bluuebunny/crossref_metadata_2025_split huggingface dataset:
The Major Attenuating Mutations of the Respiratory Syncytial Virus Vaccine Candidate
cpts
530/1009 Specify Temperature-Sensitive Defects in Transcription and Replication and a Non-Temperature-Sensitive Alteration in mRNA Termination
Authors: Juhasz Katalin , Murphy Brian R. , Collins Peter L. 
Published: December 2019

ABSTRACT

The live-attenuated respiratory syncytial virus vaccine candidate
cpts
530/1009 was previously shown to contain two separate amino acid changes in the L protein, mutations 530 and 1009 (Phe-521→Leu and Met-1169→Val, respectively, according to the amino acid sequence of the L protein). Each mutation independently specifies temperature-sensitive (
ts
) and attenuation phenotypes. In this study, we examined the effects of these mutations on transcription and RNA replication, using complete infectious recombinant virus as well as a plasmid-based minireplicon system, the latter under conditions in which effects on replication and transcription are uncoupled. In comparison with recombinant wild-type virus, the 530 and 1009 viruses were partially restricted at 37°C for RNA replication, mRNA synthesis, and virus growth. The 1009 virus was partially restricted for RNA synthesis and virus growth even at 32°C, which suggested that the 1009 mutation has a non-
ts
component in addition to the
ts
component. Interestingly, the synthesis of polycistronic readthrough mRNAs was elevated 1.6- to 3.8-fold for the 1009 virus, and this defect was non-
ts
. Studies with the minigenome system showed that the 530 and 1009 mutations each directly affect both replication and transcription, that the effect on replication was marginally greater than on transcription for the 530 mutation, and that the increase in readthrough mRNA associated with the 1009 mutation also was observed with the minigenome system.
Epstein-Barr Virus Encodes a Novel Homolog of thebcl-2Oncogene That Inhibits Apoptosis and Associates with Bax and Bak
Authors: Marshall William L. , Yim Ching , Gustafson Erik , Graf Thomas , Sage David R. , Hanify Katherine , Williams Louisa , Fingeroth Joyce , Finberg Robert W. 
Published: December 2019

ABSTRACTThe sequenced gammaherpesviruses each contain a single viralbcl-2homolog (v-bcl-2) which may encode a protein that functions in preventing the apoptotic death of virus-infected cells. Epstein-Barr virus (EBV), a gammaherpesvirus associated with several lymphoid and epithelial malignancies, encodes the v-Bcl-2 homolog BHRF1. In this report the previously uncharacterized BALF1 open reading frame in EBV is identified as having significant sequence similarity to other v-bcl-2homologs and cellularbcl-2. Transfection of cells with a BALF1 cDNA conferred apoptosis resistance. Furthermore, a recombinant green fluorescent protein-BALF1 fusion protein suppressed apoptosis and associated with Bax and Bak. These results indicate that EBV encodes a second functional v-bcl-2.
Modular Evolution of the Integrase Domain in the Ty3/Gypsy Class of LTR Retrotransposons
Authors: Malik Harmit S. , Eickbush Thomas H. 
Published: December 2019

ABSTRACT
A phylogenetic analysis of the Ty3/Gypsy group of retrotransposons identified a conserved domain (GPY/F) present in the integrases of several members of this group as well as of certain vertebrate retroviruses. The analysis suggested an evolutionary scheme for the acquisition and loss of the GPY/F domain as well as the acquisition of a chromodomain module in the integrase encoded by this group of elements that may direct targeting specificity in the host genome.
Infection of Primary Cultures of Human Kupffer Cells by Dengue Virus: No Viral Progeny Synthesis, but Cytokine Production Is Evident
Authors: Marianneau Philippe , Steffan Anne-Marie , Royer Cathy , Drouet Marie-Thérèse , Jaeck D. , Kirn André , Deubel Vincent 
Published: December 2019

ABSTRACT
We investigated the ability of dengue virus to invade human primary Kupffer cells and to complete its life cycle. The virus effectively penetrated Kupffer cells, but the infection did not result in any viral progeny. Dengue virus-replicating Kupffer cells underwent apoptosis and were cleared by phagocytosis. Infected Kupffer cells produced soluble mediators that could intervene in dengue virus pathogenesis.
In Vitro Assembly of Alphavirus Cores by Using Nucleocapsid Protein Expressed in
Escherichia coli
Authors: Tellinghuisen Timothy L. , Hamburger Agnes E. , Fisher Bonnie R. , Ostendorp Ralf , Kuhn Richard J. 
Published: December 2019

ABSTRACT

The production of the alphavirus virion is a multistep event requiring the assembly of the nucleocapsid core in the cytoplasm and the maturation of the glycoproteins in the endoplasmic reticulum and the Golgi apparatus. These components associate during the budding process to produce the mature virion. The nucleocapsid proteins of Sindbis virus and Ross River virus have been produced in a T7-based
Escherichia coli
expression system and purified. In the presence of single-stranded but not double-stranded nucleic acid, the proteins oligomerize in vitro into core-like particles which resemble the native viral nucleocapsid cores. Despite their similarities, Sindbis virus and Ross River virus capsid proteins do not form mixed core-like particles. Truncated forms of the Sindbis capsid protein were used to establish amino acid requirements for assembly. A capsid protein starting at residue 19 [CP(19–264)] was fully competent for in vitro assembly, whereas proteins with further N-terminal truncations could not support assembly. However, a capsid protein starting at residue 32 or 81 was able to incorporate into particles in the presence of CP(19–264) or could inhibit assembly if its molar ratio relative to CP(19–264) was greater than 1:1. This system provides a basis for the molecular dissection of alphavirus core assembly.
Comparative Fitness of Multi-Dideoxynucleoside-Resistant Human Immunodeficiency Virus Type 1 (HIV-1) in an In Vitro Competitive HIV-1 Replication Assay
Authors: Kosalaraksa Pope , Kavlick Mark F. , Maroun Victor , Le Richard , Mitsuya Hiroaki 
Published: December 2019

ABSTRACT

We examined whether human immunodeficiency virus type 1 (HIV-1) fitness was altered upon the acquisition of a set or subset of five mutations (A62V, V75I, F77L, F116Y, and Q151M) in the
pol
gene, which confers resistance to multiple dideoxynucleosides (MDR), as well as the zidovudine resistance-associated mutation T215Y, using a competitive HIV-1 replication assay in a setting of an HXB2D genetic background. Target H9 cells were exposed to a 50:50 mixture of paired infectious molecular clones, and HIV-1 in the culture supernatant was transmitted to new cultures every 7 to 10 days. The polymerase-encoding region of the virus was sequenced at various time points, and the relative proportion of the two viral populations was determined. In the absence of drugs, the comparative order for replicative fitness was HIV-1
62/75/77/116/151
> HIV-1
77/116/151
> HIV-1
151
> wild-type HIV-1 (HIV-1
wt
) > HIV-1
75/77/116/151
> HIV-1
151/215
> HIV-1
215
. In the presence of zidovudine or didanosine, the order was HIV-1
62/75/77/116/151
> HIV-1
77/116/151
> HIV-1
75/77/116/151
> HIV-1
151
> HIV-1
215
. HIV-1
215S(TCC)
, a putative intermediate infectious clone for HIV-1
215
, replicated comparably to HIV-1
wt
, while two putative intermediates for HIV-1
151
[HIV-1
151L(CTG)
and HIV-1
151K(AAG)
] replicated much less efficiently than HIV-1
wt
and HIV-1
151
, suggesting that for HIV-1
151
to develop, two base substitutions are likely to occur concurrently or within a short interval. These data may illustrate the molecular basis by which HIV-1
151
emerges much less frequently than HIV-1
215
. The present data also demonstrate that several MDR HIV-1 variants are more fit than HIV-1
wt
in the absence of drugs and that resistance-associated mutations and drug pressure are critical variates for HIV-1 fitness.
Induction of Human Papillomavirus-Specific CD4+and CD8+Lymphocytes by E7-Pulsed Autologous Dendritic Cells in Patients with Human Papillomavirus Type 16- and 18-Positive Cervical Cancer
Authors: Santin Alessandro D. , Hermonat Paul L. , Ravaggi Antonella , Chiriva-Internati Maurizio , Zhan Dejin , Pecorelli Sergio , Parham Groesbeck P. , Cannon Martin J. 
Published: December 2019

ABSTRACTHuman papillomavirus (HPV) type 16 (HPV 16) and HPV type 18 (HPV 18) are implicated in the induction and progression of the majority of cervical cancers. Since the E6 and E7 oncoproteins of these viruses are expressed in these lesions, such proteins might be potential tumor-specific targets for immunotherapy. In this report, we demonstrate that recombinant, full-length E7-pulsed autologous dendritic cells (DC) can elicit a specific CD8+cytotoxic T-lymphocyte (CTL) response against autologous tumor target cells in three patients with HPV 16- or HPV 18-positive cervical cancer. E7-specific CTL populations expressed strong cytolytic activity against autologous tumor cells, did not lyse autologous concanavalin A-treated lymphoblasts or autologous Epstein-Barr virus-transformed lymphoblastoid cell lines (LCL), and showed low levels of cytotoxicity against natural killer cell-sensitive K562 cells. Cytotoxicity against autologous tumor cells could be significantly blocked by anti-HLA class I (W6/32) and anti-CD11a/LFA-1 antibodies. Phenotypically, all CTL populations were CD3+/CD8+, with variable levels of CD56 expression. CTL induced by E7-pulsed DC were also highly cytotoxic against an allogeneic HLA-A2+HPV 16-positive matched cell line (CaSki). In addition, we show that specific lymphoproliferative responses by autologous CD4+T cells can also be induced by E7-pulsed autologus DC. E7-specific CD4+T cells proliferated in response to E7-pulsed LCL but not unpulsed LCL, and this response could be blocked by anti-HLA class II antibody. Finally, with two-color flow cytometric analysis of intracellular cytokine expression at the single-cell level, a marked Th1-like bias (as determined by the frequency of gamma interferon- and interleukin 4-expressing cells) was observable for both CD8+and CD4+E7-specific lymphocyte populations. Taken together, these data demonstrate that full-length E7-pulsed DC can induce both E7-specific CD4+T-cell proliferative responses and strong CD8+CTL responses capable of lysing autologous naturally HPV-infected cancer cells in patients with cervical cancer. These results may have important implications for the treatment of cervical cancer patients with active or adoptive immunotherapy.
Identification of a Cytoplasmic Targeting/Retention Signal in a Retroviral Gag Polyprotein
Authors: Choi Gyu , Park Sunyoung , Choi Bongkun , Hong Suntaek , Lee Jiyeon , Hunter Eric , Rhee Sung S. 
Published: December 2019

ABSTRACT
Retroviral capsid assembly can occur by either of two distinct morphogenic processes: in type C viruses, the capsid assembles and buds at the plasma membrane, while in type B and D viruses, the capsid assembles within the cytoplasm and is then transported to the plasma membrane for budding. We have previously reported that a single-amino-acid substitution of a tryptophan for an arginine in the matrix protein (MA) of Mason-Pfizer monkey virus (MPMV) converts its capsid assembly from that of a type D retrovirus to that of the type C viruses (S. S. Rhee and E. Hunter, Cell 63:77–86, 1990). Here we identify a region of 18 amino acids within the MA of MPMV that is responsible for type D-specific morphogenesis. Insertion of these 18 amino acids into the MA of type C Moloney murine leukemia virus causes it to assemble an immature capsid in the cytoplasm. Furthermore, fusion of the MPMV MA to the green fluorescent protein resulted in altered intracellular targeting and a punctate accumulation of the fusion protein in the cytoplasm. These 18 amino acids, which are necessary and sufficient to target retroviral Gag polyproteins to defined sites in the cytoplasm, appear to define a novel mammalian cytoplasmic targeting/retention signal.
Replication, Integration, and Packaging of Plasmid DNA following Cotransfection with Baculovirus Viral DNA
Authors: Wu Yuntao , Liu Ge , Carstens Eric B. 
Published: December 2019

ABSTRACT

Infection-dependent replication assays have been used to identify numerous putative origins of baculovirus replication. However, plasmid DNA, when cotransfected into insect cells with
Autographa californica
multinucleocapsid nucleopolyhedrovirus (Ac
M
NPV) DNA, replicates independently of any viral sequence in
cis
(11). Cotransfection of transfer plasmids and baculovirus DNA is a common procedure used in generating recombinant viruses and in measuring the level of gene expression in transient-expression assays. We have examined the fate of a series of vector plasmids in cotransfection experiments. The data reveal that these plasmids replicate following cotransfection and the replication of plasmid DNA is not due to acquisition of viral putative origin sequences. The conformation of plasmid DNA replicating in the cotransfected cells was analyzed and found to exist as high-molecular-weight concatemers. Ten to 25% of the replicated plasmid DNA was integrated into multiple locations on the viral genome and was present in progeny virions following serial passage. Sequence analysis of plasmid-viral DNA junction sites revealed no homologous or conserved sequences in the proximity of the integration sites, suggesting that nonhomologous recombination was involved during the integration process. These data suggest that while a rolling-circle mechanism could be used for baculovirus DNA replication, recombination may also be involved in this process. Plasmid integration may generate large deletions of the viral genome, suggesting that the process of DNA replication in baculovirus may be prone to generation of defective genomes.
Amelioration of Retroviral Vector Silencing in Locus Control Region β-Globin-Transgenic Mice and Transduced F9 Embryonic Cells
Authors: Osborne Cameron S. , Pasceri Peter , Singal Rakesh , Sukonnik Tanya , Ginder Gordon D. , Ellis James 
Published: December 2019

ABSTRACT

Retroviral vectors are transcriptionally silenced in hematopoietic stem cells, and this phenomenon must be overcome for effective gene therapy of blood diseases. The murine stem cell virus (MSCV) vector completely silences β-globin reporter genes regulated by locus control region (LCR) elements 5′HS2 to 5′HS4 in seven of eight transgenic mice. Here, we show that no single known MSCV silencer element is sufficient for complete LCR β-globin transgene silencing. However, partial silencing of high-copy transgenes is conveyed by the MSCV direct repeat and promoter elements. The CpG methylation pattern of silenced and expressed MSCV promoter transgenes is virtually identical, demonstrating that silencing does not absolutely correlate with methylation status. Combined mutations in all four MSCV silencer elements leads to expression of β-globin in 6 of 10 transgenic mice. The same mutations incorporated into the HSC1 retrovirus vector direct
neo
gene expression in 71% of transduced F9 embryonic carcinoma cells. These studies demonstrate that combined mutation of four retroviral silencer elements relieves complete silencing in most transgenic mice and transduced F9 cells and suggests that novel silencer elements remain. Enhanced expression of the HSC1 vector in primitive stem cells is well suited for blood gene therapy applications.
A Complex Translational Program Generates Multiple Novel Proteins from the Latently Expressed Kaposin (K12) Locus of Kaposi’s Sarcoma-Associated Herpesvirus
Authors: Sadler Robert , Wu Lijun , Forghani Bagher , Renne Rolf , Zhong Weidong , Herndier Brian , Ganem Don 
Published: December 2019

ABSTRACT
The most abundantly expressed latent transcripts encoded by the Kaposi’s sarcoma (KS)-associated herpesvirus derive from the genomic region surrounding open reading frame (ORF) K12 (kaposin A). Here we show that these transcripts, initially described as limited to ORF K12 itself, more frequently encompass upstream sequences spanning two sets of 23-nucleotide GC-rich direct repeats (DRs) (DR1 and DR2). Although the DRs lack AUG codons and were previously presumed to be noncoding, a monoclonal antibody raised to infected cells detected multiple polypeptides encoded by this region. These proteins are expressed during latency and upon induction of lytic viral replication in both primary effusion lymphoma (PEL) cell lines and KS tumors. Biochemical and genetic analyses reveal that these proteins are derived from variant translational initiation at CUG codons. The predominant translation product in the PEL cell line BCBL-1 derives from the 5′-most CUG codon in the transcript, resulting in a protein (termed kaposin B) which is encoded largely by the repeats themselves and which does not include K12 sequences. Other non-AUG codons in alternate reading frames are also used at lower efficiency, including one that initiates translation of a DR-K12 fusion protein (kaposin C) that is predicted to sort to a different subcellular locale than kaposin B. Thus, the products of the K12 region, which is the most abundantly transcribed region in latency, are surprisingly complex and may encompass multiple biological functions.
The P236L Delavirdine-Resistant Human Immunodeficiency Virus Type 1 Mutant Is Replication Defective and Demonstrates Alterations in both RNA 5′-End- and DNA 3′-End-Directed RNase H Activities
Authors: Gerondelis Peter , Archer Richard H. , Palaniappan Chockalingam , Reichman Richard C. , Fay Philip J. , Bambara Robert A. , Demeter Lisa M. 
Published: December 2019

ABSTRACT
The nonnucleoside reverse transcriptase (RT) inhibitor (NNRTI) delavirdine (DLV) selects in vitro for the human immunodeficiency virus type 1 (HIV-1) RT mutation P236L, which confers high-level resistance to DLV but not other NNRTIs. Unexpectedly, P236L has developed infrequently in HIV-1 isolates obtained from patients receiving DLV; K103N is the predominant resistance mutation observed in that setting. We characterized the replication fitness of viruses derived from pNL4-3 containing P236L or K103N in both H9 and primary human peripheral blood mononuclear cell cultures infected in parallel with the two mutants. In the absence of DLV, p24 production by wild-type virus occurred more rapidly and to higher levels than with either mutant; P236L consistently demonstrated a two- to threefold decrease in p24 relative to K103N. At low levels of DLV, growth of wild-type virus was severely inhibited, and K103N replicated two- to threefold more efficiently than P236L. At high concentrations of DLV, P236L replication and K103N replication were both inhibited. Recombinant RTs containing K103N or P236L were analyzed for DNA polymerization on heteropolymeric RNA templates and RNase H degradation of RNA-DNA hybrids. Neither mutant demonstrated defects in polymerization. K103N demonstrated normal RNA 5′-end-directed RNase H cleavage and slowed DNA 3′-end-directed RNase H cleavage compared to wild-type RT. P236L demonstrated slowing of both DNA 3′-end- and RNA 5′-end-directed RNase H cleavage, consistent with its reduced replication efficiency relative to K103N. These data suggest that NNRTI resistance mutations can lead to reductions in the efficiency of RNase H cleavage, which may contribute to a reduction in the replication fitness of HIV-1.
The Human Papillomavirus Type 16 E6 Gene Alone Is Sufficient To Induce Carcinomas in Transgenic Animals
Authors: Song Shiyu , Pitot Henry C. , Lambert Paul F. 
Published: December 2019

ABSTRACT
High-risk human papillomaviruses (HPVs) are the causative agents of certain human cancers. HPV type 16 (HPV16) is the papillomavirus most frequently associated with cervical cancer in women. The E6 and E7 genes of HPV are expressed in cells derived from these cancers and can transform cells in tissue culture. Animal experiments have demonstrated that E6 and E7 together cause tumors. We showed previously that E6 and E7 together or E7 alone could induce skin tumors in mice when these genes were expressed in the basal epithelia of the skin. In this study, we investigated the role that the E6 gene plays in carcinogenesis. We generated K14E6 transgenic mice, in which the HPV16 E6 gene was directed in its expression by the human keratin 14 promoter (hK14) to the basal layer of the epidermis. We found that E6 induced cellular hyperproliferation and epidermal hyperplasia and caused skin tumors in adult mice. Interestingly, the tumors derived from E6 were mostly malignant, as opposed to the tumors from E7 mice, which were mostly benign. This result leads us to hypothesize that E6 may contribute differently than E7 to HPV-associated carcinogenesis; whereas E7 primarily contributes to the early stages of carcinogenesis that lead to the formation of benign tumors, E6 primarily contributes to the late stages of carcinogenesis that lead to malignancy.
Identification and Rapid Quantification of Early- and Late-Lytic Human Herpesvirus 8 Infection in Single Cells by Flow Cytometric Analysis: Characterization of Antiherpesvirus Agents
Authors: Zoeteweij J. Paul , Eyes Sharon T. , Orenstein Jan M. , Kawamura Tatsuyoshi , Wu LiJun , Chandran Bala , Forghani Bagher , Blauvelt Andrew 
Published: December 2019

ABSTRACT

Human herpesvirus 8 (HHV-8) infection is associated with Kaposi’s sarcoma, primary effusion lymphoma (PEL), and multicentric Castleman’s disease. In this study, we used monoclonal antibodies (MAbs) directed against HHV-8 lytic cycle-associated proteins encoded by open reading frame (ORF) 59 (nuclear PF-8 protein) and ORF K8.1 (viral envelope glycoprotein K8.1 [gpK8.1]) to investigate HHV-8 lytic infection in single cells. Lytically infected cells were labeled with MAbs, stained with fluorescently conjugated secondary Abs, and analyzed by flow cytometry. A 3-day stimulation of HHV-8-positive PEL cell lines (BCBL-1 and BC-3) with 12-
O
-tetradecanoylphorbol-13-acetate (30 nM) or
n
-butyric acid (0.3 mM) maximized the expression of lytic-phase viral proteins and minimized cell toxicity. The absolute number of expressing cells was inducer and cell line dependent. Expression of PF-8 occurred earlier and more frequently (in up to 20% of cells) than did expression of gpK8.1. A subset of PF-8 positive cells (25%) co-expressed gpK8.1, representing the majority of gpK8.1 expressing cells. Acyclovir, foscarnet, cidofovir, and PMEA reduced the number of cells expressing gpK8.1, but not the number expressing the nonstructural early lytic gene product PF-8. By contrast, alpha interferon (IFN-α) and IFN-β reduced expression of both PF-8 and gpK8.1, implying an overall inhibitory effect on viral gene transcription or translation. In summary, we have characterized and quantified HHV-8 lytic infection in single cells by dual measurement of early- and late-lytic-cycle HHV-8 protein expression. This technique should prove useful for screening of possible antiherpesvirus agents and for detailed phenotypic characterization of HHV-8-infected cells in vitro and in patients with HHV-8-associated diseases.