bluuebunny/crossref_metadata_2025 huggingface dataset:
Quorum-Sensing Signal Synthesis by theYersinia pestisAcyl-Homoserine Lactone Synthase YspI
Authors: Kirwan J. Paul , Gould Ty A. , Schweizer Herbert P. , Bearden Scott W. , Murphy Robert C. , Churchill Mair E. A.
Published: December 2005

The acyl-homoserine lactone molecular species (AHLs) produced by theYersinia pestisAHL synthase YspI were identified by biochemical and physical/chemical techniques. Bioassays of extracts from culture supernatants of the recombinant YspI and wild-typeYersinia pestisshowed similar profiles of AHLs. Analysis by liquid chromatography-mass spectrometry revealed that the predominant AHLs wereN-3-oxooctanoyl-l-homoserine lactone andN-3-oxo-hexanoyl-l-homoserine lactone.
Autorepression of RctB, an Initiator of
Vibrio cholerae
Chromosome II Replication
Authors: Egan Elizabeth S. , Duigou Stephane , Waldor Matthew K.
Published: December 2005

The RctB protein binds to the origin of replication of
Vibrio cholerae
chromosome II (chrII) and is required for

oriCII
Vc

-based replication. Here, we found that RctB acts as an autorepressor, inhibiting
rctB
transcription. Integration host factor promotes
rctB
transcription, while Dam and DnaA, factors required for replication of both
V. cholerae
chromosomes, influence RctB autorepression. Thus, RctB appears to regulate chrII replication as both an initiator and a transcription repressor, and its synthesis is modulated by factors that govern replication of both chromosomes.
Analysis of
hemF
Gene Function and Expression in
Rhodobacter sphaeroides
2.4.1
Authors: Zeilstra-Ryalls Jill H. , Schornberg Kathryn L.
Published: December 2005

The
hemF
gene of
Rhodobacter sphaeroides
2.4.1 is predicted to code for an oxygen-dependent coproporphyrinogen III oxidase. We found that a HemF
−
mutant strain is unable to grow under aerobic conditions. We also determined that
hemF
expression is controlled by oxygen, which is mediated, at least in part, by the response regulatory protein PrrA.
Whole-Genome Comparison betweenPhotorhabdusStrains To Identify Genomic Regions Involved in the Specificity of Nematode Interaction
Authors: Gaudriault S. , Duchaud E. , Lanois A. , Canoy A.-S. , Bourot S. , DeRose R. , Kunst F. , Boemare N. , Givaudan A.
Published: December 2005

The bacteriumPhotorhabdusestablishes a highly specific association withHeterorhabditis, its nematode host.Photorhabdusstrains associated withHeterorhabditis bacteriophoraorHeterorhabditis megidiswere compared using aPhotorhabdusDNA microarray. We describe 31 regions belonging to thePhotorhabdusflexible gene pool. Distribution analysis of regions among thePhotorhabdusgenus identified loci possibly involved in nematode specificity.
Low pH-Dependent Endosomal Processing of the Incoming Parvovirus Minute Virus of Mice Virion Leads to Externalization of the VP1 N-Terminal Sequence (N-VP1), N-VP2 Cleavage, and Uncoating of the Full-Length Genome
Authors: Mani Bernhard , Baltzer Claudia , Valle Noelia , Almendral José M. , Kempf Christoph , Ros Carlos
Published: December 2005

Minute virus of mice (MVM) enters the host cell via receptor-mediated endocytosis. Although endosomal processing is required, its role remains uncertain. In particular, the effect of low endosomal pH on capsid configuration and nuclear delivery of the viral genome is unclear. We have followed the progression and structural transitions of DNA full-virus capsids (FC) and empty capsids (EC) containing the VP1 and VP2 structural proteins and of VP2-only virus-like particles (VLP) during the endosomal trafficking. Three capsid rearrangements were detected in FC: externalization of the VP1 N-terminal sequence (N-VP1), cleavage of the exposed VP2 N-terminal sequence (N-VP2), and uncoating of the full-length genome. All three capsid modifications occurred simultaneously, starting as early as 30 min after internalization, and all of them were blocked by raising the endosomal pH. In particles lacking viral single-stranded DNA (EC and VLP), the N-VP2 was not exposed and thus it was not cleaved. However, the EC did externalize N-VP1 with kinetics similar to those of FC. The bulk of all the incoming particles (FC, EC, and VLP) accumulated in lysosomes without signs of lysosomal membrane destabilization. Inside lysosomes, capsid degradation was not detected, although the uncoated DNA of FC was slowly degraded. Interestingly, at any time postinfection, the amount of structural proteins of the incoming virions accumulating in the nuclear fraction was negligible. These results indicate that during the early endosomal trafficking, the MVM particles are structurally modified by low-pH-dependent mechanisms. Regardless of the structural transitions and protein composition, the majority of the entering viral particles and genomes end in lysosomes, limiting the efficiency of MVM nuclear translocation.
Varicella-Zoster Virus ORF63 Inhibits Apoptosis of Primary Human Neurons
Authors: Hood Chantelle , Cunningham Anthony L. , Slobedman Barry , Arvin Ann M. , Sommer Marvin H. , Kinchington Paul R. , Abendroth Allison
Published: December 2005

Virus-encoded modulation of apoptosis may serve as a mechanism to enhance cell survival and virus persistence. The impact of productive varicella-zoster virus (VZV) infection on apoptosis appears to be cell type specific, as infected human sensory neurons are resistant to apoptosis, yet human fibroblasts readily become apoptotic. We sought to identify the viral gene product(s) responsible for this antiapoptotic phenotype in primary human sensory neurons. Treatment with phosphonoacetic acid to inhibit viral DNA replication and late-phase gene expression did not alter the antiapoptotic phenotype, implicating immediate-early (IE) or early genes or a virion component. Compared to the parental VZV strain (rOKA), a recombinant virus unable to express one copy of the diploid IE gene ORF63 (rOkaΔORF63) demonstrated a significant induction of apoptosis in infected neurons, as determined by three methods: annexin V staining, deoxynucleotidyltransferase-mediated dUTP-biotin nick end label staining, and transmission electron microscopy. Furthermore, neurons transfected with a plasmid expressing ORF63 resisted apoptosis induced by nerve growth factor withdrawal. These results show that ORF63 can suppress apoptosis of neurons and provide the first identification of a VZV gene encoding an antiapoptotic function. As ORF63 is expressed in neurons during both productive and latent infection, it may play a significant role in viral pathogenesis by promoting neuron survival during primary and reactivated infections.
Interferon Regulatory Factor 3 Is Necessary for Induction of Antiviral Genes during Human Cytomegalovirus Infection
Authors: DeFilippis Victor R. , Robinson Bridget , Keck Thomas M. , Hansen Scott G. , Nelson Jay A. , Früh Klaus J.
Published: December 2005

Viral infection activates interferon regulatory factor 3 (IRF3), a cofactor for the induction of interferon-stimulated genes (ISGs). The role of IRF3 in the activation of ISGs by human cytomegalovirus (HCMV) is controversial despite the fact that HCMV has consistently been shown to induce ISGs during infection of fibroblasts. To address the function of IRF3 in HCMV-mediated ISG induction, we monitored ISG expression and global gene expression in HCMV-infected cells in which IRF3 function had been depleted by small interfering RNA or blocked by dominant negative IRF3. A specific reduction of ISG induction was observed, whereas other transcripts were unaffected. We therefore conclude that IRF3 specifically regulates ISG induction during the initial phase of HCMV infection.
Rescue of Recombinant Marburg Virus from cDNA Is Dependent on Nucleocapsid Protein VP30
Authors: Enterlein Sven , Volchkov Viktor , Weik Michael , Kolesnikova Larissa , Volchkova Valentina , Klenk Hans-Dieter , Mühlberger Elke
Published: December 2005

Here we report recovery of infectious Marburg virus (MARV) from a full-length cDNA clone. Compared to the wild-type virus, recombinant MARV showed no difference in terms of morphology of virus particles, intracellular distribution in infected cells, and growth kinetics. The nucleocapsid protein VP30 of MARV and Ebola virus (EBOV) contains a Zn-binding motif which is important for the function of VP30 as a transcriptional activator in EBOV, whereas its role for MARV is unclear. It has been reported previously that MARV VP30 is able to support transcription in an EBOV-specific minigenome system. When the Zn-binding motif was destroyed, MARV VP30 was shown to be inactive in the EBOV system. While it was not possible to rescue recombinant MARV when the VP30 plasmid was omitted from transfection, MARV VP30 with a destroyed Zn-binding motif and EBOV VP30 were able to mediate virus recovery. In contrast, rescue of recombinant EBOV was not supported by EBOV VP30 containing a mutated Zn-binding domain.
Mefloquine, an Antimalaria Drug with Antiprion Activity In Vitro, Lacks Activity In Vivo
Authors: Kocisko David A. , Caughey Byron
Published: December 2005

In view of the effectiveness of antimalaria drugs inhibiting abnormal protease-resistant prion protein (PrP-res) formation in scrapie agent-infected cells, we tested other antimalarial compounds for similar activity. Mefloquine (MF), a quinoline antimalaria drug, was the most active compound tested against RML and 22L mouse scrapie agent-infected cells, with 50% inhibitory concentrations of ∼0.5 and ∼1.2 μM, respectively. However, MF administered to mice did not delay the onset of intraperitoneally inoculated scrapie agent, the result previously observed with quinacrine. While most anti-scrapie agent compounds inhibit PrP-res formation in vitro, many PrP-res inhibitors have no activity in vivo. This underscores the importance of testing promising candidates in vivo.
Longitudinal Analysis of Human Immunodeficiency Virus Type 1
nef
/Long Terminal Repeat Sequences in a Cohort of Long-Term Survivors Infected from a Single Source
Authors: Churchill Melissa J. , Rhodes David I. , Learmont Jennifer C. , Sullivan John S. , Wesselingh Steven L. , Cooke Ian R. C. , Deacon Nicholas J. , Gorry Paul R.
Published: December 2005

We studied the evolution of human immunodeficiency virus type 1 (HIV-1) in a cohort of long-term survivors infected with an attenuated strain of HIV-1 acquired from a single source. Although the cohort members experienced differing clinical courses, we demonstrate similar evolution of HIV-1
nef
/long-terminal repeat (LTR) sequences, characterized by progressive sequence deletions tending toward a minimal
nef
/LTR structure that retains only sequence elements required for viral replication. The in vivo pathogenicity of attenuated HIV-1 is therefore dictated by viral and/or host factors other than those that impose a unidirectional selection pressure on the
nef
/LTR region of the HIV-1 genome.
ATP Hydrolysis and AMP Kinase Activities of Nonstructural Protein 2C of Human Parechovirus 1
Authors: Samuilova Olga , Krogerus Camilla , Fabrichniy Igor , Hyypiä Timo
Published: December 2005

The highly conserved picornavirus 2C proteins, thought to be involved in genome replication, contain three motifs found in NTPases/helicases of superfamily III. We report that human parechovirus 1 2C displays Mg
2+
-dependent ATP diphosphohydrolase activity in vitro, whereas other nucleoside triphosphates are not substrates for the hydrolysis. We also found that the 2C protein has an enzymatic activity that converts AMP to a corresponding diphosphate using ADP or ATP as a phosphate donor. In addition, we observed that ATP hydrolysis results in 2C autophosphorylation. These findings indicate that the parechovirus 2C protein has enzymatic activities, which may contribute to several functions in the viral replication cycle.
Nucleocytoplasmic Shuttling of Bovine Herpesvirus 1 UL47 Protein in Infected Cells
Authors: Verhagen Janneke , Hutchinson Ian , Elliott Gillian
Published: December 2005

Previous studies with transfected cells have shown that the herpes simplex virus type 1 (HSV-1) and bovine herpesvirus 1 (BHV-1) UL47 proteins shuttle between the nucleus and the cytoplasm. HSV-1 UL47 has also been shown to bind RNA. Here we examine the BHV-1 UL47 protein in infected cells using a green fluorescent protein-UL47-expressing virus. We show that UL47 is detected in the nucleus early in infection. We use fluorescence loss in photobleaching to show that nuclear UL47 undergoes rapid nucleocytoplasmic shuttling. Furthermore, we demonstrate that actinomycin D inhibits the reaccumulation of UL47 in the nuclei of infected cells. These results suggest that UL47 exhibits behavior similar to that of previously characterized RNA-transporting proteins.
Characterization of a Vaccinia Virus Mutant with a Deletion of the D10R Gene Encoding a Putative Negative Regulator of Gene Expression
Authors: Parrish Susan , Moss Bernard
Published: December 2005

The D9 and D10 proteins of vaccinia virus are 25% identical to each other, contain a
mutT
motif characteristic of nudix hydrolases, and are conserved in all sequenced poxviruses. Previous studies indicated that overexpression of D10 and, to a lesser extent, D9 decreased the levels of capped mRNAs and their translation products. Here, we further characterized the D10 protein and showed that only trace amounts are associated with purified virions and that it is expressed exclusively at late times after vaccinia virus infection. A viable deletion mutant (vΔD10) produced smaller plaques and lower virus yields than either wild-type virus or a D9R deletion mutant (vΔD9). Purified vΔD10 virions appeared normal by microscopic examination and biochemical analysis but produced 6- to 10-fold-fewer plaques at the same concentration as wild-type or vΔD9 virions. When 4 PFU per cell of wild-type or vΔD9 virions or equal numbers of vΔD10 virions were used for inoculation, nearly all cells were infected in each case, but viral early and late transcription was initiated more slowly in vΔD10-infected cells than in the others. However, viral early transcripts accumulated to higher levels in vΔD10-infected cells than in cells infected with the wild type or vΔD9. In addition, viral early and late mRNAs and cellular actin mRNA persisted longer in vΔD10-infected cells than in others. Furthermore, analysis of pulse-labeled proteins indicated prolonged synthesis of cellular and viral early proteins. These results are consistent with a role for D10 in regulating RNA levels in poxvirus-infected cells.
Evolution of Broad Host Range in Retroviruses Leads to Cell Death Mediated by Highly Cytopathic Variants
Authors: Rainey G. Jonah A. , Coffin John M.
Published: December 2005

The ability of many retroviruses to cause disease can be correlated to their cytopathic effect (CPE) in tissue culture characterized by an acute period of cell death and viral DNA accumulation. Here, we show that mutants of a subgroup B avian retrovirus (
Alpharetrovirus
) cause a very dramatic CPE in certain susceptible avian cells that is coincident with elevated levels of apoptosis, as measured by nuclear morphology, and persistent viral DNA accumulation. These mutants also have a broadly extended host range that includes rodent, cat, dog, monkey, and human cells (31). Previously, we have shown that the mutants exhibit diminished resistance to superinfection. The results presented here have important implications for the process of evolution of retroviruses to use distinct cellular receptors.
Sequence Homology Required by Human Immunodeficiency Virus Type 1 To Escape from Short Interfering RNAs
Authors: Sabariegos Rosario , Giménez-Barcons Mireia , Tàpia Natalia , Clotet Bonaventura , Martínez Miguel Angel
Published: December 2005

Short interfering RNAs (siRNAs) targeting viral or cellular genes can efficiently inhibit human immunodeficiency virus type 1 (HIV-1) replication. Nevertheless, the emergence of mutations in the gene being targeted could lead to the rapid escape from the siRNA. Here, we simulate viral escape by systematically introducing single-nucleotide substitutions in all 19 HIV-1 residues targeted by an effective siRNA. We found that all mutant viruses that were tested replicated better in the presence of the siRNA than in the presence of the wild-type virus. The antiviral activity of the siRNA was completely abolished by single substitutions in 10 (positions 4 to 11, 14, and 15) out of 16 positions tested (substitution at 3 of the 19 positions explored rendered nonviable viruses). With the exception of the substitution observed at position 12, substitutions at either the 5′ end or the 3′ end (positions 1 to 3, 16, and 18) were better tolerated by the RNA interference machinery and only in part affected siRNA inhibition. Our results show that optimal HIV-1 gene silencing by siRNA requires a complete homology within most of the target sequence and that substitutions at only a few positions at the 5′ and 3′ ends are partially tolerated.
The MC160 Protein Expressed by the Dermatotropic Poxvirus Molluscum Contagiosum Virus Prevents Tumor Necrosis Factor Alpha-Induced NF-κB Activation via Inhibition of I Kappa Kinase Complex Formation
Authors: Nichols Daniel Brian , Shisler Joanna L.
Published: December 2005

The pluripotent cytokine tumor necrosis factor alpha (TNF-α) binds to its cognate TNF receptor I (TNF-RI) to stimulate inflammation via activation of the NF-κB transcription factor. To prevent the detrimental effects of TNF-α in keratinocytes infected with the molluscum contagiosum virus (MCV), this poxvirus is expected to produce proteins that block at least one step of the TNF-RI signal transduction pathway. One such product, the MC160 protein, is predicted to interfere with this cellular response because of its homology to other proteins that regulate TNF-RI-mediated signaling. We report here that expression of MC160 molecules did significantly reduce TNF-α-mediated NF-κB activation in 293T cells, as measured by gene reporter and gel mobility shift assays. Since we observed that MC160 decreased other NF-κB activation pathways, namely those activated by receptor-interacting protein, TNF receptor-associated factor 2, NF-κB-inducing kinase, or MyD88, we hypothesized that the MC160 product interfered with I kappa kinase (IKK) activation, an event common to multiple signal transduction pathways. Indeed, MC160 protein expression was associated with a reduction in in vitro IKK kinase activity and IKK subunit phosphorylation. Further, IKK1-IKK2 interactions were not detected in MC160-expressing cells, under conditions demonstrated to induce IKK complex formation, but interactions between the MC160 protein and the major IKK subunits were undetectable. Surprisingly, MC160 expression correlated with a decrease in IKK1, but not IKK2 levels, suggesting a mechanism for MC160 disruption of IKK1-IKK2 interactions. MCV has probably retained its MC160 gene to inhibit NF-κB activation by interfering with signaling via multiple biological mediators. In the context of an MCV infection in vivo, MC160 protein expression may dampen the cellular production of proinflammatory molecules and enhance persistent infections in host keratinocytes.
Point Mutations Upstream of Hepatitis B Virus Core Gene Affect DNA Replication at the Step of Core Protein Expression
Authors: Guarnieri Michael , Kim Kyun-Hwan , Bang Genie , Li Jisu , Zhou Yonghong , Tang Xiaoli , Wands Jack , Tong Shuping
Published: December 2005

The pregenomic RNA directs replication of the hepatitis B virus (HBV) genome by serving both as the messenger for core protein and polymerase and as the genome precursor following its packaging into the core particle. RNA packaging is mediated by a stem-loop structure present at its 5′ end designated the ε signal, which includes the core gene initiator AUG. The precore RNA has a slightly extended 5′ end to cover the entire precore region and, consequently, directs the translation of a precore/core protein, which is secreted as e antigen (HBeAg) following removal of precore-derived signal peptide and the carboxyl terminus. A naturally occurring G1862T mutation upstream of the core AUG affects the bulge of the ε signal and generates a “forbidden” residue at the −3 position of the signal peptide cleavage site. Transfection of this and other mutants into human hepatoma cells failed to prove their inhibition of HBeAg secretion but rather revealed great impairment of genome replication. This replication defect was associated with reduced expression of core protein and could be overcome by a G1899A covariation, or by nonsense or frameshift mutation in the precore region. All these mutations antagonized the G1862T mutation on core protein expression. Cotransfection of the G1862T mutant with a replication-deficient HBV genome that provides core protein intransalso restored genome replication. Consistent with our findings in cell culture, HBV genotype A found in African/Asian patients has T1862 and is associated with much lower viremia titers than the European subgroup of genotype A.
Inhibition of Protease-Resistant Prion Protein Formation in a Transformed Deer Cell Line Infected with Chronic Wasting Disease
Authors: Raymond Gregory J. , Olsen Emily A. , Lee Kil Sun , Raymond Lynne D. , Bryant P. Kruger , Baron Gerald S. , Caughey Winslow S. , Kocisko David A. , McHolland Linda E. , Favara Cynthia , Langeveld Jan P. M. , van Zijderveld Fred G. , Mayer Richard T. , Miller Michael W. , Williams Elizabeth S. , Caughey Byron
Published: December 2005

Chronic wasting disease (CWD) is an emerging transmissible spongiform encephalopathy (prion disease) of North American cervids, i.e., mule deer, white-tailed deer, and elk (wapiti). To facilitate in vitro studies of CWD, we have developed a transformed deer cell line that is persistently infected with CWD. Primary cultures derived from uninfected mule deer brain tissue were transformed by transfection with a plasmid containing the simian virus 40 genome. A transformed cell line (MDB) was exposed to microsomes prepared from the brainstem of a CWD-affected mule deer. CWD-associated, protease-resistant prion protein (PrP
CWD
) was used as an indicator of CWD infection. Although no PrP
CWD
was detected in any of these cultures after two passes, dilution cloning of cells yielded one PrP
CWD
-positive clone out of 51. This clone, designated MDB
CWD
, has maintained stable PrP
CWD
production through 32 serial passes thus far. A second round of dilution cloning yielded 20 PrP
CWD
-positive subclones out of 30, one of which was designated MDB
CWD2
. The MDB
CWD2
cell line was positive for fibronectin and negative for microtubule-associated protein 2 (a neuronal marker) and glial fibrillary acidic protein (an activated astrocyte marker), consistent with derivation from brain fibroblasts (e.g., meningeal fibroblasts). Two inhibitors of rodent scrapie protease-resistant PrP accumulation, pentosan polysulfate and a porphyrin compound, indium (III) meso-tetra(4-sulfonatophenyl)porphine chloride, potently blocked PrP
CWD
accumulation in MDB
CWD
cells. This demonstrates the utility of these cells in a rapid in vitro screening assay for PrP
CWD
inhibitors and suggests that these compounds have potential to be active against CWD in vivo.
Restriction of Foamy Viruses by APOBEC Cytidine Deaminases
Authors: Delebecque Frédéric , Suspène Rodolphe , Calattini Sara , Casartelli Nicoletta , Saïb Ali , Froment Alain , Wain-Hobson Simon , Gessain Antoine , Vartanian Jean-Pierre , Schwartz Olivier
Published: December 2005

Foamy viruses (FVs) are nonpathogenic retroviruses infecting many species of mammals, notably primates, cattle, and cats. We have examined whether members of the apolipoprotein B-editing catalytic polypeptide-like subunit (APOBEC) family of antiviral cytidine deaminases restrict replication of simian FV. We show that human APOBEC3G is a potent inhibitor of FV infectivity in cell culture experiments. This antiviral activity is associated with cytidine editing of the viral genome. Both molecular FV clones and primary uncloned viruses were susceptible to APOBEC3G, and viral infectivity was also inhibited by murine and simian APOBEC3G homologues, as well as by human APOBEC3F. Wild-type and
bet
-deleted viruses were similarly sensitive to this antiviral activity, suggesting that Bet does not significantly counteract APOBEC proteins. Moreover, we did not detect FV sequences that may have been targeted by APOBEC in naturally infected macaques, but we observed a few G-to-A substitutions in humans that have been accidentally contaminated by simian FV. In infected hosts, the persistence strategy employed by FV might be based on low levels of replication, as well as avoidance of cells expressing large amounts of active cytidine deaminases.
Morphological Changes in the T=3 Capsid of Flock House Virus during Cell Entry
Authors: Walukiewicz Hanna E. , Johnson John E. , Schneemann Anette
Published: December 2005

We report the identification and characterization of a viral intermediate formed during infection of
Drosophila
cells with the nodavirus Flock House virus (FHV). We observed that even at a very low multiplicity of infection, only 70% of the input virus stayed attached to or entered the cells, while the remaining 30% of the virus eluted from cells after initial binding. The eluted FHV particles did not rebind to
Drosophila
cells and, thus, could no longer initiate infection by the receptor-mediated entry pathway. FHV virus-like particles with the same capsid composition as native FHV but containing cellular RNA also exhibited formation of eluted particles when incubated with the cells. A maturation cleavage-defective mutant of FHV, however, did not. Compared to naïve FHV particles, i.e., particles that had never been incubated with cells, eluted particles showed an acid-sensitive phenotype and morphological alterations. Furthermore, eluted particles had lost a fraction of the internally located capsid protein gamma. Based on these results, we hypothesize that FHV eluted particles represent an infection intermediate analogous to eluted particles observed for members of the family
Picornaviridae
.
Addition of N-Glycans in the Stalk of the Newcastle Disease Virus HN Protein Blocks Its Interaction with the F Protein and Prevents Fusion
Authors: Melanson Vanessa R. , Iorio Ronald M.
Published: December 2005

Most paramyxovirus fusion (F) proteins require the coexpression of the homologous attachment (HN) protein to promote membrane fusion, consistent with the existence of a virus-specific interaction between the two proteins. Analysis of the fusion activities of chimeric HN proteins indicates that the stalk region of the HN spike determines its F protein specificity, and analysis of a panel of site-directed mutants indicates that the F-interactive site resides in this region. Here, we use the addition of oligosaccharides to further explore the role of the HN stalk in the interaction with F. N-glycans were individually added at several positions in the stalk to determine their effects on the activities of HN, as well as its structure. N-glycan addition at positions 69 and 77 in the stalk specifically blocks fusion and the HN-F interaction without affecting either HN structure or its other activities. N-glycans added at other positions in the stalk modulate activities that reside in the globular head of HN. This correlates with an alteration of the tetrameric structure of the protein, as indicated by sucrose gradient sedimentation analyses. Finally, N-glycan addition in another region of HN (residues 124 to 152), predicted by a peptide-based analysis to mediate the interaction with F, does not significantly reduce the level of fusion, arguing strongly against this site being part of the F-interactive domain in HN. Our data support the idea that the F-interactive site on HN is defined by the stalk region of the protein.
Perturbations of Cell Cycle Control in T Cells Contribute to the Different Outcomes of Simian Immunodeficiency Virus Infection in Rhesus Macaques and Sooty Mangabeys
Authors: Paiardini M. , Cervasi B. , Sumpter B. , McClure H. M. , Sodora D. L. , Magnani M. , Staprans S. I. , Piedimonte G. , Silvestri G.
Published: December 2005

In contrast to human immunodeficiency virus (HIV) infection of humans and experimental simian immunodeficiency virus (SIV) infection of rhesus macaques (RMs), SIV infection of sooty mangabeys (SMs), a natural host African monkey species, is typically nonpathogenic and associated with preservation of CD4+T-cell counts despite chronic high levels of viral replication. In previous studies, we have shown that the lack of SIV disease progression in SMs is related to lower levels of immune activation and bystander T-cell apoptosis compared to those of pathogenic HIV/SIV infection (G. Silvestri, D. Sodora, R. Koup, M. Paiardini, S. O’Neil, H. M. McClure, S. I. Staprans, and M. B. Feinberg, Immunity 18:441-452, 2003; G. Silvestri, A. Fedanov, S. Germon, N. Kozyr, W. J. Kaiser, D. A. Garber, H. M. McClure, M. B. Feinberg, and S. I. Staprans, J. Virol. 79:4043-4054, 2005). In HIV-infected patients, increased T-cell susceptibility to apoptosis is associated with a complex cell cycle dysregulation (CCD) that involves increased activation of the cyclin B/p34-cdc2 complex and abnormal nucleolar structure with dysregulation of nucleolin turnover. Here we report that CCD is also present during pathogenic SIV infection of RMs, and its extent correlates with the level of immune activation and T-cell apoptosis. In marked contrast, naturally SIV-infected SMs show normal regulation of cell cycle control (i.e., normal intracellular levels of cyclin B and preserved nucleolin turnover) and a low propensity to apoptosis in both peripheral blood- and lymph node-derived T cells. The absence of significant CCD in the AIDS-free, non-immune-activated SMs despite high levels of viral replication indicates that CCD is a marker of disease progression during lentiviral infection and supports the hypothesis that the preservation of cell cycle control may help to confer the disease-resistant phenotype of SIV-infected SMs.
Dynamics of Hepatitis B Virus Resistance to Lamivudine
Authors: Pallier Coralie , Castéra Laurent , Soulier Alexandre , Hézode Christophe , Nordmann Patrice , Dhumeaux Daniel , Pawlotsky Jean-Michel
Published: December 2005

Lamivudine was the first approved inhibitor of hepatitis B virus (HBV) reverse transcriptase (RT). Lamivudine resistance develops in 53% to 76% of patients after 3 years of treatment. We extensively characterized the dynamics of HBV quasispecies variant populations in four HBV-infected patients who developed lamivudine resistance. Virological breakthrough was preceded by 2 to 4 months by the emergence of quasispecies variants bearing amino acid substitutions at RT position 204, i.e., within the YMDD catalytic motif (rtM204V/I). Three patients had a gradual switch from a YMDD variant population at baseline to a 100% lamivudine-resistant variant population, whereas the remaining patient had a fluctuating pattern of resistance variant dynamics. Careful analysis of amino acid substitutions located outside domain C of HBV RT, including those known to partially restore replication capacities in vitro, showed that the in vivo replication of HBV variants is driven by multiple forces, including intrinsic replicative advantages conferred by mutations accumulating outside domain C and the changing environment in which these variants replicate. Our findings also suggest that individual treatment optimization will require sensitive methods capable of detecting the emergence of viral resistance before the relevant variants acquire optimal replicative capacities.
The Transcription Profile of Aleutian Mink Disease Virus in CRFK Cells Is Generated by Alternative Processing of Pre-mRNAs Produced from a Single Promoter
Authors: Qiu Jianming , Cheng Fang , Burger Lisa R. , Pintel David
Published: December 2005

A reevaluation of the transcription profile of Aleutian mink disease parvovirus (AMDV)-infected CRFK cells at either 32°C or 37°C has determined that strain AMDV-G encodes six species of mRNAs produced by alternative splicing and alternative polyadenylation of a pre-mRNA generated by a single promoter at the left end of the genome. Three different splicing patterns are used, and each type is found polyadenylated at either the 3′ end of the genome (the distal site) or at a site in the center of the genome (the proximal site). All spliced species accumulate similarly over the course of infection, with the R2 RNA predominant throughout. The R2 RNA, which contains and can express the NS2 coding region, encodes the viral capsid proteins VP1 and VP2.
Expanded Tissue Targets for Foamy Virus Replication with Simian Immunodeficiency Virus-Induced Immunosuppression
Authors: Murray S. M. , Picker L. J. , Axthelm M. K. , Linial M. L.
Published: December 2005

Foamy viruses (FV) are the oldest known genus of retroviruses and have persisted in nonhuman primates for over 60 million years. FV are efficiently transmitted, leading to a lifelong nonpathogenic infection. Transmission is thought to occur through saliva, but the detailed mechanism is unknown. Interestingly, this persistent infection contrasts with the rapid cytopathicity caused by FV in vitro, suggesting a host defense against FV. To better understand the tissue specificity of FV replication and host immunologic defense against FV cytopathicity, we quantified FV in tissues of healthy rhesus macaques (RM) and those severely immunosuppressed by simian immunodeficiency virus (SIV). Contrary to earlier findings, we find that all immunocompetent animals consistently have high levels of viral RNA in oral tissues but not in other tissues examined, including the small intestine. Strikingly, abundant viral transcripts were detected in the small intestine of all of the SIV-infected RM, which has been shown to be a major site of SIV (and human immunodeficiency virus)-induced CD4
+
T-cell depletion. In contrast, there was a trend to lower viral RNA levels in oropharyngeal tissues of SIV-infected animals. The expansion of FV replication to the small intestine but not to other CD4
+
T-cell-depleted tissues suggests that factors other than T-cell depletion, such as dysregulation of the jejunal microenvironment after SIV infection, likely account for the expanded tissue tropism of FV replication.
Reovirus Variants Selected for Resistance to Ammonium Chloride Have Mutations in Viral Outer-Capsid Protein σ3
Authors: Clark Kimberly M. , Wetzel J. Denise , Gu Yingqi , Ebert Daniel H. , McAbee Stephanie A. , Stoneman Emily K. , Baer Geoffrey S. , Zhu Yuwei , Wilson Gregory J. , Prasad B. V. V. , Dermody Terence S.
Published: December 2005

Mammalian reoviruses are internalized into cells by receptor-mediated endocytosis. Within the endocytic compartment, the viral outer capsid undergoes acid-dependent proteolysis resulting in removal of the σ3 protein and proteolytic cleavage of the μ1/μ1C protein. Ammonium chloride (AC) is a weak base that blocks disassembly of reovirus virions by inhibiting acidification of intracellular vacuoles. To identify domains in reovirus proteins that influence pH-sensitive steps in viral disassembly, we adapted strain type 3 Dearing (T3D) to growth in murine L929 cells treated with AC. In comparison to wild-type (wt) T3D, AC-adapted (ACA-D) variant viruses exhibited increased yields in AC-treated cells. AC resistance of reassortant viruses generated from a cross of wt type 1 Lang and ACA-D variant ACA-D1 segregated with the σ3-encoding S4 gene. The deduced σ3 amino acid sequences of six independently derived ACA-D variants contain one or two mutations each, affecting a total of six residues. Four of these mutations, I180T, A246G, I347S, and Y354H, cluster in the virion-distal lobe of σ3. Linkage of these mutations to AC resistance was confirmed in experiments using reovirus disassembly intermediates recoated with wt or mutant σ3 proteins. In comparison to wt virions, ACA-D viruses displayed enhanced susceptibility to proteolysis by endocytic protease cathepsin L. Image reconstructions of cryoelectron micrographs of three ACA-D viruses that each contain a single mutation in the virion-distal lobe of σ3 demonstrated native capsid protein organization and minimal alterations in σ3 structure. These results suggest that mutations in σ3 that confer resistance to inhibitors of vacuolar acidification identify a specific domain that regulates proteolytic disassembly.
Live-Cell Characterization and Analysis of a Clinical Isolate of Bovine Respiratory Syncytial Virus, Using Molecular Beacons
Authors: Santangelo Philip , Nitin Nitin , LaConte Leslie , Woolums Amelia , Bao Gang
Published: December 2005

Understanding viral pathogenesis is critical for prevention of outbreaks, development of antiviral drugs, and biodefense. Here, we utilize molecular beacons to directly detect the viral genome and characterize a clinical isolate of bovine respiratory syncytial virus (bRSV) in living cells. Molecular beacons are dual-labeled, hairpin oligonucleotide probes with a reporter fluorophore at one end and a quencher at the other; they are designed to fluoresce only when hybridizing to a complementary target. By imaging the fluorescence signal of molecular beacons, the spread of bRSV was monitored for 7 days with a signal-to-noise ratio of 50 to 200, and the measured time course of infection was quantified with a mathematical model for viral growth. We found that molecular beacon signal could be detected in single living cells infected with a viral titer of 2 × 103.650% tissue culture infective doses/ml diluted 1,000 fold, demonstrating high detection sensitivity. Low background in uninfected cells and simultaneous staining of fixed cells with molecular beacons and antibodies showed high detection specificity. Furthermore, using confocal microscopy to image the viral genome in live, infected cells, we observed a connected, highly three-dimensional, amorphous inclusion body structure not seen in fixed cells. Taken together, the use of molecular beacons for active virus imaging provides a powerful tool for rapid viral infection detection, the characterization of RNA viruses, and the design of new antiviral drugs.
Defining the Cellular Target(s) of Porcine Reproductive and Respiratory Syndrome Virus Blocking Monoclonal Antibody 7G10
Authors: Kim Jeong-Ki , Fahad Al-Majhdi , Shanmukhappa Kumar , Kapil Sanjay
Published: December 2005

We produced a monoclonal antibody (MAb) (7G10) that has blocking activity against porcine reproductive and respiratory syndrome virus (PRRSV). In this study, we identified the components of the 7G10 MAb-bound complex as cytoskeletal filaments: vimentin, cytokeratin 8, cytokeratin 18, actin, and hair type II basic keratin. Vimentin bound to PRRSV nucleocapsid protein and anti-vimentin antibodies showed PRRSV-blocking activity. Vimentin was expressed on the surface of MARC-145, a PRRSV-susceptible cell line. Simian vimentin rendered BHK-21 and CRFK, nonsusceptible cell lines, susceptible to PRRSV infection. These results suggest that vimentin is part of the PRRSV receptor complex and that it plays an important role in PRRSV binding with the other cytoskeletal filaments that mediate transportation of the virus in the cytosol.
Kaposi's Sarcoma-Associated Herpesvirus-Encoded Latency-Associated Nuclear Antigen Induces Chromosomal Instability through Inhibition of p53 Function
Authors: Si Huaxin , Robertson Erle S.
Published: December 2005

Kaposi's sarcoma-associated herpesvirus (KSHV) is predominantly associated with three human malignancies, KS, primary effusion lymphoma, and multicentric Castleman's disease. These disorders are linked to genomic instability, known to be a crucial component of the oncogenic process. Latency-associated nuclear antigen (LANA), encoded by open reading frame 73 of the KSHV genome, is a latent protein consistently expressed in all KSHV-associated diseases. LANA is important in viral genome maintenance and is associated with cellular and viral proteins to regulate viral and cellular gene expression. LANA interacts with the tumor suppressor genes p53 and pRb, indicating that LANA may target these proteins and promote oncogenesis. In this study, we generated cell lines which stably expressed LANA to observe the effects of LANA expression on cell phenotype. LANA expression in these stable cell lines showed a dramatic increase in chromosomal instability, indicated by the presence of increased multinucleation, micronuclei, and aberrant centrosomes. In addition, these stable cell lines demonstrated an increased proliferation rate and as well as increased entry into S phase in both stable and transiently transfected LANA-expressing cells. Additionally, p53 transcription and its transactivation activity were suppressed by LANA expression in a dose-dependent manner. LANA may therefore promote chromosomal instability by suppressing the functional activities of p53, thereby facilitating KSHV-mediated pathogenesis and cancer.
Human Cytomegalovirus Entry into Epithelial and Endothelial Cells Depends on Genes UL128 to UL150 and Occurs by Endocytosis and Low-pH Fusion
Authors: Ryckman Brent J. , Jarvis Michael A , Drummond Derek D. , Nelson Jay A. , Johnson David C.
Published: December 2005

Human cytomegalovirus (HCMV) replication in epithelial and endothelial cells appears to be important in virus spread, disease, and persistence. It has been difficult to study infection of these cell types because HCMV laboratory strains (e.g., AD169 and Towne) have lost their ability to infect cultured epithelial and endothelial cells during extensive propagation in fibroblasts. Clinical strains of HCMV (e.g., TR and FIX) possess a cluster of genes (UL128 to UL150) that are largely mutated in laboratory strains, and recent studies have indicated that these genes facilitate replication in epithelial and endothelial cells. The mechanisms by which these genes promote infection of these two cell types are unclear. We derived an HCMV UL128-to-UL150 deletion mutant from strain TR, TRΔ4, and studied early events in HCMV infection of epithelial and endothelial cells, and the role of genes UL128 to UL150. Analysis of wild-type TR indicated that HCMV enters epithelial and endothelial cells by endocytosis followed by low-pH-dependent fusion, which is different from the pH-independent fusion with the plasma membrane observed with human fibroblasts. TRΔ4 displayed a number of defects in early infection processes. Adsorption and entry of TRΔ4 on epithelial cells were poor compared with those of TR, but these defects could be overcome with higher doses of virus and the use of polyethylene glycol (PEG) to promote fusion between virion and cellular membranes. High multiplicity and PEG treatment did not promote infection of endothelial cells by TRΔ4, yet virus particles were internalized. Together, these data indicate that genes UL128 to UL150 are required for HCMV adsorption and penetration of epithelial cells and to promote some early stage of virus replication, subsequent to virus entry, in endothelial cells.
Identification of 5′ and 3′
cis
-Acting Elements of the Porcine Reproductive and Respiratory Syndrome Virus: Acquisition of Novel 5′ AU-Rich Sequences Restored Replication of a 5′-Proximal 7-Nucleotide Deletion Mutant
Authors: Choi Yu-Jeong , Yun Sang-Im , Kang Shien-Young , Lee Young-Min
Published: December 2005

We here demonstrate the successful engineering of the RNA genome of porcine reproductive and respiratory syndrome virus (PRRSV) by using an infectious cDNA as a bacterial artificial chromosome. Runoff transcription from this cDNA by SP6 polymerase resulted in capped synthetic RNAs bearing authentic 5′ and 3′ ends of the viral genome that had specific infectivities of >5 × 10
5
PFU/μg of RNA. The synthetic viruses recovered from the transfected cells were genotypically and phenotypically indistinguishable from the parental virus. Using our system, a series of genomic RNAs with nucleotide deletions in their 5′ ends produced viruses with decreased or no infectivity. Various pseudorevertants were isolated, and acquisition of novel 5′ sequences of various sizes, composed predominantly of A and U bases, restored their infectivities, providing a novel insight into functional elements of the 5′ end of the PRRSV genome. In addition, our system was further engineered to generate a panel of self-replicating, self-limiting, luciferase-expressing PRRSV viral replicons bearing various deletions. Analysis of these replicons revealed the presence and location of a 3′
cis
-acting element in the genome that was required for replication. Moreover, we produced enhanced green fluorescent protein-expressing infectious viruses, which indicates that the PRRSV cDNA/viral replicon/recombinant virus can be developed as a vector for the expression of a variety of heterologous genes. Thus, our PRRSV reverse genetics system not only offers a means of directly investigating the molecular mechanisms of PRRSV replication and pathogenesis but also can be used to generate new heterologous gene expression vectors and genetically defined antiviral vaccines.
Characterization of Ross River Virus Tropism and Virus-Induced Inflammation in a Mouse Model of Viral Arthritis and Myositis
Authors: Morrison Thomas E. , Whitmore Alan C. , Shabman Reed S. , Lidbury Brett A. , Mahalingam Suresh , Heise Mark T.
Published: December 2005

Mosquito-borne alphaviruses are a significant cause of both encephalitic and arthritic disease in humans worldwide. In contrast to the encephalitic alphaviruses, the pathogenesis of alphavirus-induced arthritic disease is not well understood. Utilizing a mouse model of Ross River virus (RRV) disease, we found that the primary targets of RRV infection are bone, joint, and skeletal muscle tissues of the hind limbs in both outbred CD-1 mice and adult C57BL/6J mice. Moreover, histological analyses demonstrated that RRV infection resulted in severe inflammation of these tissues. Characterization of the inflammatory infiltrate within the skeletal muscle tissue identified inflammatory macrophages, NK cells, and CD4+and CD8+T lymphocytes. To determine the contribution of the adaptive immune system, the outcome of RRV-induced disease was examined in C57BL/6J RAG-1−/−mice, which lack functional T and B lymphocytes. RAG-1−/−and wild-type mice developed similar disease signs, infiltration of inflammatory macrophages and NK cells, and muscle pathology, suggesting that the adaptive immune response does not play a critical role in the development of disease. These results establish the mouse model of RRV disease as a useful system for the identification of viral and host factors that contribute to alphavirus-induced arthritis and myositis.
Human Immunodeficiency Virus Type 1 Coreceptor Switching: V1/V2 Gain-of-Fitness Mutations Compensate for V3 Loss-of-Fitness Mutations
Authors: Pastore C. , Nedellec R. , Ramos A. , Pontow S. , Ratner L. , Mosier D. E.
Published: December 2005

Human immunodeficiency virus type 1 (HIV-1) entry into target cells is mediated by the virus envelope binding to CD4 and the conformationally altered envelope subsequently binding to one of two chemokine receptors. HIV-1 envelope glycoprotein (gp120) has five variable loops, of which three (V1/V2 and V3) influence the binding of either CCR5 or CXCR4, the two primary coreceptors for virus entry. Minimal sequence changes in V3 are sufficient for changing coreceptor use from CCR5 to CXCR4 in some HIV-1 isolates, but more commonly additional mutations in V1/V2 are observed during coreceptor switching. We have modeled coreceptor switching by introducing most possible combinations of mutations in the variable loops that distinguish a previously identified group of CCR5- and CXCR4-using viruses. We found that V3 mutations entail high risk, ranging from major loss of entry fitness to lethality. Mutations in or near V1/V2 were able to compensate for the deleterious V3 mutations and may need to precede V3 mutations to permit virus survival. V1/V2 mutations in the absence of V3 mutations often increased the capacity of virus to utilize CCR5 but were unable to confer CXCR4 use. V3 mutations were thus necessary but not sufficient for coreceptor switching, and V1/V2 mutations were necessary for virus survival. HIV-1 envelope sequence evolution from CCR5 to CXCR4 use is constrained by relatively frequent lethal mutations, deep fitness valleys, and requirements to make the right amino acid substitution in the right place at the right time.