bluuebunny/crossref_metadata_2025 huggingface dataset:
Frequent Reassortments May Explain the Genetic Heterogeneity of Rotaviruses: Analysis of Finnish Rotavirus Strains
Authors: Maunula Leena , von Bonsdorff Carl-Henrik
Published: November 2002

The predominant rotavirus electropherotypes (e-types) during 17 epidemic seasons (1980 through 1997) in Finland were established, and representative virus isolates were studied by nucleotide sequencing and phylogenetic analysis. The virus isolates were either P[8]G1 or P[8]G4 types. The G1 and G4 strains formed one G1 lineage (VP7-G1-1) and one G4 lineage, respectively. Otherwise, they belonged to two P[8] lineages (VP4-P[8]-1 and -2) unrelated to their G types. Phylogenetic analysis of partial sequences of all 11 RNA segments obtained from the strains also revealed genetic diversity among gene segments other than those defining P and G types. With the exception of segments 1, 3, and 10, the sequences of the other segments could be assigned to 2 to 4 different genetic clusters. The results of this study suggest that, in addition to the RNA segments encoding VP4 and VP7, the other RNA segments may segregate independently as well. In total, the 9 predominant e-types represented 7 different RNA segment combinations when the phylogenetic clusters of their 11 genes were determined. The extensive genetic diversity and number of e-types among rotaviruses are best explained by frequent genetic reassortment.
Amino Acid Residues in the Carboxy-Terminal Region of Cottontail Rabbit Papillomavirus E6 Influence Spontaneous Regression of Cutaneous Papillomas
Authors: Hu Jiafen , Cladel Nancy M. , Pickel Martin D. , Christensen Neil D.
Published: November 2002

Previous studies have identified two different strains of cottontail rabbit papillomavirus (CRPV) that differ by approximately 5% in base pair sequence and that perform quite differently when used to challenge New Zealand White (NZW) rabbit skin. One strain caused persistent lesions (progressor strain), and the other induced papillomas that spontaneously regressed (regressor strain) at high frequencies (J. Salmon, M. Nonnenmacher, S. Caze, P. Flamant, O. Croissant, G. Orth, and F. Breitburd, J. Virol. 74:10766-10777, 2000; J. Salmon, N. Ramoz, P. Cassonnet, G. Orth, and F. Breitburd, Virology 235:228-234, 1997). We generated a panel of CRPV genomes that contained chimeric and mutant progressor and regressor strain E6 genes and assessed the outcome upon infection of both outbred and EIII/JC inbred NZW rabbits. The carboxy-terminal 77-amino-acid region of the regressor CRPV strain E6, which contained 15 amino acid residues that are different from those of the equivalent region of the persistent CRPV strain E6, played a dominant role in the conversion of the persistent CRPV strain to one showing high rates of spontaneous regressions. In addition, a single amino acid change (G252E) in the E6 protein of the CRPV progressor strain led to high frequencies of spontaneous regressions in inbred rabbits. These observations imply that small changes in the amino acid sequences of papillomavirus proteins can dramatically impact the outcome of natural host immune responses to these viral infections. The data imply that intrastrain differences between separate isolates of a single papillomavirus type (such as human papillomavirus type 16) may contribute to a collective variability in host immune responses in outbred human populations.
Inhibition of Polyomavirus
ori-
Dependent DNA Replication by mSin3B
Authors: Xie An-Yong , Folk William R.
Published: November 2002

When tethered in
cis
to DNA, the transcriptional corepressor mSin3B inhibits polyomavirus (Py)
ori
-dependent DNA replication in vivo. Histone deacetylases (HDACs) appear not to be involved, since tethering class I and class II HDACs in
cis
does not inhibit replication and treating the cells with trichostatin A does not specifically relieve inhibition by mSin3B. However, the mSin3B L59P mutation that impairs mSin3B interaction with N-CoR/SMRT abrogates inhibition of replication, suggesting the involvement of N-CoR/SMRT. Py large T antigen interacts with mSin3B, suggesting an HDAC-independent mechanism by which mSin3B inhibits DNA replication.
Receptor-Induced Conformational Changes of Murine Coronavirus Spike Protein
Authors: Matsuyama Shutoku , Taguchi Fumihiro
Published: November 2002

Although murine coronavirus mouse hepatitis virus (MHV) enters cells by virus-cell membrane fusion triggered by its spike (S) protein, it is not well known how the S protein participates in fusion events. We reported that the soluble form of MHV receptor (soMHVR) transformed a nonfusogenic S protein into a fusogenic one (F. Taguchi and S. Matsuyama, J. Virol.
76:
950-958, 2002). In the present study, we demonstrate that soMHVR induces the conformational changes of the S protein, as shown by the proteinase digestion test. A cl-2 mutant, srr7, of the MHV JHM virus (JHMV) was digested with proteinase K after treatment with soMHVR, and the resultant S protein was analyzed by Western blotting using monoclonal antibody (MAb) 10G, specific for the membrane-anchored S2 subunit. A 58-kDa fragment, encompassing the two heptad repeats in S2, was detected when srr7 was digested after soMHVR treatment, while no band was seen when the virus was untreated. The appearance of the proteinase-resistant fragment was dependent on the temperature and time of srr7 incubation with soMHVR and also on the concentration of soMHVR. Coimmunoprecipitation indicated that the direct binding of soMHVR to srr7 S protein induced these conformational changes; this was also suggested by the inhibition of the changes following pretreatment of soMHVR with anti-MHVR MAb CC1. soMHVR induced conformational changes of the S proteins of wild-type (wt) JHMV cl-2, as well as revertants from srr7, srr7A and srr7B; however, a major proportion of these S proteins were resistant to proteinase K even without soMHVR treatment. The implications of this proteinase-resistant fraction are discussed. This is the first report on receptor-induced conformational changes of the membrane-anchored fragment of the coronavirus S protein.
Capture and Transfer of Simian Immunodeficiency Virus by Macaque Dendritic Cells Is Enhanced by DC-SIGN
Authors: Yu Kimata Monica T. , Cella Marina , Biggins Julia E. , Rorex Colin , White Robert , Hicks Sarah , Wilson Joelle M. , Patel Parul G. , Allan Jonathan S. , Colonna Marco , Kimata Jason T.
Published: November 2002

Dendritic cells (DCs) are among the first cells encountered by human and simian immunodeficiency virus (HIV and SIV) following mucosal infection. Because these cells efficiently capture and transmit virus to T cells, they may play a major role in mediating HIV and SIV infection. Recently, a C-type lectin protein present on DCs, DC-specific ICAM-3-grabbing nonintegrin (DC-SIGN), was shown to efficiently bind and present HIV and SIV to CD4+, coreceptor-positive cells intrans. However, the significance of DC-SIGN for virus transmission and pathogenesis in vivo remains unclear. Because SIV infection of macaques may represent the best model to study the importance of DC-SIGN in HIV infection, we cloned and characterized pig-tailed macaque DC-SIGN and generated monoclonal antibodies (MAbs) against it. We demonstrate that, like human DC-SIGN, pig-tailed macaque DC-SIGN (ptDC-SIGN) is expressed on DCs and macrophages but not on monocytes, T cells, or B cells. Moderate levels of ptDC-SIGN expression were detected on the surface of DCs, and low-level expression was found on macrophages. Additionally, we show that ptDC-SIGN efficiently binds and transmits replication-competent SIVmne variants to CD4+, coreceptor-positive cells. Moreover, transmission of virus between pig-tailed macaque DCs and CD4+T cells is largely ptDC-SIGN dependent. Interestingly, MAbs directed against ptDC-SIGN vary in the capacity to block transmission of different SIVmne variants. These data demonstrate that ptDC-SIGN plays a central role in transmitting virus from macaque DCs to T cells, and they suggest that SIVmne variants may differ in their interactions with ptDC-SIGN. Thus, SIVmne infection of pig-tailed macaques may provide an opportunity to investigate the significance of DC-SIGN in primate lentiviral infections.
Comparison of Molecular and Biological Characteristics of a Modified Live Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) Vaccine (Ingelvac PRRS MLV), the Parent Strain of the Vaccine (ATCC VR2332), ATCC VR2385, and Two Recent Field Isolates of PRRSV
Authors: Opriessnig T. , Halbur P. G. , Yoon K.-J. , Pogranichniy R. M. , Harmon K. M. , Evans R. , Key K. F. , Pallares F. J. , Thomas P. , Meng X. J.
Published: November 2002

The objectives of this study were to compare the molecular and biological characteristics of recent porcine reproductive and respiratory syndrome virus (PRRSV) field isolates to those of a modified live virus (MLV) PRRS vaccine and its parent strain. One hundred seventeen, 4-week-old pigs were randomly assigned to six groups. Group 1 (n= 20) served as sham-inoculated negative controls, group 2 (n= 19) was inoculated with Ingelvac PRRS MLV vaccine, group 3 (n= 20) was inoculated with the parent strain of the vaccine (ATCC VR2332), group 4 (n= 19) was inoculated with vaccine-like PRRSV field isolate 98-38803, group 5 (n= 19) was inoculated with PRRSV field isolate 98-37120, and group 6 (n= 20) was inoculated with known high-virulence PRRSV isolate ATCC VR2385. The levels of severity of gross lung lesions (0 to 100%) among the groups were significantly different at both 10 (P< 0.0001) and 28 days postinoculation (p.i.) (P= 0.002). At 10 days p.i., VR2332 (26.5% ± 4.64%) and VR2385 (36.4% ± 6.51%) induced gross lesions of significantly greater severity than 98-38803 (0.0% ± 0.0%), 98-37120 (0.8% ± 0.42%), Ingelvac PRRS MLV (0.9% ± 0.46%), and negative controls (2.3% ± 1.26%). At 28 days p.i., 98-37120 (17.2% ± 6.51%) induced gross lesions of significantly greater severity than any of the other viruses. Analyses of the microscopic-interstitial-pneumonia-lesion scores (0 to 6) revealed that VR2332 (2.9 ± 0.23) and VR2385 (3.1 ± 0.35) induced significantly more severe lesions at 10 days p.i. At 28 days p.i., VR2385 (2.5 ± 0.27), VR2332 (2.3 ± 0.21), 98-38803 (2.6 ± 0.29), and 98-37120 (3.0 ± 0.41) induced significantly more severe lesions than Ingelvac PRRS MLV (0.7 ± 0.17) and controls (0.7 ± 0.15). The molecular analyses and biological characterizations suggest that the vaccine-like isolate 98-38803 (99.5% amino acid homology based on the ORF5 gene) induces microscopic pneumonia lesions similar in type to, but different in severity and time of onset from, those observed with virulent strains VR2385 and the parent strain of the vaccine. Our data strongly suggest that isolate 98-38803 is a derivative of Ingelvac PRRS MLV and that the isolate is pneumovirulent.
Palmitoylation of the Murine Leukemia Virus Envelope Protein Is Critical for Lipid Raft Association and Surface Expression
Authors: Li Min , Yang Chinglai , Tong Suxiang , Weidmann Armin , Compans Richard W.
Published: November 2002

To investigate the association of the murine leukemia virus (MuLV) Env protein with lipid rafts, we compared wild-type and palmitoylation-deficient mutant Env proteins by using extraction with the mild detergent Triton X-100 (TX-100) followed by a sucrose gradient flotation assay. We found that the wild-type MuLV Env protein was resistant to ice-cold TX-100 treatment and floated to the top of the gradients. In contrast, we observed that the palmitoylation-deficient mutant Env protein was mostly soluble when extracted by ice-cold TX-100 and stayed at the bottom of the gradients. Both the wild-type and mutant Env proteins were found to be soluble when treated with methyl-β-cyclodextrin before extraction with ice-cold TX-100 or when treated with ice-cold octyl-β-glucoside instead of TX-100. These results indicate that the MuLV Env protein is associated with lipid rafts and that palmitoylation of the Env protein is critical for lipid raft association. Although the palmitoylation-deficient Env mutant was synthesized at a level similar to that of the wild-type Env, it was found to be expressed at reduced levels on the cell surface. We observed syncytium formation activity with both the wild-type and mutant Env proteins, indicating that palmitoylation or raft association is not required for MuLV viral fusion activity.
RNA Incorporation Is Critical for Retroviral Particle Integrity after Cell Membrane Assembly of Gag Complexes
Authors: Wang Shainn-Wei , Aldovini Anna
Published: November 2002

The nucleocapsid (NC) domain of retroviruses plays a critical role in specific viral RNA packaging and virus assembly. RNA is thought to facilitate viral particle assembly, but the results described here with NC mutants indicate that it also plays a critical role in particle integrity. We investigated the assembly and integrity of particles produced by the human immunodeficiency virus type 1 M1-2/BR mutant virus, in which 10 of the 13 positive residues of NC have been replaced with alanines and incorporation of viral genomic RNA is virtually abolished. We found that the mutations in the basic residues of NC did not disrupt Gag assembly at the cell membrane. The mutant Gag protein can assemble efficiently at the cell membrane, and viral proteins are detected outside the cell as efficiently as they are for the wild type. However, only ∼10% of the Gag molecules present in the supernatant of this mutant sediment at the correct density for a retroviral particle. The reduction of positive charge in the NC basic domain of the M1-2/BR virus adversely affects both the specific and nonspecific RNA binding properties of NC, and thus the assembled Gag polyprotein does not bind significant amounts of viral or cellular RNA. We found a direct correlation between the percentage of Gag associated with sedimented particles and the amount of incorporated RNA. We conclude that RNA binding by Gag, whether the RNA is viral or not, is critical to retroviral particle integrity after cell membrane assembly and is less important for Gag-Gag interactions during particle assembly and release.
Mapping of Functional Regions in the Amino-Terminal Portion of the Herpes Simplex Virus ICP27 Regulatory Protein: Importance of the Leucine-Rich Nuclear Export Signal and RGG Box RNA-Binding Domain
Authors: Lengyel Joy , Guy Chandra , Leong Vivian , Borge Sarah , Rice Stephen A.
Published: November 2002

Infected-cell protein 27 (ICP27) is an essential herpes simplex virus type 1 (HSV-1) regulatory protein that activates a subset of viral delayed-early and late genes, at least in part through posttranscriptional mechanisms. Previous studies have shown that the amino (N)-terminal half of the protein contains important functional regions, including a leucine-rich nuclear export signal (NES). However, to date, the phenotype of an HSV-1 ICP27 NES mutant has not been reported. In this study, we engineered and characterized
d
Leu, an HSV-1 deletion mutant that specifically lacks ICP27's NES (amino acids 6 to 19). The phenotype of
d
Leu was analyzed alongside those of eight other ICP27 N-terminal deletion mutants. We found that in Vero cells,
d
Leu displays modest defects in viral gene expression and an approximately 100-fold reduction in the production of viral progeny. Unlike wild-type (WT) ICP27, which exhibits a cytoplasmic distribution in addition to its predominant nuclear localization,
d
Leu ICP27 is highly restricted to the cell nucleus. This strongly suggests that the N-terminal leucine-rich sequence functions as an NES during viral infection. Our analysis of
d
Leu and the other mutants has enabled us to genetically define the regions in the N-terminal 200 residues of ICP27 which are required for efficient viral growth in Vero cells. Only two regions appear to be important: (i) the leucine-rich NES and (ii) the RGG box RNA-binding domain, encoded by residues 139 to 153. A virus lacking the RGG box-encoding sequence,
d
4-5, has a phenotype similar to that of
d
Leu in that it displays modest defects in viral gene expression and grows poorly. Interestingly, deletion of both the NES and RGG box, as well as the sequences in between, is lethal. The resulting virus,
d
1-5, displays severe defects in viral gene expression and DNA synthesis and is unable to produce significant amounts of infectious progeny. Therefore, the N-terminal portion of ICP27 contains at least two functional domains which collectively are absolutely essential for viral infection.
The Kaposi's Sarcoma-Associated Herpesvirus K12 Transcript from a Primary Effusion Lymphoma Contains Complex Repeat Elements, Is Spliced, and Initiates from a Novel Promoter
Authors: Li Hong , Komatsu Takashi , Dezube Bruce J. , Kaye Kenneth M.
Published: November 2002

Kaposi's sarcoma-associated herpesvirus (KSHV) latently infects KS tumors, primary effusion lymphomas (PELs), and PEL cell lines. K12 (T0.7) is the most abundant transcript expressed in latent KSHV infection. We characterize here the K12 transcript from a PEL tumor prior to passage in cell culture. The PEL tumor KSHV K12 transcript contained additional complex nucleotide repeat elements compared to the previously described K12 message of the BCBL-1 PEL cell line. The PEL tumor lacked kaposin B, the predominant BCBL-1 K12 protein product, but encoded kaposins A and C. The K12 transcript was spliced and the splicing event occurred in all KSHV isolates tested. The 5′ end of the K12 transcript was mapped by 5′ RACE (rapid amplification of cDNA ends) and S1 nuclease protection assays and was at the site of an active promoter. This work demonstrates that the K12 transcript contains variable, complex repeat elements, is spliced and is expressed from a novel KSHV promoter.
Mapping the Encapsidation Determinants of Feline Immunodeficiency Virus
Authors: Kemler Iris , Barraza Roman , Poeschla Eric M.
Published: November 2002

Encapsidation of retroviral RNA involves specific interactions between viral proteins and
cis
-acting genomic RNA sequences. Human immunodeficiency virus type 1 (HIV-1) RNA encapsidation determinants appear to be more complex and dispersed than those of murine retroviruses. Feline lentiviral (feline immunodeficiency virus [FIV]) encapsidation has not been studied. To gain comparative insight into lentiviral encapsidation and to optimize FIV-based vectors, we used RNase protection assays of cellular and virion RNAs to determine packaging efficiencies of FIV deletion mutants, and we studied replicative phenotypes of mutant viruses. Unlike the case for other mammalian retroviruses, the sequences between the major splice donor (MSD) and the start codon of
gag
contribute negligibly to FIV encapsidation. Moreover, molecular clones having deletions in this region were replication competent. In contrast, sequences upstream of the MSD were important for encapsidation, and deletion of the U5 element markedly reduced genomic RNA packaging. The contribution of
gag
sequences to packaging was systematically investigated with subgenomic FIV vectors containing variable portions of the
gag
open reading frame, with all virion proteins supplied in
trans
. When no
gag
sequence was present, packaging was abolished and marker gene transduction was absent. Inclusion of the first 144 nucleotides (nt) of
gag
increased vector encapsidation to detectable levels, while inclusion of the first 311 nt increased it to nearly wild-type levels and resulted in high-titer FIV vectors. However, the identified proximal
gag
sequence is necessary but not sufficient, since viral mRNAs that contain all coding regions, with or without as much as 119 nt of adjacent upstream 5′ leader, were excluded from encapsidation. The results identify a mechanism whereby FIV can encapsidate its genomic mRNA in preference to subgenomic mRNAs.
Poly(ADP-Ribose) Polymerase 1 Is Not Strictly Required for Infection of Murine Cells by Retroviruses
Authors: Siva Amara C. , Bushman Frederic
Published: November 2002

The DNA-breaking and -joining steps initiating retroviral integration are well understood, but the later steps, thought to be carried out by cellular DNA repair enzymes, have not been fully characterized. Poly(ADP-ribose) polymerase 1 (PARP-1) has been proposed to play a role late during retroviral integration, because infection by human immunodeficiency virus (HIV)-based vectors was reported to be strongly inhibited in PARP-1-deficient fibroblasts. PARP-1, a nuclear enzyme, binds tightly to nicked DNA and synthesizes poly(ADP-ribose) as an early response to DNA damage. To investigate the role of PARP-1 in retroviral integration, we infected wild-type and PARP-1-deficient mouse embryonic fibroblasts (MEFs) separately with two HIV type 1-derived, vesicular stomatitis virus G-pseudotyped lentivirus vectors. Surprisingly, infection of both wild-type and PARP-1-deficient cells was observed with both vectors. Marker gene transduction and provirus formation by one vector was reduced by 45 to 75% compared to the wild type, but the other vector was unaffected by the PARP-1 mutant. In addition, PARP-1-deficient MEFs infected with Moloney murine leukemia virus showed no decrease in virus output after infection compared to the wild type. We conclude that PARP-1 cannot be strictly required for retroviral infection because replication steps, including integration, can proceed efficiently in its absence.
Neonates Mount Robust and Protective Adult-Like CD8+-T-Cell Responses to DNA Vaccines
Authors: Zhang Jie , Silvestri Nicole , Whitton J. Lindsay , Hassett Daniel E.
Published: November 2002

Neonates are thought to mount less vigorous adaptive immune responses than adults to antigens and infectious agents. This concept has led to a delay in the administration of many currently available vaccines until late infancy or early childhood. It has recently been shown that vaccines composed of plasmid DNA can induce both humoral and cell-mediated antimicrobial immunity when administered within hours of birth. In most of these studies, immune responses were measured weeks or months after the initial vaccination, and it is therefore questionable whether the observed responses were actually the result of priming of splenocytes within the neonatal period. Here we show that DNA vaccination at birth results in the rapid induction of antigen-specific CD8+T cells within neonatal life. Analyses of T-cell effector functions critical for the resolution of many viral infections revealed that neonatal and adult CD8+T cells produce similar arrays of cytokines. Furthermore, the avidities of neonatal and adult CD8+T cells for peptide and the rapidity with which they upregulate cytokine production after recall encounters with antigen are similar. Protective immunity against the arenavirus lymphocytic choriomeningitis virus, which is mediated by CD8+cytotoxic T cells, is also rapidly acquired within the neonatal period. Collectively these data imply that, at least in the case of CD8+T cells, neonates are not as immunodeficient as previously supposed and that DNA vaccines may be an effective and safe means of providing critical cell-mediated antiviral immunity extremely early in life.
Hepatitis A Virus Inhibits Cellular Antiviral Defense Mechanisms Induced by Double-Stranded RNA
Authors: Brack Kerstin , Berk Iris , Magulski Thomas , Lederer Jörg , Dotzauer Andreas , Vallbracht Angelika
Published: November 2002

The consequences of a hepatitis A virus (HAV) infection on cell-based antiviral responses and the interactions between virus and host cells resulting in persistent infections are poorly understood. In this report, we show that HAV does inhibit double-stranded (dsRNA)-induced beta interferon (IFN-β) gene expression by influencing the IFN-β enhanceosome, as well as dsRNA-induced apoptosis, which suggests that both effects may be connected by shared viral and/or cellular factors. This ability of HAV, which preserves the sites of virus production for a longer time, may allow the virus to establish an infection and may be the presupposition for setting up persistent infections. Our results suggest that the inhibitory effect of HAV on the cellular defense mechanisms might not be sufficient to completely prevent the antiviral reactions, which may be induced by accumulating viral dsRNA, at a later stage of infection. However, HAV seems to counteract this situation by downregulation of viral replication and in the following production of viral dsRNA. This ability of noncytopathogenic HAV acts dominantly on cytopathogenic HAV in
trans
. The downregulation might ensure the moderate replication which seems necessary for inhibition of the antiviral mechanisms by HAV and therefore for the persistent state of the HAV infection.
Recombinant Respiratory Syncytial Virus with the G and F Genes Shifted to the Promoter-Proximal Positions
Authors: Krempl Christine , Murphy Brian R. , Collins Peter L.
Published: November 2002

The genome of human respiratory syncytial virus (RSV) encodes 10 mRNAs and 11 proteins in the order 3′-NS1-NS2-N-P-M-SH-G-F-M2-1/M2-2-L-5′. The G and F glycoproteins are the major RSV neutralization and protective antigens. It seems likely that a high level of expression of G and F would be desirable for a live RSV vaccine. For mononegaviruses, the gene order is a major factor controlling the level of mRNA and protein expression due to the polar gradient of sequential transcription. In order to increase the expression of G and F, recombinant RSVs based on strain A2 were constructed in which the G or F gene was shifted from the sixth or seventh position (in a genome lacking the SH gene), respectively, to the first position (rRSV-G1/ΔSH and rRSV-F1/ΔSH, respectively). Another virus was made in which G and F were shifted together to the first and second positions, respectively (rRSV-G1F2/ΔSH). Shifting one or two genes to the promoter-proximal position resulted in increased mRNA and protein expression of the shifted genes, with G and F expression increased up to 2.4-and 7.8-fold, respectively, at the mRNA level and approximately 2.5-fold at the protein level, compared to the parental virus. Interestingly, the transcription of downstream genes was not greatly affected even though shifting G or F, or G and F together, had the consequence of moving the block of genes NS1-NS2-N-P-M-(G) one or two positions further from the promoter. The efficiency of replication of the gene shift viruses in vitro was increased up to 10-fold. However, their efficiency of replication in the lower respiratory tracts of mice was statistically indistinguishable from that of the parental virus. In the upper respiratory tract, replication was slightly reduced on some days for viruses in which G was in the first position. The magnitude of the G-specific antibody response to the gene shift viruses was similar to that to the parental virus, whereas the F-specific response was increased up to fourfold, although this was not reflected in an increase of the neutralizing activity. Thus, shifting the G and F genes to the promoter-proximal position increased virus replication in vitro, had little effect on replication in the mouse, and increased the antigen-specific immunogenicity of the virus beyond that of parental RSV.
Relationship between Autophosphorylation and Phosphorylation of Exogenous Substrates by the Human Cytomegalovirus UL97 Protein Kinase
Authors: Baek Moon-Chang , Krosky Paula M. , Coen Donald M.
Published: November 2002

Human cytomegalovirus encodes an unusual protein kinase, UL97, which is a member of the HvU
L
family of protein kinases encoded by diverse herpesviruses. UL97 is able to autophosphorylate and to phosphorylate certain exogenous substrates, including nucleoside analogs such as ganciclovir. It has previously been concluded that phosphorylation of UL97 is essential for its phosphorylation of ganciclovir. We examined the relationship between autophosphorylation of UL97 and its activity on exogenous substrates. Glutathione
S
-transferase-UL97 fusion protein purified from insect cells was found to be already partially phosphorylated, but neither extensive autophosphorylation nor phosphatase treatment meaningfully altered the time course of its phosphorylation of the exogenous substrate, histone H2B. Sequencing and mass spectrometric analyses of
32
P-labeled tryptic peptides of the UL97 fusion protein identified nine sites of autophosphorylation, all within the first 200 residues of the protein, outside of conserved protein kinase subdomains. A peptide corresponding to the N-terminal UL97 segment that was most extensively autophosphorylated was readily phosphorylated by UL97, confirming that fusion protein sequences are not required for phosphorylation at this site. Deletion mutants lacking at least the first 239 residues exhibited drastically reduced autophosphorylation (<5%) but retained near-wild-type H2B phosphorylation activity. Baculoviruses expressing these mutants efficiently directed the phosphorylation of ganciclovir in insect cells. Taken together, these results identify the autophosphorylation sites of a herpesvirus protein kinase and show that autophosphorylation of UL97 is not required for phosphorylation of exogenous substrates.
Virus Population Homogenization following Acute Human Immunodeficiency Virus Type 1 Infection
Authors: Learn Gerald H. , Muthui David , Brodie Scott J. , Zhu Tuofu , Diem Kurt , Mullins James I. , Corey Lawrence
Published: November 2002

Understanding the properties of human immunodeficiency virus type 1 (HIV-1) variants capable of establishing infection is critical to the development of a vaccine against AIDS. Previous studies of men have shown that the HIV-1
env
gene is homogeneous early in infection, leading to the suggestion that infection is established by a single transmitted variant. However, we report here that all of eight homosexual men evaluated beginning 3.7 to 9 weeks following onset of symptoms of acute infection harbored diverse virus populations in their blood, with median genetic distances averaging 1.08% in the
env
C2V5 region and 0.81% in the
gag
p17 gene. Within another 4.7 to 11 weeks, the variant lineage in
env
became more homogeneous, while
gag
sequences continued to diversify. Thus, the homogenization that has been reported to characterize acute infection is actually preceded by the replication of multiple virus variants. This early selective process focuses on viral properties within Env but not Gag p17. Hence, the viral homogeneity observed early in HIV-1 infection results from a selective process that occurs during the establishment of infection.
Characterization of RNA Determinants Recognized by the Arginine- and Proline-Rich Region of Us11, a Herpes Simplex Virus Type 1-Encoded Double-Stranded RNA Binding Protein That Prevents PKR Activation
Authors: Khoo David , Perez Cesar , Mohr Ian
Published: November 2002

The herpes simplex virus Us11 gene product inhibits activation of the cellular PKR kinase and associates with a limited number of unrelated viral and cellular RNA molecules via a carboxyl-terminal 68-amino-acid segment rich in arginine and proline. To characterize the determinants underlying the recognition of an RNA target by Us11, we employed an in vitro selection technique to isolate RNA ligands that bind Us11 with high affinity from a population of molecules containing an internal randomized segment. Binding of Us11 to these RNA ligands is specific and appears to occur preferentially on conformational isoforms that possess a higher-order structure. While the addition of unlabeled poly(I · C) reduced binding of Us11 to a selected radiolabeled RNA, single-stranded homopolymers were not effective competitors. Us11 directly associates with poly(I · C), and inclusion of an unlabeled selected RNA in the reaction reduces poly(I · C) binding, while single-stranded RNA homopolymers have no effect. Finally, Us11 binds to defined, double-stranded RNA (dsRNA) molecules that exhibit greater sequence complexity. Binding to these dsRNA perfect duplexes displays a striking dependence on length, as 39-bp or shorter duplexes do not bind efficiently. Furthermore, this interaction is specific for dsRNA as opposed to dsDNA, implying that the Us11 RNA binding domain can distinguish nucleic acid duplexes containing 2′ hydroxyl groups from those that do not. These results establish that Us11 is a dsRNA binding protein. The arginine- and proline-rich Us11 RNA binding domain is unrelated to known dsRNA binding elements and thus constitutes a unique recognition motif that interacts with dsRNA. The ability of Us11 to bind dsRNA may be important for inhibiting activation of the cellular PKR kinase in response to dsRNA.
An Efficient and Versatile Mammalian Viral Vector System for Major Histocompatibility Complex Class I/Peptide Complexes
Authors: Kawana-Tachikawa Ai , Tomizawa Mariko , Nunoya Jun-ichi , Shioda Tatsuo , Kato Atsushi , Nakayama Emi E. , Nakamura Tetsuya , Nagai Yoshiyuki , Iwamoto Aikichi
Published: November 2002

We report a Sendai virus (SeV) vector system for expression of major histocompatibility complex (MHC) class I/peptide complexes. We cloned the extracellular domain of a human MHC class I heavy chain, HLA-A*2402, and human β-2 microglobulin (β2m) fused with HLA-A*2402-restricted human immunodeficiency virus type 1 (HIV-1) cytotoxic T-lymphocyte (CTL) epitopes (e-β2m) in separate SeV vectors. When we coinfected nonhuman mammalian cells with the SeVs, naturally folded human MHC class I/peptide complexes were secreted in the culture supernatants. Biotin binding peptide sequences on the C terminus of the heavy chain were used to tetramerize the complexes. These tetramers made in the SeV system recognized specific CD8-positive T cells in peripheral blood mononuclear cells of HIV-1-positive patients with a specificity and sensitivity similar to those of MHC class I tetramers made in anEscherichia colisystem. Solo infection of e-β2m/SeV produced soluble e-β2m in the culture supernatant, and cells pulsed with the soluble protein were recognized by specific CTLs. Furthermore, when cells were infected with e-β2m/SeV, these cells were recognized by the specific CTLs more efficiently than the protein pulse per se. SeV is nonpathogenic for humans, can transduce foreign genes into nondividing cells, and may be useful for immunotherapy to enhance antigen-specific immune responses. Our system can be used not only to detect but also to stimulate antigen-specific cellular immune responses.
Cell Proteins TIA-1 and TIAR Interact with the 3′ Stem-Loop of the West Nile Virus Complementary Minus-Strand RNA and Facilitate Virus Replication
Authors: Li W. , Li Y. , Kedersha N. , Anderson P. , Emara M. , Swiderek K. M. , Moreno G. T. , Brinton M. A.
Published: November 2002

It was reported previously that four baby hamster kidney (BHK) proteins with molecular masses of 108, 60, 50, and 42 kDa bind specifically to the 3′-terminal stem-loop of the West Nile virus minus-stand RNA [WNV 3′(−) SL RNA] (P. Y. Shi, W. Li, and M. A. Brinton, J. Virol. 70:6278-6287, 1996). In this study, p42 was purified using an RNA affinity column and identified as TIAR by peptide sequencing. A 42-kDa UV-cross-linked viral RNA-cell protein complex formed in BHK cytoplasmic extracts incubated with the WNV 3′(−) SL RNA was immunoprecipitated by anti-TIAR antibody. Both TIAR and the closely related protein TIA-1 are members of the RNA recognition motif (RRM) family of RNA binding proteins. TIA-1 also binds to the WNV 3′(−) SL RNA. The specificity of these viral RNA-cell protein interactions was demonstrated using recombinant proteins in competition gel mobility shift assays. The binding site for the WNV 3′(−) SL RNA was mapped to RRM2 on both TIAR and TIA-1. However, the dissociation constant (

K
d

) for the interaction between TIAR RRM2 and the WNV 3′(−) SL RNA was 1.5 × 10
−8
, while that for TIA-1 RRM2 was 1.12 × 10
−7
. WNV growth was less efficient in murine TIAR knockout cell lines than in control cells. This effect was not observed for two other types of RNA viruses or two types of DNA viruses. Reconstitution of the TIAR knockout cells with TIAR increased the efficiency of WNV growth, but neither the level of TIAR nor WNV replication was as high as in control cells. These data suggest a functional role for TIAR and possibly also for TIA-1 during WNV replication.
Cell-Free Replication of the Hepatitis C Virus Subgenomic Replicon
Authors: Ali Naushad , Tardif Keith D. , Siddiqui Aleem
Published: November 2002

The hepatitis C virus (HCV) contains a plus-strand RNA genome. The 5′ noncoding region (NCR) of the viral genome functions as an internal ribosome entry site, and its unique 3′ NCR is required for the assembly of the replication complex during initiation of HCV RNA replication. Lohmann et al. (V. Lohmann, F. Korner, J.-O. Koch, U. Herian, L. Theilman, and R. Batenschlager, Science 285:110-113, 1999) developed a subgenomic HCV replicon system, which represents an important tool in studying HCV replication in cultured cells. In this study, we describe a cell-free replication system that utilizes cytoplasmic lysates prepared from Huh-7 cells harboring the HCV subgenomic replicons. These lysates, which contain ribonucleoprotein complexes associated with cellular membranes, were capable of incorporating [α
32
P]CTP into newly synthesized RNA from subgenomic replicons in vitro. Replicative forms (RFs) and replicative intermediates (RIs) were synthesized from the endogenous HCV RNA templates. Consistent with previous observations, RFs were found to be resistant to RNase A digestion, whereas RIs were sensitive to RNase treatment. The radiolabeled HCV RF-RI complexes contained both minus and plus strands and were specific to the lysates derived from replicon-expressing cells. The availability of a cell-free replication system offers opportunities to probe the mechanism(s) of HCV replication. It also provides a novel assay for potential therapeutic agents.
Distinct Poly(rC) Binding Protein KH Domain Determinants for Poliovirus Translation Initiation and Viral RNA Replication
Authors: Walter Brandon L. , Parsley Todd B. , Ehrenfeld Ellie , Semler Bert L.
Published: November 2002

The limited coding capacity of picornavirus genomic RNAs necessitates utilization of host cell factors in the completion of an infectious cycle. One host protein that plays a role in both translation initiation and viral RNA synthesis is poly(rC) binding protein 2 (PCBP2). For picornavirus RNAs containing type I internal ribosome entry site (IRES) elements, PCBP2 binds the major stem-loop structure (stem-loop IV) in the IRES and is essential for translation initiation. Additionally, the binding of PCBP2 to the 5′-terminal stem-loop structure (stem-loop I or cloverleaf) in concert with viral protein 3CD is required for initiation of RNA synthesis directed by poliovirus replication complexes. PCBP1, a highly homologous isoform of PCBP2, binds to poliovirus stem-loop I with an affinity similar to that of PCBP2; however, PCBP1 has reduced affinity for stem-loop IV. Using a dicistronic poliovirus RNA, we were able to functionally uncouple translation and RNA replication in PCBP-depleted extracts. Our results demonstrate that PCBP1 rescues RNA replication but is not able to rescue translation initiation. We have also generated mutated versions of PCBP2 containing site-directed lesions in each of the three RNA-binding domains. Specific defects in RNA binding to either stem-loop I and/or stem-loop IV suggest that these domains may have differential functions in translation and RNA replication. These predictions were confirmed in functional assays that allow separation of RNA replication activities from translation. Our data have implications for differential picornavirus template utilization during viral translation and RNA replication and suggest that specific PCBP2 domains may have distinct roles in these activities.
Addition of Six-His-Tagged Peptide to the C Terminus of Adeno-Associated Virus VP3 Does Not Affect Viral Tropism or Production
Authors: Zhang Huang-Ge , Xie Jinfu , Dmitriev Igor , Kashentseva Elena , Curiel David T. , Hsu Hui-Chen , Mountz John D.
Published: November 2002

Production of large quantities of recombinant adeno-associated virus (AAV) is difficult and not cost-effective. To overcome this problem, we have explored the feasibility of creating a recombinant AAV encoding a 6×His tag on the VP3 capsid protein. We generated a plasmid vector containing a six-His (6×His)-tagged AAV VP3. A second plasmid vector was generated that contained the full-length AAV capsid capable of producing VP1 and VP2, but not VP3 due to a mutation at position 2809 that encodes the start codon for VP3. These plasmids, necessary for production of AAV, were transfected into 293 cells to generate a 6×His-tagged VP3mutant recombinant AAV. The 6×His-tagged VP3 did not affect the formation of AAV virus, and the physical properties of the 6×His-modified AAV were equivalent to those of wild-type particles. The 6×His-tagged AAV did not affect the production titer of recombinant AAV and could be used to purify the recombinant AAV using an Ni-nitrilotriacetic acid column. Addition of the 6×His tag did not alter the viral tropism compared to wild-type AAV. These observations demonstrate the feasibility of producing high-titer AAV containing a 6×His-tagged AAV VP3 capsid protein and to utilize the 6×His-tagged VP3 capsid to achieve high-affinity purification of this recombinant AAV.
Baculovirus
lef-12
Is Not Required for Viral Replication
Authors: Guarino Linda A. , Mistretta Toni-Ann , Dong Wen
Published: November 2002

The baculovirus
lef-12
(
orf41
) gene is required for transient expression of baculovirus late genes. To analyze the role of LEF-12 in the context of infected cells, two mutant viruses were constructed. Both mutants were viable in
Trichoplusia ni
High 5 and
Spodoptera frugiperda
Sf9 cells. Single-step growth curves, however, indicated that virus yields were reduced approximately fivefold in the absence of LEF-12. Pulse-labeling of infected cells revealed that LEF-12 mutant viruses entered the late phase and synthesized late proteins at levels equivalent to or only twofold lower than those of wild-type virus-infected cells. Western blot analyses confirmed that LEF-12 was not synthesized in cells infected with mutant virus. In wild-type virus-infected cells, LEF-12 was not detected until 18 h postinfection, and accumulation of LEF-12 peaked at 24 to 36 h postinfection. Primer extension mapping revealed that
lef-12
mRNA was synthesized by 12 h postinfection and peaked between 18 and 24 h postinfection. Furthermore, synthesis of
lef-12
mRNA and LEF-12 protein were inhibited by the addition of aphidicolin, indicating that
lef-12
is expressed after DNA replication.
Kaposi's Sarcoma-Associated Herpesvirus (Human Herpesvirus 8) Replication and Transcription Factor Activates the K9 (vIRF) Gene through Two Distinct
cis
Elements by a Non-DNA-Binding Mechanism
Authors: Ueda Keiji , Ishikawa Kayo , Nishimura Ken , Sakakibara Shuhei , Do Eunju , Yamanishi Koichi
Published: November 2002

The replication and transcription activator (RTA) of Kaposi's sarcoma-associated herpesvirus (KSHV), or human herpesvirus 8, a homologue of Epstein-Barr virus BRLF1 or Rta, is a strong transactivator and inducer of lytic replication. RTA acting alone can induce lytic replication of KSHV in infected cell lines that originated from primary effusion lymphomas, leading to virus production. During the lytic replication process, RTA activates many kinds of genes, including polyadenylated nuclear RNA, K8, K9 (vIRF), ORF57, and so on. We focused here on the mechanism of how RTA upregulates the K9 (vIRF) promoter and identified two independent
cis
-acting elements in the K9 (vIRF) promoter that responded to RTA. These elements were finally confined to the sequence 5′-TCTGGGACAGTC-3′ in responsive element (RE) I-2B and the sequence 5′-GTACTTAAAATA-3′ in RE IIC-2, both of which did not share sequence homology. Multiple factors bound specifically with these elements, and their binding was correlated with the RTA-responsive activity. Electrophoretic mobility shift assay with nuclear extract from infected cells and the N-terminal part of RTA expressed in
Escherichia coli
, however, did not show that RTA interacted directly with these elements, in contrast to the RTA responsive elements in the PAN/K12 promoter region, the ORF57/K8 promoter region. Thus, it was likely that RTA could transactivate several kinds of unique
cis
elements without directly binding to the responsive elements, probably through cooperation with other DNA-binding factors.
Complete Genomic Sequence of an Epstein-Barr Virus-Related Herpesvirus Naturally Infecting a New World Primate: a Defining Point in the Evolution of Oncogenic Lymphocryptoviruses
Authors: Rivailler Pierre , Cho Young-gyu , Wang Fred
Published: November 2002

Callitrichine herpesvirus 3 (CalHV-3) was isolated from a B-cell lymphoma arising spontaneously in the New World primate
Callithrix jacchus
, the common marmoset. Partial genomic sequence analysis definitively identified CalHV-3 as a member of the Epstein-Barr virus (EBV)-related lymphocryptovirus (LCV) genus and extended the known host range of LCVs beyond humans and Old World nonhuman primates. We have now completed the first genomic sequence of an LCV infecting a New World primate by describing the unique short region, the major internal repeat, and a portion of the unique long region. This portion of the genome contains the putative latent origin of replication and 13 additional open reading frames (ORFs), 5 of which show no homology to any viral or cell genes. One of the novel genes, C5, is a positional homologue for the transformation-essential EBV gene EBNA-2. The marmoset LCV genome is also notable for the absence of viral interleukin-10 and small nonpolyadenylated RNA homologues. Marmoset LCV transcripts encoding putative latent infection nuclear proteins have a common leader sequence that is spliced from the major internal repeat in a manner similar to that of the EBV EBNA-LP, suggesting strong conservation of a common promoter and splicing of these latent infection mRNAs. An EBV LMP2A-like spliced transcript crossing the terminal repeats encodes a unique ORF, C7, with multiple transmembrane domains and tyrosine kinase phosphorylation sites functionally reminiscent of EBV LMP2A. However, the carboxy-terminal location of the candidate phosphotyrosine residues is more reminiscent of the Kaposi's sarcoma-associated herpesvirus K15 gene and provides potential evidence of an evolutionary transition from rhadinoviruses to lymphocryptoviruses. The unusual gene repertoire of the marmoset LCV differentiates ancestral viral genes likely present in an LCV progenitor from viral genes acquired later as primates and LCV coevolved, providing a defining point in the evolution of oncogenic LCVs.
Replication Advantage and Host Factor-Independent Phenotypes Attributable to a Common Naturally Occurring Capsid Mutation (I97L) in Human Hepatitis B Virus
Authors: Suk Fat-Moon , Lin Min-Hui , Newman Margaret , Pan Shann , Chen Sheng-Hsuan , Liu Jean-Dean , Shih Chiaho
Published: November 2002

Mutations of human hepatitis B virus (HBV) occur frequently within the capsid (core) protein in natural infections. The most frequent mutation of the core protein in HBV from Southeast Asia occurs at amino acid 97, changing an isoleucine (I) to a leucine (L). In our systematic study of virus-host interactions, we have examined the replication efficiency of a site-directed mutant, I97L, and its parental wild-type HBV in several different hepatoma cell lines. Interestingly, we found that this capsid variant replicated in human Huh7 hepatoma cells approximately 4.8-fold better than its parental wild-type HBV. A similar phenomenon was observed in another hepatoma cell line, J3. In addition, the level of encapsidated RNA pregenome in mutant I97L was about 5.7-fold higher than that of the wild-type HBV in Huh7 cells. Unlike Huh7 cells, no significant difference in viral DNA replication between the same I97L mutant and its parental wild-type HBV was observed in HepG2, a human hepatoblastoma cell line. This finding of a profound replication advantage for mutant I97L in Huh7 and J3 cells but not in HepG2 cells may have important implications for the emergence of this mutant in chronic HBV carriers. We speculate here that the mutation confers a host factor-independent growth advantage for the survival of HBV variants in gradually dedifferentiating hepatocytes and thus helps prolong viral persistence.