bluuebunny/crossref_metadata_2025 huggingface dataset:
Genetic Diversity among Lassa Virus Strains
Authors: Bowen Michael D. , Rollin Pierre E. , Ksiazek Thomas G. , Hustad Heather L. , Bausch Daniel G. , Demby Austin H. , Bajani Mary D. , Peters Clarence J. , Nichol Stuart T.
Published: July 2002

The arenavirus Lassa virus causes Lassa fever, a viral hemorrhagic fever that is endemic in the countries of Nigeria, Sierra Leone, Liberia, and Guinea and perhaps elsewhere in West Africa. To determine the degree of genetic diversity among Lassa virus strains, partial nucleoprotein (NP) gene sequences were obtained from 54 strains and analyzed. Phylogenetic analyses showed that Lassa viruses comprise four lineages, three of which are found in Nigeria and the fourth in Guinea, Liberia, and Sierra Leone. Overall strain variation in the partial NP gene sequence was found to be as high as 27% at the nucleotide level and 15% at the amino acid level. Genetic distance among Lassa strains was found to correlate with geographic distance rather than time, and no evidence of a “molecular clock” was found. A method for amplifying and cloning full-length arenavirus S RNAs was developed and used to obtain the complete NP and glycoprotein gene (GP1 and GP2) sequences for two representative Nigerian strains of Lassa virus. Comparison of full-length gene sequences for four Lassa virus strains representing the four lineages showed that the NP gene (up to 23.8% nucleotide difference and 12.0% amino acid difference) is more variable than the glycoprotein genes. Although the evolutionary order of descent within Lassa virus strains was not completely resolved, the phylogenetic analyses of full-length NP, GP1, and GP2 gene sequences suggested that Nigerian strains of Lassa virus were ancestral to strains from Guinea, Liberia, and Sierra Leone. Compared to the New World arenaviruses, Lassa and the other Old World arenaviruses have either undergone a shorter period of diverisification or are evolving at a slower rate. This study represents the first large-scale examination of Lassa virus genetic variation.
Hairy Leukoplakia: an Unusual Combination of Transforming and Permissive Epstein-Barr Virus Infections
Authors: Webster-Cyriaque J. , Middeldorp J. , Raab-Traub N.
Published: July 2002

Human herpesviruses are characterized by distinct states of infection. Typically in permissive herpesvirus infection, abundant virus production results in cell lysis. In latent transforming Epstein-Barr virus (EBV) infection, viral proteins that induce cell growth are expressed. The immunodeficiency-associated hairy leukoplakia (HLP) lesion is the only pathologic manifestation of permissive EBV infection; however, within HLP, viral proteins characteristic of latent infection have also been detected. In this study, we further analyzed expression of EBV latent genes and investigated their contribution to the unique histologic phenotype of HLP. Coexpression of lytic and transforming viral proteins was detected simultaneously within individual HLP keratinocytes. LMP1 has now been shown to be uniformly expressed in the affected tissue, and it is associated and colocalizes with tumor necrosis factor receptor-associated factor (TRAF) signaling molecules. Effects induced by activated TRAF signaling that were detected in HLP included activation of NF-κB and c-Jun terminal kinase 1 (JNK1) and upregulated expression of epidermal growth factor receptor (EGFR), CD40, A20, and TRAFs. This study identifies a novel state of EBV infection with concurrent expression of replicative and transforming proteins. It is probable that both replicative and latent proteins contribute to HLP development and induce many of the histologic features of HLP, such as acanthosis and hyperproliferation. In contrast to other permissive herpesvirus infections, expression of EBV transforming proteins within the permissively infected HLP tissue enables epithelial cell survival and may enhance viral replication.
Infection of Nonhuman Primate Cells by Pig Endogenous Retrovirus
Authors: Blusch Juergen H. , Patience Clive , Takeuchi Yasuhiro , Templin Christian , Roos Christian , Von Der Helm Klaus , Steinhoff Gustav , Martin Ulrich
Published: July 2002

The ongoing shortage of human donor organs for transplantation has catalyzed new interest in the application of pig organs (xenotransplantation). One of the biggest concerns about the transplantation of porcine grafts into humans is the transmission of pig endogenous retroviruses (PERV) to the recipients or even to other members of the community. Although nonhuman primate models are excellently suited to mimic clinical xenotransplantation settings, their value for risk assessment of PERV transmission at xenotransplantation is questionable since all of the primate cell lines tested so far have been found to be nonpermissive for PERV infection. Here we demonstrate that human, gorilla, and
Papio hamadryas
primary skin fibroblasts and also baboon B-cell lines are permissive for PERV infection. This suggests that a reevaluation of the suitability of the baboon model for risk assessment in xenotransplantation is critical at this point.
Infection of Human Cells by Dengue Virus Is Modulated by Different Cell Types and Viral Strains
Authors: Diamond Michael S. , Edgil Dianna , Roberts T. Guy , Lu Betty , Harris Eva
Published: July 2002

Although prior studies have investigated cellular infection by dengue virus (DV), many have used highly passaged strains. We have reassessed cellular infection by DV type 2 (DV2) using prototype and low-passage isolates representing genotypes from different geographic areas. We observed marked variation in the susceptibility to infection among cell types by different DV2 strains. HepG2 hepatoma cells were susceptible to infection by all DV2 strains assayed. Although the prototype strain generated higher titers of secreted virus than the low-passage isolates, this difference did not correspond to positive- or negative-strand viral RNA levels and thus may reflect variation in efficiency among DV2 isolates to translate viral proteins or package and/or secrete virus. In contrast, human foreskin fibroblasts were susceptible to the prototype and low-passage Thai isolates but not to five Nicaraguan strains tested, as reflected by the absence of accumulation of negative-strand viral RNA, viral antigen, and infectious virus. A similar pattern was observed with the antibody-dependent pathway of infection. U937 and THP-1 myeloid cells and peripheral blood monocytes were infected in the presence of enhancing antibodies by the prototype strain but not by low-passage Nicaraguan isolates. Again, the barrier appeared to be prior to negative-strand accumulation. Thus, depending on the cell type and viral isolate, blocks that limit the production of infectious virus in vitro may occur at distinct steps in the pathway of cellular infection.
The 5′ RNA Terminus of Spleen Necrosis Virus Stimulates Translation of Nonviral mRNA
Authors: Roberts Tiffiney M. , Boris-Lawrie Kathleen
Published: July 2002

The RU5 region at the 5′ RNA terminus of spleen necrosis virus (SNV) has been shown to facilitate expression of human immunodeficiency virus type 1 (HIV) unspliced RNA independently of the Rev-responsive element (RRE) and Rev. The SNV sequences act as a distinct posttranscriptional control element to stimulate
gag
RNA nuclear export and association with polyribosomes. Here we sought to determine whether RU5 functions to neutralize the
cis
-acting inhibitory sequences (INSs) in HIV RNA that confer RRE/Rev dependence or functions as an independent stimulatory sequence. Experiments with HIV
gag
reporter plasmids that contain inactivated INS-1 indicated that neutralization of INSs does not account for RU5 function. Results with luciferase reporter gene (
luc
) plasmids further indicated that RU5 stimulates expression of a nonretroviral RNA that lacks INSs. Northern blot and RT-PCR analyses indicated that RU5 does not increase the steady-state levels or nuclear export of the
luc
transcript but rather that the U5 region facilitates efficient polyribosomal association of the mRNA. RU5 does not function as an internal ribosome entry site in bicistronic reporter plasmids, and it requires the 5′-proximal position for efficient function. Our results indicate that RU5 contains stimulatory sequences that function in a 5′-proximal position to enhance initiation of translation of a nonretroviral reporter gene RNA. We speculate that RU5 evolved to overcome the translation-inhibitory effect of the highly structured encapsidation signal and other replication motifs in the 5′ untranslated region of the retroviral RNA.
Characterization of a Novel Human Herpesvirus 8-Encoded Protein, vIRF-3, That Shows Homology to Viral and Cellular Interferon Regulatory Factors
Authors: Lubyova Barbora , Pitha Paula M.
Published: July 2002

The genome of the human herpesvirus 8 (HHV-8) contains a cluster of open reading frames (ORFs) encoding proteins with homology to the cellular transcription factors of the interferon regulatory factor (IRF) family. Two of these homologues, vIRF-1 and vIRF-2, were previously identified and functionally analyzed. In this study, we have characterized a novel gene, designated vIRF-3, encoded within the previously predicted ORF K10.5 and our newly identified ORF K10.6. Northern blotting of RNA extracted from BCBL-1 cells with a vIRF-3-specific probe and reverse transcription-PCR analyses revealed a single transcript of 2.2 kb with a splice present in the coding region. The vIRF-3 mRNA levels in BCBL-1 cells were increased upon 12-O-tetradecanoylphorbol-13-acetate treatment, with kinetics of expression similar to those of the early immediate genes. The vIRF-3 ORF encodes a 73-kDa protein with homology to cellular IRF-4 and HHV-8-encoded vIRF-2 and K11. In transient transfection assays with the IFNACAT reporter, vIRF-3 functioned as a dominant-negative mutant of both IRF-3 and IRF-7 and inhibited virus-mediated transcriptional activity of the IFNA promoter. Similarly, the overexpression of vIRF-3 in mouse L929 cells resulted in inhibition of virus-mediated synthesis of biologically active interferons. These results suggest that by targeting IRF-3 and IRF-7, which play a critical role in the activation of alpha/beta interferon (IFN) genes, HHV-8 has evolved a mechanism by which it directly subverts the functions of IRFs and down-regulates the induction of the IFN genes that are important components of the innate immunity.
A Monomeric GTPase-Negative MxA Mutant with Antiviral Activity
Authors: Janzen Christian , Kochs Georg , Haller Otto
Published: July 2002

MxA is a large, interferon-induced GTPase with antiviral activity against RNA viruses. It forms large oligomers, but whether oligomerization and GTPase activity are important for antiviral function is not known. The mutant protein MxA(L612K) carries a lysine-for-leucine substitution at position 612 and fails to form oligomers. Here we show that monomeric MxA(L612K) lacks detectable GTPase activity but is capable of inhibiting Thogoto virus in transiently transfected Vero cells or in a Thogoto virus minireplicon system. Likewise, MxA(L612K) inhibited vesicular stomatitis virus multiplication. These findings indicate that MxA monomers are antivirally active and suggest that GTP hydrolysis may not be required for antiviral activity. MxA(L612K) is rapidly degraded in cells, whereas wild-type MxA is stable. We propose that high-molecular-weight MxA oligomers represent a stable intracellular pool from which active MxA monomers are recruited.
Avian Nephritis Virus (ANV) as a New Member of the Family
Astroviridae
and Construction of Infectious ANV cDNA
Authors: Imada Tadao , Yamaguchi Shigeo , Mase Masaji , Tsukamoto Kenji , Kubo Masanori , Morooka Akira
Published: July 2002

The complete RNA genome of the avian nephritis virus (ANV) associated with acute nephritis in chickens has been molecularly cloned and sequenced. Excluding the poly(A) tail, the genome comprises 6,927 nucleotides and contains three sequential open reading frames (ORFs). The first ORF (ORF 1a) contains a sequence encoding a serine protease motif, and the second ORF (ORF 1b) has a sequence encoding an RNA-dependent RNA polymerase. ORF 1a may be linked to the second ORF by a ribosomal frameshifting mechanism. The third ORF (ORF 2) may encode the virion structural proteins as a polyprotein precursor. Two RNAs, probably genonic and subgenonic RNA (7.5 and 3.0 kb), were detected in the cytoplasm of ANV-infected cells. ANV and human astroviruses have the same genonic organization, and both are characterized by the presence of two RNA bands. The amino acid homologies of the products of ORF 1a, 1b, and 2 were 20.3, 41.9, and 25.8% to products of the corresponding ORFs of human astrovirus serotype 1 (A/88 Newcastle strain). We have constructed a genonic-length cDNA clone of ANV to test whether the in vitro transcript is infectious. When a chicken kidney cell culture was transfected with RNA transcribed in vitro and the cDNA clone, infectious virus was produced with cytopathic effects in the absence of trypsin. These observations suggested that the ANV (G-4260 strain) is a new genus of the family
Astroviridae
.
Localization of Human Immunodeficiency Virus Type 1 Gag and Env at the Plasma Membrane by Confocal Imaging
Authors: Hermida-Matsumoto Luz , Resh Marilyn D.
Published: July 2002

Budding of lentiviruses occurs at the plasma membrane, but the preceding steps involved in particle assembly are poorly understood. Since the Gag polyprotein mediates virion assembly and budding, studies on the localization of Gag within the cell should provide insight into the mechanism of particle assembly. Here, we utilize biochemical fractionation techniques as well as high-resolution confocal imaging of live cells to demonstrate that Gag is localized at the plasma membrane in a striking punctate pattern. Mutation of the N-terminal myristoylation site results in the formation of large cytosolic complexes, whereas mutation of the N-terminal basic residue cluster in the matrix domain redirects the Gag protein to a region partially overlapping the Golgi apparatus. In addition, we show that Gag and Env colocalize at the plasma membrane and that mistargeting of a mutant Gag to the Golgi apparatus alters the pattern of surface expression of Env.
Zinc Finger Structures in the Human Immunodeficiency Virus Type 1 Nucleocapsid Protein Facilitate Efficient Minus- and Plus-Strand Transfer
Authors: Guo Jianhui , Wu Tiyun , Anderson Jada , Kane Bradley F. , Johnson Donald G. , Gorelick Robert J. , Henderson Louis E. , Levin Judith G.
Published: July 2002

The nucleocapsid protein (NC) of human immunodeficiency virus type 1 (HIV-1) has two zinc fingers, each containing the invariant metal ion binding residues CCHC. Recent reports indicate that mutations in the CCHC motifs are deleterious for reverse transcription in vivo. To identify reverse transcriptase (RT) reactions affected by such changes, we have probed zinc finger functions in NC-dependent RT-catalyzed HIV-1 minus- and plus-strand transfer model systems. Our approach was to examine the activities of wild-type NC and a mutant in which all six cysteine residues were replaced by serine (SSHS NC); this mutation severely disrupts zinc coordination. We find that the zinc fingers contribute to the role of NC in complete tRNA primer removal from minus-strand DNA during plus-strand transfer. Annealing of the primer binding site sequences in plus-strand strong-stop DNA [(+) SSDNA] to its complement in minus-strand acceptor DNA is not dependent on NC zinc fingers. In contrast, the rate of annealing of the complementary R regions in (−) SSDNA and 3′ viral RNA during minus-strand transfer is approximately eightfold lower when SSHS NC is used in place of wild-type NC. Moreover, unlike wild-type NC, SSHS NC has only a small stimulatory effect on minus-strand transfer and is essentially unable to block TAR-induced self-priming from (−) SSDNA. Our results strongly suggest that NC zinc finger structures are needed to unfold highly structured RNA and DNA strand transfer intermediates. Thus, it appears that in these cases, zinc finger interactions are important components of NC nucleic acid chaperone activity.
The LMP2A ITAM Is Essential for Providing B Cells with Development and Survival Signals In Vivo
Authors: Merchant Mark , Caldwell Robert G. , Longnecker Richard
Published: July 2002

In Epstein-Barr virus-transformed B cells, known as lymphoblastoid cell lines (LCLs), LMP2A binds the tyrosine kinases Syk and Lyn, blocking B-cell receptor (BCR) signaling and viral lytic replication. SH2 domains in Syk mediate binding to a phosphorylated immunoreceptor tyrosine-based activation motif (ITAM) in LMP2A. Mutation of the LMP2A ITAM in LCLs eliminates Syk binding and allows for full BCR signaling, thereby delineating the significance of the LMP2A-Syk interaction. In transgenic mice, LMP2A causes a developmental alteration characterized by a block in surface immunoglobulin rearrangement resulting in BCR-negative B cells. Normally B cells lacking cognate BCR are rapidly apoptosed; however, LMP2A transgenic B cells develop and survive without a BCR. When bred into the recombinase activating gene 1 null (RAG−/−) background, all LMP2A transgenic lines produce BCR-negative B cells that develop and survive in the periphery. These data indicate that LMP2A imparts developmental and survival signals to B cells in vivo. In this study, LMP2A ITAM mutant transgenic mice were generated to investigate whether the LMP2A ITAM is essential for the survival phenotype in vivo. LMP2A ITAM mutant B cells develop normally, although transgene expression is comparable to that in previously described nonmutated LMP2A transgenic B cells. Additionally, LMP2A ITAM mutant mice are unable to promote B-cell development or survival when bred into the RAG−/−background or when grown in methylcellulose containing interleukin-7. These data demonstrate that the LMP2A ITAM is required for LMP2A-mediated developmental and survival signals in vivo.
Large-Plaque Mutants of Sindbis Virus Show Reduced Binding to Heparan Sulfate, Heightened Viremia, and Slower Clearance from the Circulation
Authors: Byrnes Andrew P. , Griffin Diane E.
Published: July 2002

Laboratory strains of Sindbis virus must bind to the negatively charged glycosaminoglycan heparan sulfate in order to efficiently infect cultured cells. During infection of mice, however, we have frequently observed the development of large-plaque viral mutants with a reduced ability to bind to heparan sulfate. Sequencing of these mutants revealed changes of positively charged amino acids in putative heparin-binding domains of the E2 glycoprotein. Recombinant viruses were constructed with these changes as single amino acid substitutions in a strain Toto 1101 background. All exhibited decreased binding to heparan sulfate and had larger plaques than Toto 1101. When injected subcutaneously into neonatal mice, large-plaque viruses produced higher-titer viremia and often caused higher mortality. Because circulating heparin-binding proteins are known to be rapidly sequestered by tissue heparan sulfate, we measured the kinetics of viral clearance following intravenous injection. Much of the parental small-plaque Toto 1101 strain of Sindbis virus was cleared from the circulation by the liver within minutes, in contrast to recombinant large-plaque viruses, which had longer circulating half-lives. These findings indicate that a decreased ability to bind to heparan sulfate allows more efficient viral production in vivo, which may in turn lead to increased mortality. Because Sindbis virus is only one of a growing number of viruses from many families which have been shown to bind to heparan sulfate, these results may be generally applicable to the pathogenesis of such viruses.
H9N2 Influenza Viruses Possessing H5N1-Like Internal Genomes Continue To Circulate in Poultry in Southeastern China
Authors: Guan Y. , Shortridge K. F. , Krauss S. , Chin P. S. , Dyrting K. C. , Ellis T. M. , Webster R. G. , Peiris M.
Published: July 2002

The transmission of H9N2 influenza viruses to humans and the realization that the A/Hong Kong/156/97-like (H5N1) (abbreviated HK/156/97) genome complex may be present in H9N2 viruses in southeastern China necessitated a study of the distribution and characterization of H9N2 viruses in poultry in the Hong Kong SAR in 1999. Serological studies indicated that H9N2 influenza viruses had infected a high proportion of chickens and other land-based birds (pigeon, pheasant, quail, guinea fowl, and chukka) from southeastern China. Two lineages of H9N2 influenza viruses present in the live-poultry markets were represented by A/Quail/Hong Kong/G1/97 (Qa/HK/G1/97)-like and A/Duck/Hong Kong/Y280/97 (Dk/HK/Y280/97)-like viruses. Up to 16% of cages of quail in the poultry markets contained Qa/HK/G1/97-like viruses, while about 5% of cages of other land-based birds were infected with Dk/HK/Y280/97-like viruses. No reassortant between the two H9N2 virus lineages was detected despite their cocirculation in the poultry markets. Reassortant viruses represented by A/Chicken/Hong Kong/G9/97 (H9N2) were the major H9N2 influenza viruses circulating in the Hong Kong markets in 1997 but have not been detected since the chicken slaughter in 1997. The Qa/HK/G1/97-like viruses were frequently isolated from quail, while Dk/HK/Y280/97-like viruses were predominately associated with chickens. The Qa/HK/G1/97-like viruses were evolving relatively rapidly, especially in their PB2, HA, NP, and NA genes, suggesting that they are in the process of adapting to a new host. Experimental studies showed that both H9N2 lineages were primarily spread by the aerosol route and that neither quail nor chickens showed evidence of disease. The high prevalence of quail infected with Qa/HK/G1/97-like virus that contains six gene segments genetically highly related to HK/156/97 (H5N1) virus emphasizes the need for surveillance of mammals including humans.
Recombinant Adenovirus Induces Maturation of Dendritic Cells via an NF-κB-Dependent Pathway
Authors: Morelli Adrian E. , Larregina Adriana T. , Ganster Raymond W. , Zahorchak Alan F. , Plowey Jeffrey M. , Takayama Takuya , Logar Alison J. , Robbins Paul D. , Falo Louis D. , Thomson Angus W.
Published: July 2002

Recombinant adenovirus (rAd) infection is one of the most effective and frequently employed methods to transduce dendritic cells (DC). Contradictory results have been reported recently concerning the influence of rAd on the differentiation and activation of DC. In this report, we show that, as a result of rAd infection, mouse bone marrow-derived immature DC upregulate expression of major histocompatibility complex class I and II antigens, costimulatory molecules (CD40, CD80, and CD86), and the adhesion molecule CD54 (ICAM-1). rAd-transduced DC exhibited increased allostimulatory capacity and levels of interleukin-6 (IL-6), IL-12p40, IL-15, gamma interferon, and tumor necrosis factor alpha mRNAs, without effects on other immunoregulatory cytokine transcripts such as IL-10 or IL-12p35. These effects were not related to specific transgenic sequences or to rAd genome transcription. The rAd effect correlated with a rapid increase (1 h) in the NF-κB–DNA binding activity detected by electrophoretic mobility shift assays. rAd-induced DC maturation was blocked by the proteasome inhibitorNα-p-tosyl-l-lysine chloromethyl ketone (TLCK) or by infection with rAd-IκB, an rAd-encoding the dominant-negative form of IκB. In vivo studies showed that after intravenous administration, rAds were rapidly entrapped in the spleen by marginal zone DC that mobilized to T-cell areas, a phenomenon suggesting that rAd also induced DC differentiation in vivo. These findings may explain the immunogenicity of rAd and the difficulties in inducing long-term antigen-specific T-cell hyporesponsiveness with rAd-transduced DC.
Retroviral cDNA Integration: Stimulation by HMG I Family Proteins
Authors: Li Ling , Yoder Kristine , Hansen Mark S. T. , Olvera Jennifer , Miller Michael D. , Bushman Frederic D.
Published: July 2002

To replicate, a retrovirus must synthesize a cDNA copy of the viral RNA genome and integrate that cDNA into a chromosome of the host. We have investigated the role of a host cell cofactor, HMG I(Y) protein, in integration of human immunodeficiency virus type 1 (HIV-1) and Moloney murine leukemia virus (MoMLV) cDNA. Previously we reported that HMG I(Y) cofractionates with HIV-1 preintegration complexes (PICs) isolated from freshly infected cells. PICs depleted of required components by treatment with high concentrations of salt could be reconstituted by addition of purified HMG I(Y) in vitro. Here we report studies using immunoprecipitation that indicate that HMG I(Y) is associated with MoMLV preintegration complexes. In mechanistic studies, we show for both HIV-1 and MoMLV that each HMG I(Y) monomer must contain multiple DNA binding domains to stimulate integration by HMG I(Y)-depleted PICs. We also find that HMG I(Y) can condense model HIV-1 or MoMLV cDNA in vitro as measured by stimulation of intermolecular ligation. This reaction, like reconstitution of integration, depends on the presence of multiple DNA binding domains in each HMG I(Y) monomer. These data suggest that binding of multivalent HMG I(Y) monomers to multiple cDNA sites compacts retroviral cDNA, thereby promoting formation of active integrase-cDNA complexes.
Template Requirements for RNA Synthesis by a Recombinant Hepatitis C Virus RNA-Dependent RNA Polymerase
Authors: Kao C. Cheng , Yang Xueyong , Kline Allen , Wang Q. May , Barket Donna , Heinz Beverly A.
Published: July 2002

The RNA-dependent RNA polymerase (RdRp) from hepatitis C virus (HCV), nonstructural protein 5B (NS5B), has recently been shown to direct de novo initiation using a number of complex RNA templates. In this study, we analyzed the features in simple RNA templates that are required to direct de novo initiation of RNA synthesis by HCV NS5B. NS5B was found to protect RNA fragments of 8 to 10 nucleotides (nt) from RNase digestion. However, NS5B could not direct RNA synthesis unless the template contained a stable secondary structure and a single-stranded sequence that contained at least one 3′ cytidylate. The structure of a 25-nt template, named SLD3, was determined by nuclear magnetic resonance spectroscopy to contain an 8-bp stem and a 6-nt single-stranded sequence. Systematic analysis of changes in SLD3 revealed which features in the stem, loop, and 3′ single-stranded sequence were required for efficient RNA synthesis. Also, chimeric molecules composed of DNA and RNA demonstrated that a DNA molecule containing a 3′-terminal ribocytidylate was able to direct RNA synthesis as efficiently as a sequence composed entirely of RNA. These results define the template sequence and structure sufficient to direct the de novo initiation of RNA synthesis by HCV RdRp.
The Major Immediate-Early Gene
ie3
of Mouse Cytomegalovirus Is Essential for Viral Growth
Authors: Angulo Ana , Ghazal Peter , Messerle Martin
Published: July 2002

The significance of the major immediate-early gene
ie3
of mouse cytomegalovirus (MCMV) and that of the corresponding
ie2
gene of human cytomegalovirus to viral replication are not known. To investigate the function of the MCMV IE3 regulatory protein, we generated two different MCMV recombinants that contained a large deletion in the IE3 open reading frame (ORF). The mutant genomes were constructed by the bacterial artificial chromosome mutagenesis technique, and MCMV
ie3
deletion mutants were reconstituted on a mouse fibroblast cell line that expresses the MCMV major immediate-early genes. The
ie3
deletion mutants failed to replicate on normal mouse fibroblasts even when a high multiplicity of infection was used. The replication defect was rescued when the IE3 protein was provided in
trans
by a complementing cell line. A revertant virus in which the IE3 ORF was restored was able to replicate with wild-type kinetics in normal mouse fibroblasts, providing evidence that the defective growth phenotype of the
ie3
mutants was due to disruption of the
ie3
gene. To characterize the point of restriction in viral replication that is controlled by
ie3
, we analyzed the pattern of expression of selective early (β) and late (γ) genes. While we could detect transcripts for the immediate-early gene
ie1
in cells infected with the
ie3
mutants, we failed to detect transcripts for representative β and γ genes. These data demonstrate that the MCMV transactivator IE3 plays an indispensable role during viral replication in tissue culture, implicating a similar role for the human CMV
ie2
gene product. To our knowledge, the
ie3
deletion mutants represent the first MCMV recombinants isolated that contain a disruption of an essential gene.
The Replication Activity of Influenza Virus Polymerase Is Linked to the Capacity of the PA Subunit To Induce Proteolysis
Authors: Perales Beatriz , Sanz-Ezquerro Juan J. , Gastaminza Pablo , Ortega Joaquin , Santarén Juan Férnandez , Ortín Juan , Nieto Amelia
Published: July 2002

The PA subunit of the influenza virus polymerase complex is a phosphorylated protein that induces a proteolytic process that decreases its own accumulation levels and those of coexpressed proteins. The amino-terminal third of the protein is responsible for the induction of proteolysis. We mutated five potential casein kinase II phosphorylation sites located in the amino-terminal third of the protein. Mutations affecting position 157 almost completely abrogated proteolysis induction, whereas a mutation at position 162 produced a moderate decrease and mutations at positions 151, 200, and 224 did not affect proteolysis induction. Reconstitution of the influenza virus polymerase in vivo with viral model RNA containing the chloramphenicol acetyltransferase (CAT) gene indicated that the CAT activity obtained correlated with the capacity of each PA mutant to induce proteolysis. RNA protection assays of the products obtained with viral polymerase, reconstituted in vivo with model RNAs, indicated that mutations at position 157 led to a selective loss of the ability to synthesize cRNA from the viral RNA template but not to transcribe viral RNA, while a mutation affecting position 162 showed an intermediate phenotype. Collectively, these data provide a link between PA-mediated induction of proteolysis and the replication activity of the polymerase.
Productive Measles Virus Brain Infection and Apoptosis in CD46 Transgenic Mice
Authors: Evlashev Alexey , Moyse Emmanuel , Valentin Hélène , Azocar Olga , Trescol-Biémont Marie-Claude , Marie Julien C. , Rabourdin-Combe Chantal , Horvat Branka
Published: July 2002

Measles virus (MV) infection causes acute childhood disease, associated in certain cases with infection of the central nervous system (CNS) and development of neurological disease. To develop a murine model of MV-induced pathology, we generated several lines of transgenic mice ubiquitously expressing as the MV receptor a human CD46 molecule with either a Cyt1 or Cyt2 cytoplasmic tail. All transgenic lines expressed CD46 protein in the brain. Newborn transgenic mice, in contrast to nontransgenic controls, were highly sensitive to intracerebral infection by the MV Edmonston strain. Signs of clinical illness (lack of mobility, tremors, and weight loss) appeared within 5 to 7 days after infection, followed by seizures, paralysis, and death of the infected animals. Virus replication was detected in neurons from infected mice, and virus was reproducibly isolated from transgenic brain tissue. MV-induced apoptosis observed in different brain regions preceded the death of infected animals. Similar results were obtained with mice expressing either a Cyt1 or Cyt2 cytoplasmic tail, demonstrating the ability of different isoforms of CD46 to function as MV receptors in vivo. In addition, maternally transferred immunity delayed death of offspring given a lethal dose of MV. These results document a novel CD46 transgenic murine model where MV neuronal infection is associated with the production of infectious virus, similarly to progressive infectious measles encephalitis seen in immunocompromised patients, and provide a new means to study pathogenesis of MV infection in the CNS.
The Central Proline of an Internal Viral Fusion Peptide Serves Two Important Roles
Authors: Delos S. E. , Gilbert J. M. , White J. M.
Published: July 2002

The fusion peptide of the avian sarcoma/leukosis virus (ASLV) envelope protein (Env) is internal, near the N terminus of its transmembrane (TM) subunit. As for most internal viral fusion peptides, there is a proline near the center of this sequence. Robson-Garnier structure predictions of the ASLV fusion peptide and immediate surrounding sequences indicate a region of order (β-sheet), a tight reverse turn containing the proline, and a second region of order (α-helix). Similar motifs (order, turn or loop, order) are predicted for other internal fusion peptides. In this study, we made and analyzed 12 Env proteins with substitutions for the central proline of the fusion peptide. Env proteins were expressed in 293T cells and in murine leukemia virus pseudotyped virions. We found the following. (i) All mutant Envs form trimers, but when the bulky hydrophobic residues phenylalanine or leucine are substituted for proline, trimerization is weakened. (ii) Surprisingly, the proline is required for maximal processing of the Env precursor into its surface and TM subunits; the amount of processing correlates linearly with the propensity of the substituted residue to be found in a reverse turn. (iii) Nonetheless, proteolytically processed forms of all Envs are preferentially incorporated into pseudotyped virions. (iv) All Envs bind receptor with affinity greater than or equal to wild-type affinity. (v) Residues that support high infectivity cluster with proline at intermediate hydrophobicity. Infectivity is not supported by mutant Envs in which charged residues are substituted for proline, nor is it supported by the trimerization-defective phenylalanine and leucine mutants. Our findings suggest that the central proline in the ASLV fusion peptide is important for the formation of the native (metastable) Env structure as well as for membrane interactions that lead to fusion.
Analysis of Capsid Formation of Human Polyomavirus JC (Tokyo-1 Strain) by a Eukaryotic Expression System: Splicing of Late RNAs, Translation and Nuclear Transport of Major Capsid Protein VP1, and Capsid Assembly
Authors: Shishido-Hara Yukiko , Hara Yoshinobu , Larson Theresa , Yasui Kotaro , Nagashima Kazuo , Stoner Gerald L.
Published: July 2002

Human polyomavirus JC (JCV) can encode the three capsid proteins VP1, VP2, and VP3, downstream of the agnoprotein in the late region. JCV virions are identified in the nucleus of infected cells. In this study, we have elucidated unique features of JCV capsid formation by using a eukaryotic expression system. Structures of JCV polycistronic late RNAs (M1 to M4 and possibly M5 and M6) generated by alternative splicing were determined. VP1 would be synthesized from M2 RNA, and VP2 and VP3 would be synthesized from M1 RNA. The presence of the open reading frame of the agnoprotein or the leader sequence (nucleotides 275 to 409) can decrease the expression level of VP1. VP1 was efficiently transported to the nucleus in the presence of VP2 and VP3 but distributed both in the cytoplasm and in the nucleus in their absence. Mutation analysis indicated that inefficiency in nuclear transport of VP1 is due to the unique structure in the N-terminal sequence, KRKGERK. Within the nucleus, VP1 was localized discretely and identified as speckles in the presence of VP2 and VP3 but distributed diffusely in their absence. These results suggest that VP1 was efficiently transported to the nucleus and localized in the discrete subnuclear regions, possibly with VP2 and VP3. By electron microscopy, recombinant virus particles were identified in the nucleus, and their intranuclear distribution was consistent with distribution of speckles. This system provides a useful model with which to understand JCV capsid formation and the structures and functions of the JCV capsid proteins.
A Chimeric Protein Containing the N Terminus of the Adeno-Associated Virus Rep Protein Recognizes Its Target Site in an In Vivo Assay
Authors: Cathomen Toni , Collete Delphine , Weitzman Matthew D.
Published: July 2002

The Rep78 and Rep68 proteins of adeno-associated virus (AAV) type 2 are involved in DNA replication, regulation of gene expression, and targeting site-specific integration. They bind to a specific Rep recognition sequence (RRS) found in both the viral inverted terminal repeats and the AAVS1 integration locus on human chromosome 19. Previous in vitro studies implied that an N-terminal segment of Rep is involved in DNA recognition, while additional domains might stabilize binding and mediate multimerization. In order to define the minimal requirements for Rep to recognize its target site in the human genome, we developed one-hybrid assays in which DNA-protein interactions are detected in vivo. Chimeric proteins consisting of the N terminus of Rep fused to different oligomerization motifs and a transcriptional activation domain were analyzed for oligomerization, DNA binding, and activation of reporter gene expression. Expression of reporter genes was driven from RRS motifs cloned upstream of minimal promoters and examined in mammalian cells from transfected plasmids and in
Saccharomyces cerevisiae
from a reporter cassette integrated into the yeast genome. Our results show for the first time that chimeric proteins containing the amino-terminal 244 residues of Rep are able to target the RRS in vitro and in vivo when incorporated into artificial multimers. These studies suggest that chimeric proteins may be used to harness the unique targeting feature of AAV for gene therapy applications.
The Decreased Replicative Capacity of Simian Immunodeficiency Virus SIVmac239ΔnefIs Manifest in Cultures of Immature Dendritic Cells and T Cells
Authors: Messmer Davorka , Ignatius Ralf , Santisteban Christine , Steinman Ralph M. , Pope Melissa
Published: July 2002

Transmission of simian immunodeficiency virus SIVmac239Δnef(Δnef) to macaques results in attenuated replication of the virus in most animals and ultimately induces protection against challenge with some pathogenic, wild-type SIV strains. It has been difficult, however, to identify a culture system in which the replication of Δnefis severely reduced relative to that of the wild type. We have utilized a primary culture system consisting of blood-derived dendritic cells (DCs) and autologous T cells. When the DCs were fully differentiated or mature, the DC–CD4+T-cell mixtures supported replication of both the parental SIV strain, 239 (the wild type), and its mutant withnefdeleted (Δnef), irrespective of virus dose and the cell type introducing the virus to the coculture. In contrast, when immature DCs were exposed to Δnefand cocultured with T cells, virus replication was significantly lower than that of the wild type. Activation of the cultures with a superantigen allowed both Δnefand the wild type to replicate comparably in immature DC–T-cell cultures. Immature DCs, which, it has been hypothesized, capture and transmit SIV in vivo, are deficient in supporting replication of Δnefin vitro and may contribute to the reduced pathogenicity of Δnefin vivo.
Interaction of Yellow Fever Virus French Neurotropic Vaccine Strain with Monkey Brain: Characterization of Monkey Brain Membrane Receptor Escape Variants
Authors: Ni Haolin , Ryman Kate D. , Wang Heiman , Saeed Mohammad F. , Hull Robin , Wood David , Minor Philip D. , Watowich Stanley J. , Barrett Alan D. T.
Published: July 2002

Binding of yellow fever virus wild-type strains Asibi and French viscerotropic virus and vaccine strains 17D and FNV to monkey brain and monkey liver cell membrane receptor preparations (MRPs) was investigated. Only FNV bound to monkey brain MRPs, while French viscerotropic virus, Asibi, and FNV all bound to monkey liver MRPs. Four monkey brain and two mouse brain MRP escape (MRP
R
) variants of FNV were selected at pH 7.6 and 6.0. Three monkey brain MRP
R
variants selected at pH 7.6 each had only one amino acid substitution in the envelope (E) protein in domain II (E-237, E-260, or E274) and were significantly attenuated in mice following intracerebral inoculation. Two of the variants were tested in monkeys and retained parental neurotropism following intracerebral inoculation at the dose tested. We speculate that this region of domain II is involved in binding of FNV E protein to monkey brain and is, in part, responsible for the enhanced neurotropism of FNV for monkeys. A monkey brain MRP
R
variant selected at pH 6.0 and two mouse brain MRP
R
variants selected at pH 7.6 were less attenuated in mice, and each had an amino acid substitution in the transmembrane region of the E protein (E-457 or E-458).
A Protein Kinase Activity Associated with Epstein-Barr Virus BGLF4 Phosphorylates the Viral Early Antigen EA-D In Vitro
Authors: Chen Mei-Ru , Chang Shin-Jye , Huang Hsiaowen , Chen Jen-Yang
Published: July 2002

The Epstein-Barr virus (EBV) open reading frame BGLF4 was identified as a potential Ser/Thr protein kinase gene through the recognition of amino acid sequence motifs characteristic of conserved regions within the catalytic domains of protein kinases. In order to investigate this potential kinase activity, BGLF4 was expressed inEscherichia coliand the purified protein was used to generate a specific antiserum. Recombinant vaccinia virus vTF7-3, which expresses the T7 RNA polymerase, was used to infect 293 and 293T cells after transient transfection with a plasmid containing BGLF4 under the control of the T7 promoter. Autophosphorylation of the BGLF4 protein was demonstrated using the specific antiserum in an immune complex kinase assay. In addition, EBNA-1-tagged BGLF4 and EBNA-1 monoclonal antibody 5C11 were used to demonstrate the specificity of the kinase activity and to locate BGLF4 in the cytoplasm of transfected cells. Manganese ions were found to be essential for autophosphorylation of BGLF4, and magnesium can stimulate the activity. BGLF4 can utilize GTP, in addition to ATP, as a phosphate donor in this assay. BGLF4 can phosphorylate histone and casein in vitro. Among the potential viral protein substrates we examined, the EBV early antigen (EA-D, BMRF1), a DNA polymerase accessory factor and an important transactivator during lytic infection, was found to be phosphorylated by BGLF4 in vitro. Amino acids 1 to 26 of BGLF4, but not the predicted conserved catalytic domain, were found to be essential for autophosphorylation of BGLF4.
Ultrastructural Organization of Recombinant Marburg Virus Nucleoprotein: Comparison with Marburg Virus Inclusions
Authors: Kolesnikova Larissa , Mühlberger Elke , Ryabchikova Elena , Becker Stephan
Published: July 2002

HeLa cells expressing the recombinant Marburg virus (MBGV) nucleoprotein (NP) have been studied by immunoelectron microscopy. It was found that MBGV NPs assembled into large aggregates which were in close association with membranes of the rough endoplasmic reticulum. Further analysis of these aggregates revealed that NPs formed tubule-like structures which were arranged in a hexagonal pattern. A similar pattern of preformed nucleocapsids was detected in intracellular inclusions induced by MBGV infection. Our data indicated that MBGV NP is able to form nucleocapsid-like structures in the absence of the authentic viral genome and other nucleocapsid-associated proteins.
Alphavirus Nucleocapsid Protein Contains a Putative Coiled Coil α-Helix Important for Core Assembly
Authors: Perera Rushika , Owen Katherine E. , Tellinghuisen Timothy L. , Gorbalenya Alexander E. , Kuhn Richard J.
Published: July 2002

The alphavirus nucleocapsid core is formed through the energetic contributions of multiple noncovalent interactions mediated by the capsid protein. This protein consists of a poorly conserved N-terminal region of unknown function and a C-terminal conserved autoprotease domain with a major role in virion formation. In this study, an 18-amino-acid conserved region, predicted to fold into an α-helix (helix I) and embedded in a low-complexity sequence enriched with basic and Pro residues, has been identified in the N-terminal region of the alphavirus capsid proteins. In Sindbis virus, helix I spans residues 38 to 55 and contains three conserved leucine residues, L38, L45, and L52, conforming to the heptad amino acid organization evident in leucine zipper proteins. Helix I consists of an N-terminally truncated heptad and two complete heptad repeats with β-branched residues and conserved leucine residues occupying the
a
and
d
positions of the helix, respectively. Complete or partial deletion of helix I, or single-site substitutions at the conserved leucine residues (L45 and L52), caused a significant decrease in virus replication. The mutant viruses were more sensitive to elevated temperature than wild-type virus. These mutant viruses also failed to accumulate cores in the cytoplasm of infected cells, although they did not have defects in protein translation or processing. Analysis of these mutants using an in vitro assembly system indicated that the majority were defective in core particle assembly. Furthermore, mutant proteins showed a
trans
-dominant negative phenotype in in vitro assembly reactions involving mutant and wild-type proteins. We propose that helix I plays a central role in the assembly of nucleocapsid cores through coiled coil interactions. These interactions may stabilize subviral intermediates formed through the interactions of the C-terminal domain of the capsid protein and the genomic RNA and contribute to the stability of the virion.
Oligomerization Mediated by a Helix-Loop-Helix-Like Domain of Baculovirus IE1 Is Required for Early Promoter Transactivation
Authors: Olson Victoria A. , Wetter Justin A. , Friesen Paul D.
Published: July 2002

IE1 is a principal transcriptional regulator of
Autographa californica
multicapsid nucleopolyhedrovirus (Ac
M
NPV). Transactivation by IE1 is stimulated when early viral promoters are
cis
linked to homologous-region (
hr
) enhancer sequences of Ac
M
NPV. This transcriptional enhancement is correlated with the binding of IE1 as a dimer to the 28-bp palindromic repeats comprising the
hr
enhancer. To define the role of homophilic interactions in IE1 transactivation, we have mapped the IE1 domains required for oligomerization. We report here that IE1 oligomerizes by a mechanism independent of enhancer binding, as demonstrated by in vitro pull-down assays using fusions of IE1 (582 residues) to the C terminus of glutathione
S
-transferase. In vivo oligomerization of IE1 was verified by immunoprecipitation of IE1 complexes from extracts of plasmid-transfected SF21 cells. Analyses of a series of site-directed IE1 insertion mutations indicated that a helix-loop-helix (HLH)-like domain extending from residue 543 to residue 568 is the primary determinant of oligomerization. Replacement of residues within the hydrophobic face of the putative dimerization domain disrupted IE1 homophilic interactions and caused loss of IE1 transactivation of
hr
-dependent promoters in plasmid transfection assays. Thus, oligomerization is required for IE1 transcriptional stimulation. HLH mutations also reduced IE1 stability and abrogated transactivation of non-
hr
-dependent promoters. These data support a model wherein IE1 oligomerizes prior to DNA binding to facilitate proper interaction with the symmetrical recognition sites within the
hr
enhancer and thereby promote the transcription of early viral genes.