bluuebunny/crossref_metadata_2025 huggingface dataset:
Multiple Enzymatic Activities Associated with Severe Acute Respiratory Syndrome Coronavirus Helicase
Authors: Ivanov Konstantin A. , Thiel Volker , Dobbe Jessika C. , van der Meer Yvonne , Snijder Eric J. , Ziebuhr John
Published: May 2004

Severe acute respiratory syndrome coronavirus (SARS-CoV), a newly identified group 2 coronavirus, is the causative agent of severe acute respiratory syndrome, a life-threatening form of pneumonia in humans. Coronavirus replication and transcription are highly specialized processes of cytoplasmic RNA synthesis that localize to virus-induced membrane structures and were recently proposed to involve a complex enzymatic machinery that, besides RNA-dependent RNA polymerase, helicase, and protease activities, also involves a series of RNA-processing enzymes that are not found in most other RNA virus families. Here, we characterized the enzymatic activities of a recombinant form of the SARS-CoV helicase (nonstructural protein [nsp] 13), a superfamily 1 helicase with an N-terminal zinc-binding domain. We report that nsp13 has both RNA and DNA duplex-unwinding activities. SARS-CoV nsp13 unwinds its substrates in a 5′-to-3′ direction and features a remarkable processivity, allowing efficient strand separation of extended regions of double-stranded RNA and DNA. Characterization of the nsp13-associated (deoxy)nucleoside triphosphatase ([dNTPase) activities revealed that all natural nucleotides and deoxynucleotides are substrates of nsp13, with ATP, dATP, and GTP being hydrolyzed slightly more efficiently than other nucleotides. Furthermore, we established an RNA 5′-triphosphatase activity for the SARS-CoV nsp13 helicase which may be involved in the formation of the 5′ cap structure of viral RNAs. The data suggest that the (d)NTPase and RNA 5′-triphosphatase activities of nsp13 have a common active site. Finally, we established that, in SARS-CoV-infected Vero E6 cells, nsp13 localizes to membranes that appear to be derived from the endoplasmic reticulum and are the likely site of SARS-CoV RNA synthesis.
Increased Sensitivity to CD4 Binding Site-Directed Neutralization following In Vitro Propagation on Primary Lymphocytes of a Neutralization-Resistant Human Immunodeficiency Virus IIIB Strain Isolated from an Accidentally Infected Laboratory Worker
Authors: Beaumont Tim , Quakkelaar Esther , van Nuenen Ad , Pantophlet Ralph , Schuitemaker Hanneke
Published: May 2004

We previously described the adaptation of the neutralization-sensitive human immunodeficiency virus type 1 (HIV-1) strain IIIB to a neutralization-resistant phenotype in an accidentally infected laboratory worker. During long-term propagation of this resistant isolate, designated FF3346, on primary peripheral blood leukocytes in vitro, an HIV-1 variant appeared that had regained sensitivity to neutralization by soluble CD4 (sCD4) and the broadly neutralizing monoclonal antibody b12. When an early passage of FF3346 was subjected to limiting-dilution culture in peripheral blood mononuclear cells, eight virus variants with various degrees of neutralization resistance were isolated. Two of them, the sCD4 neutralization-resistant variant LW_H8resand the sCD4 neutralization-sensitive variant LW_G9sens, were selected for further study. Interestingly, these two viruses were equally resistant to neutralization by agents that recognize domains other than the CD4 binding site. Site-directed mutagenesis revealed that the increased neutralization sensitivity of variant LW_G9sensresulted from only two changes, an Asn-to-Ser substitution at position 164 in the V2 loop and an Ala-to-Glu substitution at position 370 in the C3 domain of gp120. In agreement with this notion, the affinity of b12 for monomeric gp120 containing the N164S and A370E substitutions in the background of the molecular clone LW_H8reswas higher than its affinity for the parental gp120. Surprisingly, no correlation was observed between CD4 binding affinity for monomeric gp120 and the level of neutralization resistance, suggesting that differences in sCD4 neutralization sensitivity between these viruses are only manifested in the context of the tertiary or quaternary structure of gp120 on the viral surface. The results obtained here indicate that the neutralization-sensitive strain IIIB can become neutralization resistant in vivo under selective pressure by neutralizing antibodies but that this resistance may be easily reversed in the absence of immunological pressure.
Capsid Is a Dominant Determinant of Retrovirus Infectivity in Nondividing Cells
Authors: Yamashita Masahiro , Emerman Michael
Published: May 2004

A major difference between lentiviruses such as human immunodeficiency virus (HIV) and most other retroviruses is their ability to productively infect nondividing cells. We present here genetic evidence for involvement of the capsid protein (CA) in the infectious phenotype in nondividing cells. A chimeric HIV type 1 (HIV-1) in which the MA and CA of HIV-1 are replaced with the MA, p12, and CA encoding sequences from murine leukemia virus (MLV) loses the ability to efficiently infect nondividing cells. Analysis of the accumulation of two-long-terminal-repeat circles implies that the impairment of nuclear transport of preintegration complexes is responsible for the restricted infection of this chimeric virus in nondividing cells. Incorporation of MLV MA and MLV p12 into HIV virions alone does not exert any adverse effects on viral infection in interphase cells. These results suggest that CA is the dominant determinant for the difference between HIV and MLV in the ability to transduce nondividing cells.
Passive and Active Inclusion of Host Proteins in Human Immunodeficiency Virus Type 1 Gag Particles during Budding at the Plasma Membrane
Authors: Hammarstedt Maria , Garoff Henrik
Published: May 2004

Human immunodeficiency virus type 1 particles form by budding at the surface of most cell types. In this process, a piece of the plasma membrane is modified into an enveloped virus particle. The process is driven by the internal viral protein Pr55

gag

. We have studied how host proteins in the membrane are dealt with by Pr55

gag

during budding. Are they included in or excluded from the particle? The question was approached by measuring the relative concentrations of host and viral proteins in the envelope of Pr55

gag

particles and in their donor membranes in the cell. We observed that the bulk of the host proteins, including actin and clathrin, were passively included into the virus-like Gag particles. This result suggests that budding by Pr55

gag

proceeds without significant alteration of the original host protein composition at the cell membrane. Nevertheless, some proteins were concentrated in the particles, and a few were excluded. The concentrated proteins included cyclophilin A and Tsg-101. These were recruited to the plasma membrane by Pr55

gag

. The membrane-bound cyclophilin A was concentrated into particles as efficiently as Pr55

gag

, whereas Tsg-101 was concentrated more efficiently. The latter finding is consistent with a role for Tsg-101 in Gag particle release.
Degradation of p53, Not Telomerase Activation, by E6 Is Required for Bypass of Crisis and Immortalization by Human Papillomavirus Type 16 E6/E7
Authors: McMurray H. R. , McCance D. J.
Published: May 2004

Bypass of two arrest points is essential in the process of cellular immortalization, one of the components of the transformation process. Expression of human papillomavirus type 16 E6 and E7 together can escape both senescence and crisis, processes which normally limit the proliferative capacity of primary human keratinocytes. Crisis is thought to be mediated by telomere shortening. Because E6 stimulates telomerase activity and exogenous expression of the TERT gene with E7 can immortalize keratinocytes, this function is thought to be important for E6 to cooperate with E7 to bypass crisis. However, it has also been reported that E6 dissociates increased telomerase activity from maintenance of telomere length and that a dominant-negative p53 molecule can substitute for E6 in cooperative immortalization of keratinocytes with E7. Thus, to determine which functions of E6 are required to allow bypass of crisis and immortalization of keratinocytes with E7, immortalization assays were performed using specific mutants of E6, in tandem with E7. In these experiments, every clone expressing an E6 mutant capable of degrading p53 was able to bypass crisis and immortalize, regardless of telomerase induction. All clones containing E6 mutants incapable of degrading p53 died at crisis. These results suggest that the ability of E6 to induce degradation of p53 compensates for continued telomere shortening in E6/E7 cells and demonstrate that degradation of p53 is required for immortalization by E6/E7, while increased telomerase activity is dispensable.
IGFBP-3, a Marker of Cellular Senescence, Is Overexpressed in Human Papillomavirus-Immortalized Cervical Cells and Enhances IGF-1-Induced Mitogenesis
Authors: Baege Astrid C. , Disbrow Gary L. , Schlegel Richard
Published: May 2004

Human ectocervical cells, following retroviral transduction with the human papillomavirus type 16 E6/E7 oncogenes, are altered in their array of transcribed cellular genes, including increased mRNA for the insulin-like growth factor binding protein 3 (IGFBP-3). IGFBP-3 expression is associated with cellular senescence, and its addition to many cell types inhibits growth or induces apoptosis. By immunoblotting and enzyme-linked immunosorbent assay methods, we demonstrate that late-passage, immortalized E6/E7-transduced cells secrete high levels of IGFBP-3 (25 ng/ml), which represent a 500-fold increase compared to levels in early-passage, nonimmortalized transduced cells (<0.05 ng/ml). Concomitantly, these late-passage cervical cells exhibit an increase in sensitivity to IGF-1, including enhanced phosphorylation of the IGF receptor (IGF-R) and insulin receptor substrate as well as increased DNA synthesis (5-fold) and cell proliferation (3.7-fold). However, there was no change in the level of IGF-R in these cells (surface or total), and the cells did not synthesize IGF-1, indicating that these arms of the IGF pathway were independently regulated and not responsible for the augmented signaling. Consistent with a causal relationship between IGFBP-3 expression and enhanced IGF-1 responses, we found that early-passage cells could be converted to the late-passage, IGF-1-responsive phenotype by preincubation with IGFBP-3. Thus, in contrast to findings with some cell types, IGFBP-3 expression in cervical cells is associated with augmented IGF-1 signaling and cell proliferation and correlates with the timing of cellular immortalization.
Features Affecting the Ability of Hepatitis Delta Virus RNAs To Initiate RNA-Directed RNA Synthesis
Authors: Gudima Severin O. , Chang Jinhong , Taylor John M.
Published: May 2004

In models of the replication of human hepatitis delta virus (HDV) RNA, it is generally assumed that circular RNAs are the only templates. However, noncircular HDV RNAs are also produced during replication, and it is known that replication can be initiated by transfection with noncircular RNAs. Therefore, strategies were devised to determine the relative ability of different HDV RNA species to initiate RNA replication. One strategy used in vivo intermolecular competition following cotransfection into cells, between two sequence-marked HDV RNA species. Circular RNA templates were found to be at least severalfold more efficient than a dimeric linear template. Unit-length linear species, that is, equivalent to circles opened at different sites, were in most cases but not always of efficiency comparable to that of each other. Greater-than-unit-length linear species were more efficient than unit-length species, presumably because of the increased opportunities for template switching. Genomic linear RNAs were generally of initiation ability comparable to that of antigenomic RNAs. A second strategy measured the ability of initiation to occur on different regions of HDV RNAs that were twice the unit length. In summary, results from these two experimental strategies make clear that linear HDV RNA species, as well as circles, can contribute to the overall process of HDV genome replication. In addition, the results from the two experimental strategies provided information on the impact of template switching during RNA-directed transcription.
The Dominant-Negative Herpes Simplex Virus Type 1 (HSV-1) Recombinant CJ83193 Can Serve as an Effective Vaccine against Wild-Type HSV-1 Infection in Mice
Authors: Augustinova Hanka , Hoeller Daniela , Yao Feng
Published: May 2004

By selectively regulating the expression of thetrans-dominant-negative mutant polypeptide UL9-C535C, of herpes simplex virus type 1 (HSV-1) origin binding protein UL9 with the tetracycline repressor (tetR)-mediated gene switch, we recently generated a novel replication-defective and anti-HSV-specific HSV-1 recombinant, CJ83193. The UL9-C535C peptides expressed by CJ83193 can function as a potent intracellular therapy against its own replication, as well as the replication of wild-type HSV-1 and HSV-2 in coinfected cells. In this report, we demonstrate that CJ83193 cannot initiate acute productive infection in corneas of infected mice nor can it reactivate from trigeminal ganglia of mice latently infected by CJ83193 in a mouse ocular model. Given that CJ83193 is capable of expressing the viral α, β, and γ1 genes but little or no γ2 genes, we tested the vaccine potential of CJ83193 against HSV-1 infection in a mouse ocular model. Our studies showed that immunization with CJ83193 significantly reduced the yields of challenge HSV in the eyes and trigeminal ganglia on days 3, 5, and 7 postchallenge. Like in mice immunized with the wild-type HSV-1 strain KOS, immunization of mice with CJ83193 prevents the development of keratitis and encephalitis induced by corneal challenge with wild-type HSV-1 strain mP. Delayed-type hypersensitivity (DTH) assays demonstrate that CJ83193 can elicit durable cell-mediated immunity at the same level as that of wild-type HSV-1 and is more effective than that induced byd27, an HSV-1 ICP27 deletion mutant. Moreover, mice immunized with CJ83193 developed strong, durable HSV-1-neutralizing antibodies at levels at least twofold higher than those induced byd27. The results presented in this report have shed new light on the development of effective HSV viral vaccines that encode a unique safety mechanism capable of inhibiting the mutant's own replication and that of wild-type virus.
Complementation of a Binding-Defective Retrovirus by a Host Cell Receptor Mutant
Authors: Qian Zhaohui , Wang Hongzhe , Empig Cyril , Anderson W. French , Albritton Lorraine M.
Published: May 2004

The entry of ecotropic murine leukemia virus (MLV) into cells requires the interaction of the envelope protein (Env) with its receptor, mouse cationic amino acid transporter 1 (mATRC1). An aspartic acid-to-lysine change at position 84 (D84K) of ecotropic Moloney MLV Env abolishes virus binding and infection. We recently identified lysine 234 (rK234) in mATRC1 as a residue that influences virus binding and infection. Here we show that D84K virus infection increased 3,000-fold on cells expressing receptor with an rK234A change and 100,000-fold on cells expressing an rK234D change. The stronger complementation of D84K virus infection by rK234D than by the rK234A receptor suggests that although the major reason for loss of infection of D84K and D84R virus is due to steric hindrance and charge repulsion, the loss of an interaction of D84 with receptor appears to contribute as well. Taken together, these results indicate that D84 is very close to rK234 of mATRC1 in the bound complex and there is likely an interaction between them. The definitive localization of the receptor binding site on SU should facilitate the design of chimeric envelope proteins that target infection to new receptors by replacing the receptor binding site with an exogenous ligand sequence.
Recombinant Respiratory Syncytial Viruses Lacking the C-Terminal Third of the Attachment (G) Protein Are Immunogenic and Attenuated In Vivo and In Vitro
Authors: Elliott Matthew B. , Pryharski Karin S. , Yu Qingzhong , Parks Christopher L. , Laughlin Todd S. , Gupta C. Kanta , Lerch Robert A. , Randolph Valerie B. , LaPierre Natisha A. , Dack Kristen M. Heers , Hancock Gerald E.
Published: May 2004

The design of attenuated vaccines for respiratory syncytial virus (RSV) historically focused on viruses made sensitive to physiologic temperature through point mutations in the genome. These prototype vaccines were not suitable for human infants primarily because of insufficient attenuation, genetic instability, and reversion to a less-attenuated phenotype. We therefore sought to construct novel attenuated viruses with less potential for reversion through genetic alteration of the attachment G protein. Complete deletion of G protein was previously shown to result in RSV strains overly attenuated for replication in mice. Using reverse genetics, recombinant RSV (rRSV) strains were engineered with truncations at amino acid 118, 174, 193, or 213 and respectively designated rA2cpΔG118, rA2cpΔG174, rA2cpΔG193, and rA2cpΔG213. All rA2cpΔG strains were attenuated for growth in vitro and in the respiratory tracts of BALB/c mice but not restricted for growth at 37°C. The mutations did not significantly affect nascent genome synthesis in human lung epithelial (A549) cells, but infectious rA2cpΔG virus shed into the culture medium was dramatically diminished. Hence, the data suggested that a site within the C-terminal 85 amino acids of G protein is important for efficient genome packaging or budding of RSV from the infected cell. Vaccination with the rA2cpΔG strains also generated efficacious immune responses in mice that were similar to those elicited by the temperature-sensitive
cpts
248/404 strain previously tested in human infants. Collectively, the data indicate that the rA2cpΔG strains are immunogenic, not likely to revert to the less-attenuated phenotype, and thus candidates for further development as vaccines against RSV.
Multiple Groups of Endogenous Betaretroviruses in Mice, Rats, and Other Mammals
Authors: Baillie Gregory J. , van de Lagemaat Louie N. , Baust Corinna , Mager Dixie L.
Published: May 2004

Betaretroviruses
exist in endogenous and exogenous forms in hosts that are widely
distributed and evolutionarily distantly related. Here we report the
discovery and characterization of several previously unknown
betaretrovirus groups in the genomes of
Mus musculus
and
Rattus norvegicus
. Each group contains both mouse and rat
elements, and several of the groups are more closely related to
previously known betaretroviruses from nonmurine hosts. Some of the
groups also include members from hosts which were not previously known
to harbor betaretroviruses, such as the gray mouse lemur
(
Microcebus murinus
) and Seba's short-tailed bat
(
Carollia perspicillata
). Some of the mouse and rat elements
possess intact open reading frames for
gag
,
pro
,
pol
, and/or
env
genes and display characteristics of
having retrotransposed recently. We propose a model whereby
betaretroviruses have been evolving within the genomes of murid rodents
for at least the last 20 million years and, subsequent to (or
concomitant with) the global spread of their murid hosts, have
occasionally been transmitted to other
species.
Density-Dependent Selection in Vesicular Stomatitis Virus
Authors: Novella Isabel S. , Reissig Daniel D. , Wilke Claus O.
Published: May 2004

We used vesicular stomatitis virus to test the effect of complementation on the relative fitness of a deleterious mutant, monoclonal antibody-resistant mutant (MARM) N, in competition with its wild-type ancestor. We carried out competitions of MARM N and wild-type populations at different multiplicities of infection (MOIs) and initial ratios of the wild type to the mutant and found that the fitness of MARM N relative to that of the wild type is very sensitive to changes in the MOI (i.e., the degree of complementation) but depends little, if at all, on the initial frequencies of MARM N and the wild type. Further, we developed a mathematical model under the assumption that during coinfection both viruses contribute to a common pool of protein products in the infected cell and that they both exploit this common pool equally. Under such conditions, the fitness of all virions that coinfect a cell is the average fitness in the absence of coinfection of that group of virions. In the absence of coinfection, complementation cannot take place and the relative fitness of each competitor is only determined by the selective value of its own products. We found good agreement between our experimental results and the model predictions, which suggests that the wild type and MARM N freely share all of their gene products under coinfection.
Porcine Endogenous Retrovirus Transmission Characteristics of Galactose α1-3 Galactose-Deficient Pig Cells
Authors: Quinn Gary , Wood James C. , Ryan David J. J. , Suling Kristen M. , Moran Kathleen M. , Kolber-Simonds Donna L. , Greenstein Julia L. , Schuurman Henk-Jan , Hawley Robert J. , Patience Clive
Published: May 2004

Galactose α1-3 galactose (Gal) trisaccharides are present on the surface of wild-type pig cells, as well as on viruses particles produced from such cells. The recognition of Gal sugars by natural anti-Gal antibodies (NAb) in human and Old World primate serum can cause the lysis of the particles via complement-dependent mechanisms and has therefore been proposed as an important antiviral mechanism. Recently, pigs have been generated that possess disrupted galactosyl-transferase (GGTA1) genes. The cells of these pigs do not express Gal sugars on their surface, i.e., are Gal null. Concerns have been raised that the risk of virus transmission from such pigs may be increased due to the absence of the Gal sugars. We investigated the sensitivity of porcine endogenous retrovirus (PERV) produced from Gal-null and Gal-positive pig cells to inactivation by purified NAb and human serum. PERV produced in Gal-null pig cells was resistant to inactivation by either NAb or human serum. In contrast, although Gal-positive PERV particles were sensitive to inactivation by NAb and human serum, they required markedly higher concentrations of NAb for inactivation compared to the Gal-positive cells from which they were produced. Complete inactivation of Gal-positive PERV particles was not achievable despite the use of high levels of NAb, indicating that NAb-mediated inactivation of cell-free PERV particles is an inefficient process.
Proteomics of Herpes Simplex Virus Replication Compartments: Association of Cellular DNA Replication, Repair, Recombination, and Chromatin Remodeling Proteinswith ICP8
Authors: Taylor Travis J. , Knipe David M.
Published: May 2004

In
this study, we have used immunoprecipitation and mass spectrometry to
identify over 50 cellular and viral proteins that are associated with
the herpes simplex virus 1 (HSV-1) ICP8 single-stranded DNA-binding
protein. Many of the coprecipitating cellular proteins are known
members of large cellular complexes involved in (i) DNA replication or
damage repair, including RPA and MSH6; (ii) nonhomologous and
homologous recombination, including the catalytic subunit of the
DNA-dependent protein kinase, Ku86, and Rad50; and (iii) chromatin
remodeling, including BRG1, BRM, hSNF2H, BAF155, mSin3a, and histone
deacetylase 2. It appears that DNA mediates the association of certain
proteins with ICP8, while more direct protein-protein interactions
mediate the association with other proteins. A number of these proteins
accumulate in viral replication compartments in the infected cell
nucleus, indicating that these proteins may have a role in
viral replication. WRN, which functions in cellular recombination
pathways via its helicase and exonuclease activities, is not absolutely
required for viral replication, as viral yields are only very slightly,
if at all, decreased in WRN-deficient human primary fibroblasts
compared to control cells. In Ku70-deficient murine embryonic
fibroblasts, viral yields are increased by almost 50-fold, suggesting
that the cellular nonhomologous end-joining pathway inhibits HSV
replication. We hypothesize that some of the proteins coprecipitating
with ICP8 are involved in HSV replication and may give new insight into
viral replication
mechanisms.
Hepatitis C Virus Persistence after Spontaneous or Treatment-Induced Resolution of Hepatitis C
Authors: Pham Tram N. Q. , MacParland Sonya A. , Mulrooney Patricia M. , Cooksley Helen , Naoumov Nikolai V. , Michalak Tomasz I.
Published: May 2004

It is presumed that resolution of hepatitis C, as evidenced by normalization of liver function tests and disappearance of hepatitis C virus (HCV) RNA from serum, as determined by conventional laboratory assays, reflects virus eradication. In this study, we examined the expression of the HCV genome in the sera, peripheral blood mononuclear cells (PBMC), and, on some occasions, monocyte-derived dendritic cells (DC) long after resolution of hepatitis C by using a highly sensitive reverse transcription (RT)-PCR-nucleic acid hybridization (RT-PCR-NAH) assay. The samples obtained from 16 randomly selected patients (5 with spontaneous and 11 with treatment-induced resolution), monitored for up to 5 years, were studied by qualitative and semiquantitative RT-PCR-NAH and by real-time RT-PCR to detect the HCV RNA positive strand. The replicative HCV RNA negative strand was examined in PBMC after culture with a T-cell proliferation stimulating mitogen. The findings show that HCV RNA was carried in the convalescent-phase sera and/or PBMC in all 16 individuals investigated. Also, DC from six of seven patients were reactive for the HCV genome. Importantly, traces of the HCV RNA negative strand, suggesting progressing virus replication, were detected in the majority of mitogen-stimulated PBMC, including four samples collected 5 years after recovery. Sequencing of the HCV 5′ untranslated region fragment revealed genotype 1b in four of nine individuals examined and genotypes 1a and 2a in three and two patients, respectively. These results imply that HCV RNA can persist at very low levels in the serum and peripheral lymphoid cells and that an intermediate replicative form of the HCV genome can persist in PBMC for many years after apparently complete spontaneous or antiviral therapy-induced resolution of chronic hepatitis C.
Antisense Morpholino-Oligomers Directed against the 5′ End of the Genome Inhibit Coronavirus Proliferation and Growth†
Authors: Neuman Benjamin W. , Stein David A. , Kroeker Andrew D. , Paulino Amy D. , Moulton Hong M. , Iversen Patrick L. , Buchmeier Michael J.
Published: May 2004

Conjugation of a peptide related to the human immunodeficiency virus type 1 Tat represents a novel method for delivery of antisense morpholino-oligomers. Conjugated and unconjugated oligomers were tested to determine sequence-specific antiviral efficacy against a member of theCoronaviridae,Mouse hepatitis virus(MHV). Specific antisense activity designed to block translation of the viral replicase polyprotein was first confirmed by reduction of luciferase expression from a target sequence-containing reporter construct in both cell-free and transfected cell culture assays. Peptide-conjugated morpholino-oligomers exhibited low toxicity in DBT astrocytoma cells used for culturing MHV. Oligomer administered at micromolar concentrations was delivered to >80% of cells and inhibited virus titers 10- to 100-fold in a sequence-specific and dose-responsive manner. In addition, targeted viral protein synthesis, plaque diameter, and cytopathic effect were significantly reduced. Inhibition of virus infectivity by peptide-conjugated morpholino was comparable to the antiviral activity of the aminoglycoside hygromycin B used at a concentration fivefold higher than the oligomer. These results suggest that this composition of antisense compound has therapeutic potential for control of coronavirus infection.
Human Papillomavirus Type 16 E6 Amino Acid 83 Variants Enhance E6-Mediated MAPK Signaling and Differentially Regulate Tumorigenesis by Notch Signaling and Oncogenic Ras
Authors: Chakrabarti Oishee , Veeraraghavalu Karthikeyan , Tergaonkar Vinay , Liu Yun , Androphy Elliot J. , Stanley Margaret A. , Krishna Sudhir
Published: May 2004

Oncogenically high-risk human papillomaviruses (HPVs) are causally associated with the progression of major human neoplasia-like cancers of the cervix. Several studies have defined functions of the key E6 and E7 oncoproteins in epithelial cell immortalization. The roles of these oncogenes in the progression of immortalized epithelial cells to invasive tumors are still poorly understood. Here, we establish a novel link between the E6 oncoprotein and activation of mitogen-activated protein kinase (MAPK) signaling and show that this signaling involves Rap1. We find that activated MAPK signaling cooperates with deregulated Notch1 signaling to recreate features of HPV-driven invasive cervical carcinomas. We extend our analysis to evaluate an E6 (amino acid [aa] 83) variant that has been linked to invasive tumors. The variant enhances MAPK signaling and cooperative transformation with deregulated Notch1 signaling. Unlike E6, this variant surprisingly inhibits oncogenic Ras-mediated transformation. Our data reveal that the quantitative differences in activation of MAPK signaling by E6 and its variant correlate with differences in cooperative transformation with other signaling pathways, thus suggesting that thresholds of MAPK activation may define permissive conditions for other signaling pathways in tumorigenesis. Epidemiological studies have suggested the importance of E6 aa 83 variants in invasive carcinomas; our data support a key deterministic role for this variant in human cervical tumorigenesis. These observations, along with our recent data showing that deregulated Notch signaling activates phosphatidylinositol 3-kinase signaling, strengthen the possibility of the existence of Ras-independent mechanisms to recreate signaling through classical Ras effector pathways.
Helper-Dependent Adenovirus Vectors Elicit Intact Innate but Attenuated Adaptive Host Immune Responses In Vivo
Authors: Muruve Daniel A. , Cotter Matthew J. , Zaiss Anne K. , White Lindsay R. , Liu Qiang , Chan Trevor , Clark Sharon A. , Ross P. Joel , Meulenbroek Robert A. , Maelandsmo Gunhild M. , Parks Robin J.
Published: May 2004

Helper-dependent adenovirus (HD-Ad) vectors with all adenoviral genes deleted mediate very long-term expression of therapeutic transgenes in a variety of animal models of disease. These vectors are associated with reduced toxicity and improved safety relative to traditional early region 1 deletion first-generation Ad (FG-Ad) vectors. Many studies have clearly demonstrated that FG-Ad vectors induce innate and adaptive immune responses in vivo; however, a comprehensive analysis of host immune responses to HD-Ad vectors has not yet been performed. In DBA/2 mice, intravenous injection of HD-Ad vectors encoding LacZ (HD-AdLacZ) or a murine secreted alkaline phosphatase (HD-AdSEAP) induced an early expression of inflammatory cytokine and chemokine genes in the liver, including interferon-inducible protein 10, macrophage inflammatory protein 2, and tumor necrosis factor alpha, and were expressed in a pattern similar to that induced by FG-Ad vectors encoding AdSEAP. Like AdSEAP, and consistent with the pattern of cellular gene expression, HD-AdLacZ and HD-AdSEAP induced the recruitment of CD11b-positive leukocytes to the transduced liver within hours of administration. AdSEAP also induced a second phase of liver inflammation, consisting of inflammatory gene expression and CD3-positive lymphocytic infiltrates 7 days posttransduction. In contrast, beyond 24 h no infiltrates or expression of inflammatory genes was detected in the livers of mice receiving HD-AdSEAP. Despite the lack of liver inflammation at 7 days, Ad-specific cytotoxic T lymphocytes could be detected in mice receiving HD-AdSEAP. This lack of liver inflammation was not due to reduced transduction since levels of transgene expression and the amounts of vector DNA in the liver were equivalent in mice receiving HD-AdSEAP and AdSEAP. These results demonstrate that HD-Ad vectors induce intact innate but attenuated adaptive immune responses in vivo.
Passive Immunotherapy in Simian Immunodeficiency Virus-Infected Macaques Accelerates the Development of Neutralizing Antibodies
Authors: Haigwood Nancy L. , Montefiori David C. , Sutton William F. , McClure Janela , Watson Andrew J. , Voss Gerald , Hirsch Vanessa M. , Richardson Barbra A. , Letvin Norman L. , Hu Shiu-Lok , Johnson Philip R.
Published: May 2004

Passively transferred neutralizing antibodies can block lentivirus infection, but their role in postexposure prophylaxis is poorly understood. In this nonhuman-primate study, the effects of short-term antibody therapy on 5-year disease progression, virus load, and host immunity were explored. We reported previously that postinfection passive treatment with polyclonal immune globulin with high neutralizing titers against SIVsmE660 (SIVIG) significantly improved the 67-week health of SIVsmE660-infectedMacaca mulattamacaques. Four of six treated macaques maintained low or undetectable levels of virus in plasma, compared with one of ten controls, while two rapid progressors controlled viremia only as long as the SIVIG was present. SIVIG treatment delayed the de novo production of envelope (Env)-specific antibodies by 8 weeks (13). We show here that differences in disease progression were also significant at 5 years postinfection, excluding rapid progressors (P= 0.05). Macaques that maintained ≤103virus particles per ml of plasma and ≤30 infectious virus particles per 106mononuclear cells from peripheral blood and lymph nodes had delayed disease onset. All macaques that survived beyond 18 months had measurable Gag-specific CD8+cytotoxic T cells, regardless of treatment. Humoral immunity in survivors beyond 20 weeks was strikingly different in the SIVIG and control groups. Despite a delay in Env-specific binding antibodies, de novo production of neutralizing antibodies was significantly accelerated in SIVIG-treated macaques. Titers of de novo neutralizing antibodies at week 12 were comparable to levels achieved in controls only by week 32 or later. Acceleration of de novo simian immunodeficiency virus immunity in the presence of passively transferred neutralizing antibodies is a novel finding with implications for postexposure prophylaxis and vaccines.
Cell-Cell Fusion Induced by the Avian Reovirus Membrane Fusion Protein Is Regulated by Protein Degradation
Authors: Shmulevitz Maya , Corcoran Jennifer , Salsman Jayme , Duncan Roy
Published: May 2004

The p10 fusion-associated small transmembrane protein of avian reovirus induces extensive syncytium formation in transfected cells. Here we show that p10-induced cell-cell fusion is restricted by rapid degradation of the majority of newly synthesized p10. The small ectodomain of p10 targets the protein for degradation following p10 insertion into an early membrane compartment. Paradoxically, conservative amino acid substitutions in the p10 ectodomain hydrophobic patch that eliminate fusion activity also increase p10 stability. The small amount of p10 that escapes intracellular degradation accumulates at the cell surface in a relatively stable form, where it mediates cell-cell fusion as a late-stage event in the virus replication cycle. The unusual relationship between a nonstructural viral membrane fusion protein and the replication cycle of a nonenveloped virus has apparently contributed to the evolution of a novel mechanism for restricting the extent of virus-induced cell-cell fusion.
Species-Specific Tropism Determinants in the Human Immunodeficiency Virus Type 1 Capsid
Authors: Hatziioannou Theodora , Cowan Simone , von Schwedler Uta K. , Sundquist Wesley I. , Bieniasz Paul D.
Published: May 2004

Retroviral tropism is determined in part by cellular restriction factors that block infection by targeting the incoming viral capsid. Indeed, human immunodeficiency virus type 1 (HIV-1) infection of many nonhuman primate cells is inhibited by one such factor, termed Lv1. In contrast, a restriction factor in humans, termed Ref1, does not inhibit HIV-1 infection unless nonnatural mutations are introduced into the HIV-1 capsid protein (CA). Here, we examined the infectivity of a panel of mutant HIV-1 strains carrying substitutions in the N-terminal CA domain in cells that exhibit restriction attributable to Lv1 or Ref1. Manipulation of HIV-1 CA could alter HIV-1 tropism, and several mutations were identified that increased or decreased HIV-1 infectivity in a target-cell-specific manner. Many residues that affected HIV-1 tropism were located in the three variable loops that lie on the outer surface of the modeled HIV-1 conical capsid. Some tropism determinants, including the CypA binding site, coincided with residues whose mutation conferred on HIV-1 CA the ability to saturate Ref1 in human cells. Notably, a mutation that reverses the infectivity defect in human cells induced by CypA binding site mutation inhibits recognition by Ref1. Overall, these findings demonstrate that exposed variable loops in CA and a partial CypA “coat” can modulate restriction and HIV-1 tropism and suggest a model in which the exposed surface of the incoming retroviral capsid is the target for inhibition by host cell-specific restriction factors.
Human Immunodeficiency Virus Type 1 Gag Contains a Dileucine-Like Motif That Regulates Association with Multivesicular Bodies
Authors: Lindwasser O. Wolf , Resh Marilyn D.
Published: May 2004

Multivesicular bodies (MVBs) are cholesterol-enriched organelles formed by the endocytic pathway. The topology of vesicle formation in MVBs is identical to that of retroviral budding from the plasma membrane, and budding of human immunodeficiency virus type 1 (HIV-1) into MVBs in macrophages has recently been visualized. The Gag proteins from HIV-1, as well as many other retroviruses, contain short motifs that mediate interactions with MVBs and other endocytic components, suggesting that Gag proteins directly interface with the endocytic pathway. Here, we show that HIV-1 Gag contains an internalization signal that promotes endocytosis of a chimeric transmembrane fusion protein. Mutation of this motif within Gag strongly inhibits virus-like particle production. Moreover, wild-type Gag, but not the internalization-defective mutation, can be induced to accumulate within CD63-positive MVBs by treatment of cells with U18666A, a drug that redistributes cholesterol from the plasma membrane to MVBs. We propose that HIV-1 Gag contains a signal that promotes interaction with the cellular endocytic machinery and that the site of particle production is regulated by the subcellular distribution of cholesterol.
Respiratory Syncytial Virus (RSV) G Glycoprotein Is Not Necessary for Vaccine-Enhanced Disease Induced by Immunization with Formalin-Inactivated RSV
Authors: Johnson Teresa R. , Teng Michael N. , Collins Peter L. , Graham Barney S.
Published: May 2004

Following respiratory syncytial virus (RSV) challenge, mice immunized with RSV G or with formalin-inactivated RSV (FI-RSV) exhibit severe disease associated with type 2 cytokine production and pulmonary eosinophilia. This has led to the proposal that the presence of RSV G is the factor in FI-RSV that induces disease-enhancing T-cell responses. Therefore, we evaluated the role of RSV G and its immunodominant region in the induction of aberrant immune responses during FI-RSV immunization. BALB/c mice were immunized with FI preparations of wild-type (wt) RSV or recombinant RSV (rRSV) containing deletions of (i) the entire G gene, (ii) the region of the G gene encoding amino acids 187 to 197 of the immunodominant region, or (iii) the entire SH gene. After challenge, illness, RSV titers, cytokine levels, and pulmonary eosinophilia were measured. Peak RSV titers postchallenge were significantly greater in mice immunized with FI preparations of the deletion viruses than in those immunized with FI-rRSV wt, suggesting that the absence of G or SH in FI-RSV reduced its protective efficacy. Deletion of G or its epitope did not reduce illness, cytokine production, or eosinophilia relative to that in mice immunized with FI-rRSV wt. While cytokine levels and eosinophilia were similar, illness was reduced in mice immunized with SH-deleted FI-RSV. These data suggest that G-specific immune responses may be important for vaccine-induced protection and are not solely the basis for FI-RSV vaccine-enhanced illness. These data suggest that the method of RSV antigen delivery, rather than the protein composition, influences the phenotype of the induced immune responses and that RSV G should not necessarily be excluded from potential vaccine strategies.
Recombinant Sendai Virus Expressing the G Glycoprotein of Respiratory Syncytial Virus (RSV) Elicits Immune Protection against RSV
Authors: Takimoto Toru , Hurwitz Julia L. , Coleclough Chris , Prouser Cecilia , Krishnamurthy Sateesh , Zhan Xiaoyan , Boyd Kelli , Scroggs Ruth A. , Brown Brita , Nagai Yoshiyuki , Portner Allen , Slobod Karen S.
Published: May 2004

Although RSV causes serious pediatric respiratory disease, an effective vaccine does not exist. To capture the strengths of a live virus vaccine, we have used the murine parainfluenza virus type 1 (Sendai virus [SV]) as a xenogeneic vector to deliver the G glycoprotein of RSV. It was previously shown (J. L. Hurwitz, K. F. Soike, M. Y. Sangster, A. Portner, R. E. Sealy, D. H. Dawson, and C. Coleclough, Vaccine 15:533-540, 1997) that intranasal SV protected African green monkeys from challenge with the related human parainfluenza virus type 1 (hPIV1), and SV has advanced to clinical trials as a vaccine for hPIV1 (K. S. Slobod, J. L. Shenep, J. Lujan-Zilbermann, K. Allison, B. Brown, R. A. Scroggs, A. Portner, C. Coleclough, and J. L. Hurwitz, Vaccine, in press). Recombinant SV expressing RSV G glycoprotein was prepared by using reverse genetics, and intranasal inoculation of cotton rats elicited RSV-specific antibody and elicited protection from RSV challenge. RSV G-recombinant SV is thus a promising live virus vaccine candidate for RSV.
Cleavage Inhibition of the Murine Coronavirus Spike Protein by a Furin-Like Enzyme Affects Cell-Cell but Not Virus-Cell Fusion
Authors: de Haan Cornelis A. M. , Stadler Konrad , Godeke Gert-Jan , Bosch Berend Jan , Rottier Peter J. M.
Published: May 2004

Cleavage of the mouse hepatitis coronavirus strain A59 spike protein was blocked in a concentration-dependent manner by a peptide furin inhibitor, indicating that furin or a furin-like enzyme is responsible for this process. While cell-cell fusion was clearly affected by preventing spike protein cleavage, virus-cell fusion was not, indicating that these events have different requirements.
CD40-Associated TRAF 6 Signaling Is Required for Disease Induction in a Retrovirus-Induced Murine Immunodeficiency
Authors: Green Kathy A. , Ahonen Cory L. , Cook W. James , Green William R.
Published: May 2004

LP-BM5 retrovirus-infected C57BL/6 mice develop splenomegaly, lymphadenopathy, hypergammaglobulinemia, and immunodeficiency; thus, this disease has been named mouse AIDS. In this syndrome, CD154/CD40 interactions are required for but do not mediate disease by upregulation of CD80 or CD86. We report here that there is nonetheless a necessity for CD40 signaling competence, specifically an intact tumor necrosis factor receptor-associated factor 6 (TRAF 6) binding site.
CKII Site in Epstein-Barr Virus Nuclear Protein 2 Controls Binding to hSNF5/Ini1 and Is Important for Growth Transformation
Authors: Kwiatkowski Bogaslaw , Chen Szu Yu Jenny , Schubach William H.
Published: May 2004

Substitution mutagenesis of EBNA2 shows that its interaction with hSNF5/Ini1 involves two sites (286IPP and DQQ313), and a mutation at a CKII phosphorylation site (SS469) is essential for the interaction. An alanine substitution (SS469AA) prevents binding to EBNA2 and diminishes the growth-promotion potential of EBNA2 in the transcomplementation assay.
Human APOBEC3F Is Another Host Factor That Blocks Human Immunodeficiency Virus Type 1 Replication
Authors: Zheng Yong-Hui , Irwin Dan , Kurosu Takeshi , Tokunaga Kenzo , Sata Tetsutaro , Peterlin B. Matija
Published: May 2004

Recently, APOBEC3G has been identified as a host factor that blocks retroviral replication. It introduces G to A hypermutations in newly synthesized minus strand viral cDNA at the step of reverse transcription in target cells. Here, we identified the human APOBEC3F protein as another host factor that blocks human immunodeficiency virus type 1 (HIV-1) replication. Similar to APOBEC3G, APOBEC3F also induced G to A hypermutations in HIV genomic DNA, and the viral Vif protein counteracted its activity. Thus, APOBEC family members might have evolved as a general defense mechanism of the body against retroviruses, retrotransposons, and other mobile genetic elements.
Information Content and Complexity in the High-Order Organization of DNA
Authors: Minsky Abraham
Published: May 2004

▪ Abstract  Nucleic acids are characterized by a vast structural variability. Secondary structural conformations include the main polymorphs A, B, and Z, cruciforms, intrinsic curvature, and multistranded motifs. DNA secondary motifs are stabilized and regulated by the primary base sequence, contextual effects, environmental factors, as well as by high-order DNA packaging modes. The high-order modes are, in turn, affected by secondary structures and by the environment. This review is concerned with the flow of structural information among the hierarchical structural levels of DNA molecules, the intricate interplay between the various factors that affect these levels, and the regulation and physiological significance of DNA high-order structures.
Three-Dimensional Electron Microscopy at Molecular Resolution
Authors: Subramaniam Sriram , Milne Jacqueline L.S.
Published: May 2004

▪ Abstract  Emerging methods in cryo-electron microscopy allow determination of the three-dimensional architectures of objects ranging in size from small proteins to large eukaryotic cells, spanning a size range of more than 12 orders of magnitude. Advances in determining structures by “single particle” microscopy and by “electron tomography” provide exciting opportunities to describe the structures of subcellular assemblies that are either too large or too heterogeneous to be investigated by conventional crystallographic methods. Here, we review selected aspects of progress in structure determination by cryo-electron microscopy at molecular resolution, with a particular emphasis on topics at the interface of single particle and tomographic approaches. The rapid pace of development in this field suggests that comprehensive descriptions of the structures of whole cells and organelles in terms of the spatial arrangements of their molecular components may soon become routine.
Mass Spectral Analysis in Proteomics
Authors: Yates John R.
Published: May 2004

▪ Abstract  Mass spectrometry provides key tools for the analysis of proteins. New types of mass spectrometers that provide enhanced capability to discover protein identities and perform improved proteomic experiments are discussed. Handling the complex mixtures of peptides and proteins generated from protein complexes and whole cells requires multidimensional separations; several forms of separation are discussed. Applications of mass spectrometry–based approaches for contemporary proteomic analyses are described.