, although the histological grade between the groups was not different. During early comedonecrosis, cells undergo apoptosis and subsequent necrosis. p53 was higher in CN+ tumours than in CN− tumours and normal ducts, whereas Bcl-2 was lower in CN+ tumours than in CN− tumours and normal ducts. Both tumours had higher Ki-67 than in normal ducts, but no difference was evident between the tumours. CN+ tumours had slightly higher E-cadherin than that in CN− tumours, but lower than that in normal ducts. The level of comedonecrosis was positively correlated with p53, but inversely correlated with Bcl-2 in all tumours, and p53 and Bcl-2 were inversely correlated with each other. Furthermore, comedonecrosis and p53 were correlated with Ki-67 in CN+ tumours, and Bcl-2 was correlated with Ki-67 in CN− tumours.

Conclusion:Comedonecrosis may be actively regulated through an apoptotic procedure in massive cancers for their survival and progression, and the above proteins may be associated cooperatively in this process. CN+ and CN− tumours may have opposite proliferative systems under the p53–Bcl-2 pathway.