bluuebunny/crossref_metadata_2025_split huggingface dataset:
Identification of Phosphorylation Sites within the Herpes Simplex Virus Tegument Protein VP22
Authors: Elliott Gillian , O’Reilly Dawn , O’Hare Peter 
Published: December 2019

ABSTRACT
The herpes simplex virus protein VP22 is a major phosphoprotein of infected cells. In this study, we identify two serine phosphorylation sites within VP22 and show that the N-terminal site is a substrate for casein kinase II, while the extreme C-terminal site is a substrate for another, as yet unidentified, cellular kinase. Furthermore, we show that a mutant of VP22 which has both sites altered is unable to incorporate phosphate in vivo, confirming that there are no other phosphorylation sites within VP22.
Effect of Inserting Paramyxovirus Simian Virus 5 Gene Junctions at the HN/L Gene Junction: Analysis of Accumulation of mRNAs Transcribed from Rescued Viable Viruses
Authors: He Biao , Lamb Robert A. 
Published: December 2019

ABSTRACTSimian parainfluenza virus 5 (SV5) is a prototype of theParamyxoviridaefamily of nonsegmented negative-sense RNA viruses. The single-stranded RNA genomes of these viruses contain a series of tandemly linked genes separated by intergenic (IG) sequences flanked by gene-end (GE) and gene-start (GS) sequences. The viral RNA polymerase (vRNAP) complex is thought to enter the genome at its 3′ end, and synthesis of mRNAs is thought to occur by a stop-start mechanism in a sequential and polar manner, with transcriptional attenuation occurring primarily at the intergenic regions. As a result, multiple nonoverlapping mRNA species are generated for each single entry of the vRNAP. To investigate the functions of GE, IG, and GS sequences in transcription, we constructed plasmids containing cDNAs of the full-length SV5 genome in which the gene junction sequences (GE, IG, and GS sequences) located between the hemagglutinin-neuraminidase (HN) and the polymerase (L) genes were replaced with the counterpart sequences from other gene junctions. By using reverse genetics, we recovered viable viruses from each cDNA construct, although their growth characteristics varied. Analysis of the HN and L mRNAs by quantitative RNase protection assay indicated that the ratios of HN to L mRNAs varied over a fourfold range. The alteration of the gene junction sequences also permitted examination of the hypothesized requirement for hexamer nucleotide position of the GS sites. The recovery of infectious viruses with transcription initiation sites that occurred at nucleotide positions 1, 2, 3, 5, and 6 of the hexamer suggest that the requirement is nonstringent.
Bicyclams, Selective Antagonists of the Human Chemokine Receptor CXCR4, Potently Inhibit Feline Immunodeficiency Virus Replication
Authors: Egberink Herman F. , De Clercq Erik , Van Vliet Arno L. W. , Balzarini Jan , Bridger Gary J. , Henson Geoffrey , Horzinek Marian C. , Schols Dominique 
Published: December 2019

ABSTRACT

Bicyclams are low-molecular-weight anti-human immunodeficiency virus (HIV) agents that have been shown to act as potent and selective CXC chemokine receptor 4 (CXCR4) antagonists. Here, we demonstrate that bicyclams are potent inhibitors of feline immunodeficiency virus (FIV) replication when evaluated in Crandell feline kidney (CRFK) cells. With a series of bicyclam derivatives, 50% inhibitory concentrations (IC
50
s) against FIV were obtained in this cell system that were comparable to those obtained for HIV-1 IIIB replication in the human CD4
+
MT-4 T-cell line. The bicyclams were also able to block FIV replication in feline thymocytes, albeit at higher concentrations than in the CRFK cells. The prototype bicyclam AMD3100, 1-1′-[1,4-phenylene-bis(methylene)]-bis(1,4,8,11-tetraazacyclotetradecane), was only fourfold less active in feline thymocytes (IC
50
, 62 ng/ml) than in CRFK cells (IC
50
, 14 ng/ml). AMD2763, 1,1′-propylene-bis(1,4,8,11-tetraazacyclotetradecane), which is a less potent CXCR4 antagonist, was virtually inactive against FIV in feline thymocytes (IC
50
, >66.5 μg/ml), while it was clearly active in CRFK cells (IC
50
, 0.9 μg/ml). The CXC chemokine stromal-cell-derived factor 1α had anti-FIV activity in CRFK cells (IC
50
, 200 ng/ml) but not in feline thymocytes (IC
50
, >2.5 μg/ml). When primary FIV isolates were evaluated for their drug susceptibility in feline thymocytes, the bicyclams AMD3100 and its Zn
2+
complex, AMD3479, inhibited all six primary isolates at equal potency. The marked susceptibility of FIV to the bicyclams suggests that FIV predominantly uses feline CXCR4 for entering its target cells.
Subcellular Localization and Rolling Circle Replication of Peach Latent Mosaic Viroid: Hallmarks of Group A Viroids
Authors: Bussière F. , Lehoux J. , Thompson D. A. , Skrzeczkowski L. J. , Perreault J.-P. 
Published: December 2019

ABSTRACT
We characterized the peach latent mosaic viroid (PLMVd) replication intermediates that accumulate in infected peach leaves and determined the tissue and subcellular localization of the RNA species. Using in situ hybridization, we showed that PLMVd strands of both plus and minus polarities concentrate in the cells forming the palisade parenchyma. At the cellular level, PLMVd was found to accumulate predominantly in chloroplasts. Northern blot analyses demonstrated that PLMVd replicates via a symmetric mode involving the accumulation of both circular and linear monomeric strands of both polarities. No multimeric conformer was detected, indicating that both strands self-cleave efficiently via their hammerhead sequences. Dot blot hybridizations revealed that PLMVd strands of both polarities accumulate equally but that the relative concentrations vary by more than 50-fold between peach cultivars. Taken together these results establish two hallmarks for the classification of viroids. Group A viroids (e.g., PLMVd), which possess hammerhead structures, replicate in the chloroplasts via the symmetric mode. By contrast, group B viroids, which share a conserved central region, replicate in the nucleus via an asymmetric mechanism. This is an important difference between self-cleaving and non-self-cleaving viroids, and the implications for the evolutionary origin and replication are discussed.
Use of DNA, RNA, and Chimeric Templates by a Viral RNA-Dependent RNA Polymerase: Evolutionary Implications for the Transition from the RNA to the DNA World
Authors: Siegel Robert W. , Bellon Laurent , Beigelman Leonid , Kao C. Cheng 
Published: December 2019

ABSTRACT
All polynucleotide polymerases have a similar structure and mechanism of catalysis, consistent with their evolution from one progenitor polymerase. Viral RNA-dependent RNA polymerases (RdRp) are expected to have properties comparable to those from this progenitor and therefore may offer insight into the commonalities of all classes of polymerases. We examined RNA synthesis by the brome mosaic virus RdRp on DNA, RNA, and hybrid templates and found that precise initiation of RNA synthesis can take place from all of these templates. Furthermore, initiation can take place from either internal or penultimate initiation sites. Using a template competition assay, we found that the BMV RdRp interacts with DNA only three- to fourfold less well than it interacts with RNA. Moreover, a DNA molecule with a ribonucleotide at position −11 relative to the initiation nucleotide was able to interact with RdRp at levels comparable to that observed with RNA. These results suggest that relatively few conditions were needed for an ancestral RdRp to replicate DNA genomes.
Both Memory and CD45RA+/CD62L+Naive CD4+T Cells Are Infected in Human Immunodeficiency Virus Type 1-Infected Individuals
Authors: Ostrowski Mario A. , Chun Tae-Wook , Justement Shawn J. , Motola Ivette , Spinelli Michael A. , Adelsberger Joseph , Ehler Linda A. , Mizell Stephanie B. , Hallahan Claire W. , Fauci Anthony S. 
Published: December 2019

ABSTRACTCellular activation is critical for the propagation of human immunodeficiency virus type 1 (HIV-1) infection. It has been suggested that truly naive CD4+T cells are resistant to productive HIV-1 infection because of their constitutive resting state. Memory and naive CD4+T-cell subsets from 11 HIV-1-infected individuals were isolated ex vivo by a combination of magnetic bead depletion and fluorescence-activated cell sorting techniques with stringent criteria of combined expression of CD45RA and CD62L to identify naive CD4+T-cell subsets. In all patients HIV-1 provirus could be detected within naive CD45RA+/CD62L+CD4+T cells; in addition, replication-competent HIV-1 was isolated from these cells upon CD4+T-cell stimulation in tissue cultures. Memory CD4+T cells had a median of fourfold more replication-competent virus and a median of sixfold more provirus than naive CD4+T cells. Overall, there was a median of 16-fold more integrated provirus identified in memory CD4+T cells than in naive CD4+T cells within a given patient. Interestingly, there was a trend toward equalization of viral loads in memory and naive CD4+T-cell subsets in those patients who harbored CXCR4-using (syncytium-inducing) viruses. Within any given patient, there was no selective usage of a particular coreceptor by virus isolated from memory versus naive CD4+T cells. Our findings suggest that naive CD4+T cells may be a significant viral reservoir for HIV, particularly in those patients harboring CXCR4-using viruses.
Lymphocyte Deficiencies Increase Susceptibility to Friend Virus-Induced Erythroleukemia inFv-2Genetically Resistant Mice
Authors: Hasenkrug Kim J. 
Published: December 2019

ABSTRACTThe study of genetic resistance to retroviral diseases provides insights into the mechanisms by which organisms overcome potentially lethal infections.Fv-2resistance to Friend virus-induced erythroleukemia acts through nonimmunological mechanisms to prevent early virus spread, but it does not completely block infection. The current experiments were done to determine whether Fv-2 alone could provide resistance or whether immunological mechanisms were also required to bring infection under control.Fv-2-resistant mice that were CD4+T-cell deficient were able to restrict early virus replication and spread as well as normalFv-2-resistant mice, but they could not maintain control and developed severe Friend virus-induced splenomegaly and erythroleukemia by 6 to 8 weeks postinfection. Mice deficient in CD8+T cells and, to a lesser extent, B cells were also susceptible to late Friend virus-induced disease. Thus,Fv-2resistance does not independently prevent FV-induced erythroleukemia but works in concert with the immune system by limiting early infection long enough to allow virus-specific immunity time to develop and facilitate recovery.
Reverse Transcriptase Inhibitors Can Selectively Block the Synthesis of Differently Sized Viral DNA Transcripts in Cells Acutely Infected with Human Immunodeficiency Virus Type 1
Authors: Quan Yudong , Rong Liwei , Liang Chen , Wainberg Mark A. 
Published: December 2019

ABSTRACT
We have recently reported that the in vitro inhibition of human immunodeficiency virus type 1 (HIV-1) reverse transcription by inhibitors of reverse transcriptase (RT) occurred most efficiently when the expected DNA products of RT reactions were long (Quan et al., Nucleic Acids Res. 26:5692–5698, 1998). Here, we have used a quantitative PCR to analyze HIV-1 reverse transcription within acutely infected cells treated with RT inhibitors. We found that levels of minus-strand strong-stop DNA [(−)ssDNA] formed in acutely infected MT2 cells were only slightly reduced if cells were infected with viruses that had been generated in the presence of either azidothymidine or nevirapine (5 μM) and maintained in the presence of this drug throughout the viral adsorption period and thereafter. Control experiments in which virus inoculation of cells was performed at 4°C, followed directly by cell extraction, showed that less than 1% of total (−)ssDNA within acutely infected cells was attributable to its presence within adsorbed virions. In contrast, synthesis of intermediate-length reverse-transcribed DNA products decreased gradually as viral DNA strand elongation took place in the presence of either of these inhibitors. This establishes that nucleoside and nonnucleoside RT inhibitors can exert similar temporal impacts in regard to inhibition of viral DNA synthesis. Generation of full-length viral DNA, as expected, was almost completely blocked in the presence of these antiviral drugs. These results provide insight into the fact that high concentrations of drugs are often needed to yield inhibitory effects in cell-free RT assays performed with short templates, whereas relatively low drug concentrations are often strongly inhibitory in cellular systems.
Association between Virus-Specific Cytotoxic T-Lymphocyte and Helper Responses in Human Immunodeficiency Virus Type 1 Infection
Authors: Kalams Spyros A. , Buchbinder S. P. , Rosenberg E. S. , Billingsley J. M. , Colbert D. S. , Jones N. G. , Shea A. K. , Trocha A. K. , Walker B. D. 
Published: December 2019

ABSTRACTCellular immune responses are thought to be an important antiviral host defense, but the relationship between virus-specific T-helper and cytotoxic-T-lymphocyte (CTL) responses has not been defined. To investigate a potential link between these responses, we examined functional human immunodeficiency virus type 1 (HIV-1)-specific memory CTL precursor frequencies and p24-specific proliferative responses in a cohort of infected untreated persons with a wide range of viral loads and CD4 cell counts. Levels of p24-specific proliferative responses positively correlated with levels of Gag-specific CTL precursors and negatively correlated with levels of plasma HIV-1 RNA. These data linking the levels of HIV-specific CTL with virus-specific helper cell function during chronic viral infection provide cellular immunologic parameters to guide therapeutic and prophylactic vaccine development.
Levels of Human Immunodeficiency Virus Type 1-Specific Cytotoxic T-Lymphocyte Effector and Memory Responses Decline after Suppression of Viremia with Highly Active Antiretroviral Therapy
Authors: Kalams Spyros A. , Goulder Philip J. , Shea Amy K. , Jones Norman G. , Trocha Alicja K. , Ogg Graham S. , Walker Bruce D. 
Published: December 2019

ABSTRACTTherapeutic suppression of human immunodeficiency virus type 1 (HIV-1) replication may help elucidate interactions between the host cellular immune responses and HIV-1 infection. We performed a detailed longitudinal evaluation of two subjects before and after the start of highly active antiretroviral therapy (HAART). Both subjects had evidence of in vivo-activated and memory cytotoxic T-lymphocyte precursor (CTLp) activity against multiple HIV-1 gene products. After the start of therapy, both subjects had declines in the levels of in vivo-activated HIV-1-specific CTLs and had immediate increases in circulating HIV-1-specific CTL memory cells. With continued therapy, and continued suppression of viral load, levels of memory CTLps declined. HLA A*0201 peptide tetramer staining demonstrated that declining levels of in vivo-activated CTL activity were associated with a decrease in the expression of the CD38+activation marker. Transient increases in viral load during continued therapy were associated with increases in the levels of virus-specific CTLps in both individuals. The results were confirmed by measuring CTL responses to discrete optimal epitopes. These studies illustrate the dynamic equilibrium between the host immune response and levels of viral antigen burden and suggest that efforts to augment HIV-1-specific immune responses in subjects on HAART may decrease the incidence of virologic relapse.
Identification of a Spliced Gene from Kaposi’s Sarcoma-Associated Herpesvirus Encoding a Protein with Similarities to Latent Membrane Proteins 1 and 2A of Epstein-Barr Virus
Authors: Glenn Mark , Rainbow Lucille , Auradé Frédéric , Davison Andrew , Schulz Thomas F. 
Published: December 2019

ABSTRACT

Kaposi’s sarcoma-associated herpesvirus (KSHV) or human herpesvirus 8 (HHV-8) is a novel herpesvirus implicated as the causative agent of Kaposi’s sarcoma (KS), primary effusion lymphoma, and some cases of multicentric Castleman’s disease. KSHV persists in the majority of KS spindle (endothelial tumor) cells and lymphoid cells in a latent form, with only a limited set of viral genes expressed in a tissue-specific manner. Here, we report the identification of a family of alternatively-spliced transcripts of approximately 7.5 kb expressed in latently infected body cavity-based lymphoma (BCBL) cell lines which are predicted to encode membrane proteins with similarities to the LMP2A and LMP1 proteins of Epstein-Barr virus. In two highly divergent sequence variants of the right end of the KSHV genome, alternative splicing of eight exons located between KSHV ORF 75 and the terminal repeats yields transcripts appropriate for proteins with up to 12 transmembrane domains, followed by a hydrophilic C-terminal, presumably cytoplasmic, domain. This C-terminal domain contains several YxxI/L motifs reminiscent of LMP2A and a putative TRAF binding site as in LMP1. In latently (persistently) infected BCBL cells the predominant transcript utilizes all eight exons, whereas in phorbol-ester-induced cells, a shorter transcript, lacking exons 4 and 5, is also abundant. We also found evidence for an alternative use of exon 1. Transfection of an epitope-tagged cDNA construct containing all exons indicates that the encoded protein is localized on cell surface and intracellular membranes, and glutathione
S
-transferase pull-down experiments indicate that its cytoplasmic domain, like that of LMP1, interacts with TRAF1, -2, and -3. Two of 20 KS patients had antibodies to the hydrophilic C-terminal domain, suggesting that the protein is expressed in vivo.
Mutations within Four Distinct Gag Proteins Are Required To Restore Replication of Human Immunodeficiency Virus Type 1 after Deletion Mutagenesis within the Dimerization Initiation Site
Authors: Liang Chen , Rong Liwei , Quan Yudong , Laughrea Michael , Kleiman Lawrence , Wainberg Mark A. 
Published: December 2019

ABSTRACT
Human immunodeficiency virus type 1 (HIV-1) genomic RNA segments at nucleotide (nt) positions +240 to +274 are thought to form a stem-loop secondary structure, termed SL1, that serves as a dimerization initiation site for viral genomic RNA. We have generated two distinct deletion mutations within this region, termed BH10-LD3 and BH10-LD4, involving nt positions +238 to +253 and +261 to +274, respectively, and have shown that each of these resulted in significant diminutions in levels of viral infectiousness. However, long-term culture of each of these viruses in MT-2 cells resulted in a restoration of infectiousness, due to a series of compensatory point mutations within four distinct proteins that are normally cleaved from the Gag precursor. In the case of BH10-LD3, these four mutations were MA1, CA1, MP2, and MNC, and they involved changes of amino acid Val-35 to Ile within the matrix protein (MA), Ile-91 to Thr within the capsid (CA), Thr-12 to Ile within p2, and Thr-24 to Ile within the nucleocapsid (NC). The order in which these mutations were acquired by the mutated BH10-LD3 was MNC > CA1 > MP2 > MA1. The results of site-directed mutagenesis studies confirmed that each of these four substitutions contributed to the increased viability of the mutated BH10-LD3 viruses and that the MNC substitution, which was acquired first, played the most important role in this regard. Three point mutations, MP2, MNC, and MA2, were also shown to be sequentially acquired by viruses that had emerged in culture from the BH10-LD4 deletion. The first two of these were identical to those described above, while the last involved a change of Val-35 to Leu. All three of these substitutions were necessary to restore the infectiousness of mutated BH10-LD4 viruses to wild-type levels, although the MP2 mutation alone, but neither of the other two substitutions, was able to confer some viability on BH10-LD4 viruses. Studies of viral RNA packaging showed that the BH10-LD4 deletion only marginally impaired encapsidation while the BH10-LD3 deletion caused a severe deficit in this regard.
Novel Endogenous Type C Retrovirus in Baboons: Complete Sequence, Providing Evidence for Baboon Endogenous Virus
gag-pol
Ancestry
Authors: Mang Rui , Goudsmit Jaap , van der Kuyl Antoinette C. 
Published: December 2019

ABSTRACT

A complete endogenous type C viral genome has been isolated from a baboon genomic library. The provirus,
Papio cynocephalus
endogenous retrovirus (PcEV), is 8,572 nucleotides long, and 38 to 59 proviral copies per baboon genome are found. The PcEV provirus possesses the typical simple retroviral gene organization, including two long terminal repeats and genes encoding gag, pol, and env proteins. The open reading frames for
gag-pol
and
env
are complete but have premature stop codons or frameshift mutations. The primer binding site of PcEV is complementary to tRNA
Gly
. The
gag
and
pol
genes of PcEV are closely related to those of the baboon endogenous virus (BaEV). The
env
coding region of PcEV is related to the
env
genes of type C retroviruses. This suggests that PcEV is one of the ancestors of BaEV contributing the type C
gag-pol
genome fragment to the type C/D recombinant virus BaEV. Earlier it was shown that another endogenous type D virus (simian endogenous retrovirus) provided the
env
gene for BaEV (A. C. van der Kuyl et al., J. Virol. 71:3666–3676, 1997).
Cytomegalovirus (CMV) DNA Load Is an Independent Predictor of CMV Disease and Survival in Advanced AIDS
Authors: Spector Stephen A. , Hsia Karen , Crager Michael , Pilcher Marshall , Cabral Sheila , Stempien Mary Jean 
Published: December 2019

ABSTRACT
The impact of cytomegalovirus (CMV) on human immunodeficiency virus type 1 (HIV-1) disease progression has been controversial. In this study, we sought to determine if CMV viral load is independent of HIV-1 viral load in predicting CMV disease and survival. Our findings indicate that in patients with advanced AIDS, CMV DNA load is an independent marker of CMV disease and survival and is more predictive than HIV-1 RNA load. Moreover, patients who respond to preemptive therapy with oral ganciclovir, with resulting undetectable levels of CMV DNA, in their plasma, have a significantly lower risk of developing CMV disease and higher rates of survival, despite stable or increasing HIV-1 RNA loads. These data provide support for CMV as an independent risk factor for mortality in persons with advanced AIDS and further suggest that effective preemptive therapy for CMV can improve patient survival rates.
E4ORF3 Requirement for Achieving Long-Term Transgene Expression from the Cytomegalovirus Promoter in Adenovirus Vectors
Authors: Armentano Donna , Smith Michael P. , Sookdeo Cathleen C. , Zabner Joseph , Perricone Michael A. , St. George Judith A. , Wadsworth Samuel C. , Gregory Richard J. 
Published: December 2019

ABSTRACT
Analysis of transgene expression under the control of the cytomegalovirus (CMV) promoter from adenovirus vectors in which the E4 region was modified indicated that E4ORF3 is required for long-term expression in the murine lung. CMV promoter truncation led to the persistence of expression in the absence of E4, thus eliminating the ORF3 requirement.