bluuebunny/crossref_metadata_2025_split huggingface dataset:
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

ABSTRACTDendritic 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

ABSTRACTThe 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

ABSTRACT
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

ABSTRACT
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

ABSTRACT

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

ABSTRACT
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

ABSTRACT

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

ABSTRACT
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

ABSTRACTNeonates 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

ABSTRACT

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

ABSTRACT
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

ABSTRACT

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.