bluuebunny/crossref_metadata_2025 huggingface dataset:
Retrograde, Transneuronal Spread of Pseudorabies Virus in Defined Neuronal Circuitry of the Rat Brain Is Facilitated by gE Mutations That Reduce Virulence
Authors: Yang M. , Card J. P. , Tirabassi R. S. , Miselis R. R. , Enquist L. W.
Published: December 2019

The pseudorabies virus (PRV) gE gene encodes a multifunctional membrane protein found in infected cell membranes and in the virion envelope. Deletion of the gE gene results in marked attenuation of the virus in almost every animal species tested that is permissive for PRV. A common inference is that gE mutants are less virulent because they have reduced ability to spread from cell to cell; e.g., gE mutants infect fewer cells and, accordingly, animals live longer. In this report, we demonstrate that this inference does not hold in a rat experimental model for virus invasion of the brain. We find that animals infected with gE mutants live longer despite extensive retrograde, transneuronal spread of virus in the rat brain. In this model of brain infection, virus is injected into the stomach musculature and virions spread to the brain in long axons of brain stem neurons that give rise to the tenth cranial nerve (the vagus). The infection then spreads from neuron to neuron in well-defined, and physically separated, areas of the brain involved in autonomic regulation of the viscera. We examined the progression of infection of five PRV strains in this circuitry: the wild-type PRV-Becker strain, the attenuated PRV-Bartha vaccine strain, and three gE mutants isogenic with the PRV-Becker strain. By 60 to 67 h after infection, all PRV-Becker-infected animals were dead. Analysis of Becker-infected rats killed prior to virus-induced death demonstrated that the virus had established an infection only in the primary vagal neurons connected directly to the stomach and synaptically linked neurons in the immediate vicinity of the caudal brain stem. There was little spread to other neurons in the vagus circuitry. In contrast, rats infected with PRV-Bartha or PRV-Becker gE mutants survived to at least 96 h and exhibited few overt signs of disease. Despite this long survival and the lack of symptoms, brains of animals sacrificed at this time revealed extensive transsynaptic infection not only of the brain stem but also of areas of the forebrain synaptically linked to neurons in the brain stem. This finding provides evidence that the gE protein plays a role in promoting symptoms of infection and death in animals that is independent of neuron-to-neuron spread during brain infection. When this early virulence function is not active, animals live longer, resulting in more extensive spread of virus in the brain.
Cyanovirin-N Binds to gp120 To Interfere with CD4-Dependent Human Immunodeficiency Virus Type 1 Virion Binding, Fusion, and Infectivity but Does Not Affect the CD4 Binding Site on gp120 or Soluble CD4-Induced Conformational Changes in gp120
Authors: Esser Mark T. , Mori Toshiyuki , Mondor Isabelle , Sattentau Quentin J. , Dey Barna , Berger Edward A. , Boyd Michael R. , Lifson Jeffrey D.
Published: December 2019

Cyanovirin-N (CV-N), an 11-kDa protein isolated from the cyanobacterium
Nostoc ellipsosporum
, potently inactivates diverse strains of human immunodeficiency virus type 1 (HIV-1), HIV-2, and simian immunodeficiency virus. While it has been well established that the viral surface envelope glycoprotein gp120 is a molecular target of CV-N, the detailed mechanism of action is of further interest. We compared matched native and CV-N-treated virus preparations in a panel of assays that measure viral replication, assessing successive stages of the viral life cycle. CV-N-treated virions failed to infect cells as detected by p24 production and quantitative PCR for HIV-1 reverse transcription products, whereas treatment of the target cells did not block infection, confirming that CV-N acts at the level of the virus, not the target cell, to abort the initial infection process. Compared to native HIV-1 preparations, CV-N-treated HIV-1 virions showed impaired CD4-dependent binding to CD4
+
T cells and did not mediate “fusion from without” of CD4
+
target cells. CV-N also blocked HIV envelope glycoprotein Env-induced, CD4-dependent cell-cell fusion. Mapping studies with monoclonal antibodies (MAbs) to defined epitopes on the HIV-1 envelope glycoprotein indicated that CV-N binds to gp120 in a manner that does not occlude or alter the CD4 binding site or V3 loop or other domains on gp120 recognized by defined MAbs and does not interfere with soluble CD4-induced conformational changes in gp120. Binding of CV-N to soluble gp120 or virions inhibited subsequent binding of the unique neutralizing MAb 2G12, which recognizes a glycosylation-dependent epitope. However, prior binding of 2G12 MAb to gp120 did not block subsequent binding by CV-N. These results help clarify the mechanism of action of CV-N and suggest that the compound may act in part by preventing essential interactions between the envelope glycoprotein and target cell receptors. This proposed mechanism is consistent with the extensive activity profile of CV-N against numerous isolates of HIV-1 and other lentiviruses and supports the potential broad utility of this protein as a microbicide to prevent the sexual transmission of HIV.
Subdomain Folding and Biological Activity of the Core Structure from Human Immunodeficiency Virus Type 1 gp41: Implications for Viral Membrane Fusion
Authors: Lu Min , Ji Hong , Shen Steven
Published: December 2019

The envelope glycoprotein of human immunodeficiency virus type 1 (HIV-1) consists of two subunits, gp120 and gp41. The extraviral portion (ectodomain) of gp41 contains an α-helical domain that likely represents the core of the fusion-active conformation of the molecule. Here we report the identification and characterization of a minimal, autonomous folding subdomain that retains key determinants in specifying the overall fold of the gp41 ectodomain core. This subdomain, designated N34(L6)C28, is formed by covalent attachment of peptides N-34 and C-28 by a short flexible linker in place of the normal disulfide-bonded loop sequence. N34(L6)C28 forms a highly thermostable, α-helical trimer. Point mutations within the envelope protein complex that abolish membrane fusion and HIV-1 infectivity also impede the formation of the N34(L6)C28 core. Moreover, N34(L6)C28 is capable of inhibiting HIV-1 envelope-mediated membrane fusion. Taken together, these results indicate that the N34(L6)C28 core plays a direct role in the membrane fusion step of HIV-1 infection and thus provides a molecular target for the development of antiviral pharmaceutical agents.
Oncogene Activation in Myeloid Leukemias by Graffi Murine Leukemia Virus Proviral Integration
Authors: Denicourt Catherine , Edouard Elsy , Rassart Eric
Published: December 2019

The Graffi murine leukemia virus (MuLV) is a nondefective retrovirus that induces granulocytic leukemia in BALB/c and NFS mice. To identify genes involved in Graffi MuLV-induced granulocytic leukemia, tumor cell DNAs were examined for genetic alterations at loci described as common proviral integration sites in MuLV-induced myeloid, lymphoid, and erythroid leukemias. Southern blot analysis revealed rearrangements in c-
myc
,
Fli-1
,
Pim-1
, and
Spi-1
/
PU.1
genes in 20, 10, 3.3, and 3.3% of the tumors tested, respectively. These results demonstrate for the first time the involvement of those genes in granulocytic leukemia.
Immunization with Potato Plants Expressing VP60 Protein Protects against Rabbit Hemorrhagic Disease Virus
Authors: Castañón S. , Marín M. S. , Martín-Alonso J. M. , Boga J. A. , Casais R. , Humara J. M. , Ordás R. J. , Parra F.
Published: December 2019

The major structural protein VP60 of rabbit hemorrhagic disease virus (RHDV) has been produced in transgenic potato plants under the control of a cauliflower mosaic virus 35S promoter or a modified 35S promoter that included two copies of a strong transcriptional enhancer. Both types of promoters allowed the production of specific mRNAs and detectable levels of recombinant VP60, which were higher for the constructs carrying the modified 35S promoter. Rabbits immunized with leaf extracts from plants carrying this modified 35S promoter showed high anti-VP60 antibody titers and were fully protected against the hemorrhagic disease.
Highly Efficient Induction of Protective Immunity by a Vaccinia Virus Vector Defective in Late Gene Expression
Authors: Holzer G. W. , Remp G. , Antoine G. , Pfleiderer M. , Enzersberger O. M. , Emsenhuber W. , Hämmerle T. , Gruber F. , Urban C. , Falkner F. G. , Dorner F.
Published: December 2019

Vaccinia viruses defective in the essential gene coding for the enzyme uracil DNA glycosylase (UDG) do not undergo DNA replication and do not express late genes in wild-type cells. A UDG-deficient vaccinia virus vector carrying the tick-borne encephalitis (TBE) virus prM/E gene, termed vD4-prME, was constructed, and its potential as a vaccine vector was evaluated. High-level expression of the prM/E antigens could be demonstrated in infected complementing cells, and moderate levels were found under noncomplementing conditions. The vD4-prME vector was used to vaccinate mice; animals receiving single vaccination doses as low as 10
4
PFU were fully protected against challenge with high doses of virulent TBE virus. Single vaccination doses of 10
3
PFU were sufficient to induce significant neutralizing antibody titers. With the corresponding replicating virus, doses at least 10-fold higher were needed to achieve protection. The data indicate that late gene expression of the vaccine vector is not required for successful vaccination; early vaccinia virus gene expression induces a potent protective immune response. The new vaccinia virus-based defective vectors are therefore promising live vaccines for prophylaxis and cancer immunotherapy.
Human Cytomegalovirus Infection of Caco-2 Cells Occurs at the Basolateral Membrane and Is Differentiation State Dependent
Authors: Jarvis Michael A. , Wang C. Edward , Meyers Heather L. , Smith Patricia P. , Corless Christopher L. , Henderson Gavin J. , Vieira Jeffrey , Britt William J. , Nelson Jay A.
Published: December 2019

Epithelial cells are known to be a major target for human cytomegalovirus (HCMV) infection; however, the analysis of virus-cell interactions has been difficult to approach due to the lack of in vitro models. In this study, we established a polarized epithelial cell model using a colon epithelial cell-derived cell line (Caco-2) that is susceptible to HCMV infection at early stages of cellular differentiation. Infection of polarized cells was restricted to the basolateral surface whereas virus was released apically, which was consistent with the apical and not basolateral surface localization of two essential viral glycoproteins, gB and gH. HCMV infection resulted in the development of a cytopathology characteristic of HCMV infection of colon epithelium in vivo, and infection did not spread from cell to cell. The inability of HCMV to infect Caco-2 cells at late stages of differentiation was due to a restriction at the level of viral entry and was consistent with the sequestration of a cellular receptor for HCMV. These observations provide the first evidence that restriction of HCMV replication in epithelial cells is due to a receptor-mediated phenomenon.
The Role of Human T-Lymphotropic Virus Type 1 (HTLV-1)-Infected Dendritic Cells in the Development of HTLV-1-Associated Myelopathy/Tropical Spastic Paraparesis
Authors: Makino Masahiko , Shimokubo Satoshi , Wakamatsu Shin-Ichi , Izumo Shuji , Baba Masanori
Published: December 2019

The development of human T-lymphotropic virus type 1 (HTLV-1)-associated myelopathy/tropical spastic paraparesis (HAM/TSP) is closely associated with the activation of T cells which are HTLV-1 specific but may cross-react with neural antigens (Ags). Immature dendritic cells (DCs), differentiated from normal donor monocytes by using recombinant granulocyte-macrophage colony-stimulating factor and recombinant interleukin-4, were pulsed with HTLV-1 in vitro. The pulsed DCs contained HTLV-1 proviral DNA and expressed HTLV-1 Gag Ag on their surface 6 days after infection. The DCs matured by lipopolysaccharides stimulated autologous CD4
+
T cells and CD8
+
T cells in a viral dose-dependent manner. However, the proliferation level of CD4
+
T cells was five- to sixfold higher than that of CD8
+
T cells. In contrast to virus-infected DCs, DCs pulsed with heat-inactivated virions activated only CD4
+
T cells. To clarify the role of DCs in HAM/TSP development, monocytes from patients were cultured for 4 days in the presence of the cytokines. The expression of CD86 Ag on DCs was higher and that of CD1a Ag was more down-regulated than in DCs generated from normal monocytes. DCs from two of five patients expressed HTLV-1 Gag Ag. Furthermore, both CD4
+
and CD8
+
T cells from the patients were greatly stimulated by contact with autologous DCs pulsed with inactivated viral Ag as well as HTLV-1-infected DCs. These results suggest that DCs are susceptible to HTLV-1 infection and that their cognate interaction with T cells may contribute to the development of HAM/TSP.
Genetic Interaction of Flavivirus Nonstructural Proteins NS1 and NS4A as a Determinant of Replicase Function
Authors: Lindenbach Brett D. , Rice Charles M.
Published: December 2019

Nonstructural protein 1 (NS1) of yellow fever virus (YF) is a glycoprotein localized to extracytoplasmic compartments within infected cells. We have previously shown that NS1 can be supplied in
trans
and is required for viral RNA replication, a process thought to occur in membrane-bound cytoplasmic complexes. Here we report that the NS1 gene from a related virus, dengue virus (DEN), is unable to function in the process of YF RNA replication. This virus-specific incompatibility leads to a lack of initial minus-strand accumulation, suggesting that DEN NS1 is unable to productively interact with the YF replicase. Based on a YF deletion mutant that requires NS1 in
trans
, a genetic screen for suppressor mutants was used to select virus variants able to utilize DEN NS1. In three independent selections, a single mutation was mapped to the NS4A gene, which encodes a putative transmembrane replicase component. This mutation, as well as several additional mutations, was engineered into the NS1-deficient genome and confirmed a genetic interaction between NS1 and NS4A. These findings suggest a potential mechanism for integrating NS1 into the cytoplasmic process of RNA replication.
Control of Cell Cycle Entry and Apoptosis in B Lymphocytes Infected by Epstein-Barr Virus
Authors: Spender Lindsay C. , Cannell Emma J. , Hollyoake Martine , Wensing Barbara , Gawn Jonathan M. , Brimmell Matthew , Packham Graham , Farrell Paul J.
Published: December 2019

Infection of human B cells with Epstein-Barr virus (EBV) results in activation of the cell cycle and cell growth. To interpret the mechanisms by which EBV activates the cell, we have assayed many proteins involved in control of the G0and G1phases of the cell cycle and regulation of apoptosis. In EBV infection most of the changes, including the early induction of cyclin D2, are dependent on expression of EBV genes, but an alteration in the E2F-4 profile was partly independent of viral gene expression, presumably occurring in response to signal transduction activated when the virus binds to its receptor, CD21. By comparing the expression of genes controlling apoptosis, including those encoding several members of the BCL-2 family of proteins, the known relative resistance of EBV-immortalized B-cell lines to apoptosis induced by low serum was found to correlate with expression of both BCL-2 and A20. A20 can be regulated by the NF-κB transcription factor, which is known to be activated by the EBV LMP-1 protein. Quantitative assays demonstrated a direct temporal relationship between LMP-1 protein levels and active NF-κB during the time course of infection.
Human Herpesvirus 8-Encoded Thymidine Kinase and Phosphotransferase Homologues Confer Sensitivity to Ganciclovir
Authors: Cannon Jennifer S. , Hamzeh Fayez , Moore Stacy , Nicholas John , Ambinder Richard F.
Published: December 2019

Human herpesvirus 8 (HHV-8) sensitivity to the nucleoside analog ganciclovir (GCV) suggests the presence of a virally encoded kinase that catalyzes the initial phosphorylation of GCV. Analysis of the HHV-8 genome identified two candidate kinases: proteins encoded by open reading frame (ORF) 21, with homology to the herpesvirus thymidine kinases (TK), and ORF 36, with homology to the herpesvirus phosphotransferases (PT). Experiments presented here show that both ORF 21 and ORF 36 encode GCV kinase activities as demonstrated by GCV phosphorylation and GCV-mediated cell death. In both regards the PT homologue ORF 36 was more active than the TK homologue ORF 21. ORF 21, but not ORF 36, weakly sensitized cells to killing by penciclovir. Neither ORF sensitized cells to killing by (
E
)-5-(2-bromovinyl)-2′-deoxyuridine.
Characterization of a Chinese Hamster Ovary Cell Line Developed by Retroviral Insertional Mutagenesis That Is Resistant to Sindbis Virus Infection
Authors: Jan Jia-Tsrong , Byrnes Andrew P. , Griffin Diane E.
Published: December 2019

The alphavirus Sindbis virus (SV) has a wide host range and infects many types of cultured cells in vitro. The outcome of infection is dependent on the strain of virus used for infection and the properties of the cells infected. To identify cellular determinants of susceptibility to SV infection we mutagenized Chinese hamster ovary (CHO) cells by retroviral insertion with a vector containing the neomycin resistance gene that allowed selection for integration into transcriptionally active genes. Cells were then selected for survival after infection with SV. The most resistant cell line (CHO-18.4m) exhibited delayed virus replication and virus-induced cell death, had a single retroviral insertion, and was defective in SV binding to the cell surface. Further analysis revealed that CHO-18.4m cells were deficient in the expression of the sulfated glycosaminoglycans heparan sulfate and chondroitin sulfate. This further confirms the importance of heparan sulfate as an attachment molecule for SV in vitro and demonstrates the usefulness of this technique for identifying cellular genes that are important for virus replication.
Highly Attenuated Vaccine Strains of Simian Immunodeficiency Virus Protect against Vaginal Challenge: Inverse Relationship of Degree of Protection with Level of Attenuation
Authors: Johnson R. Paul , Lifson Jeffrey D. , Czajak Susan C. , Cole Kelly Stefano , Manson Kelledy H. , Glickman Rhona , Yang Janet , Montefiori David C. , Montelaro Ronald , Wyand Michael S. , Desrosiers Ronald C.
Published: December 2019

Three different deletion mutants of simian immunodeficiency virus (SIV) that vary in their levels of attenuation were tested for the ability to protect against mucosal challenge with pathogenic SIV. Four female rhesus monkeys were vaccinated by intravenous inoculation with SIVmac239Δ3, four with SIVmac239Δ3X, and four with SIVmac239Δ4. These three vaccine strains exhibit increasing levels of attenuation: Δ3 < Δ3X <Δ4. The vaccinated monkeys were challenged by vaginal exposure to uncloned, pathogenic SIVmac251 at 61 weeks after the time of vaccination. On the basis of viral RNA loads in plasma, cell-associated virus loads in peripheral blood, and CD4 cell counts, strong protective effects were observed in all three groups of vaccinated monkeys. However, the degree of protection correlated inversely with the level of attenuation; the least-attenuated strain, SIVmac239Δ3, gave the greatest protection. One monkey in the Δ3X group and two in the Δ4 group clearly became superinfected by the challenge virus, but these animals had levels of SIV RNA in plasma that were considerably lower than those of naive animals that were challenged in parallel. Protection against vaginal challenge appears easier to achieve than protection against intravenous challenge, since four other SIVmac239Δ4-vaccinated monkeys showed no protection when challenged intravenously with a much lower inoculum of the same challenge virus stock. Protection against vaginal challenge in the Δ4-vaccinated group occurred in the absence of detectable serum neutralizing activities and appeared to be associated with the development of an early SIV-specific cytotoxic-T-lymphocyte response. Our results demonstrate that mucosal protection can be achieved by systemic immunization with the highly attenuated SIVmac239Δ4 more than 1 year prior to the time of challenge.
Adenovirus-Mediated p21
(WAF1/SDII/CIP1)
Gene Transfer Induces Apoptosis of Human Cervical Cancer Cell Lines
Authors: Tsao Yeou-Ping , Huang Shyh-Jer , Chang Junn-Liang , Hsieh Jer-Tsong , Pong Rey-Chen , Chen Show-Li
Published: December 2019

p21
(WAF1/SDII/CIP1)
(p21) arrests cell growth by inhibiting cyclin-depend kinases. To explore the potential of using p21 for the gene therapy of cervical cancer, we infected human papillomavirus (HPV)-positive cervical cancer cells (HeLa, SiHa, and Z172) and HPV-negative cervical cancer cells (C33A) with recombinant adenovirus encoding p21 cDNA. The results revealed that effective inhibition of cell growth could be achieved by sense p21 adenovirus but not antisense p21 adenovirus infection and occurred through apoptosis as measured by DNA fragmentation and chromatin condensation. Apoptosis was also observed in xenografts of human cervical cancer cells infected with sense p21 adenovirus, as confirmed by in situ terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL). The apoptosis was not prevented by overexpression of the
bcl-2
transgene. To sum up, the apoptotic effect suggests that p21 should be a tumoricidal agent instead of a tumoristatic agent in preventing cervical cancers. In addition, our report substantiates the combination of the high efficiency of adenovirus vector-mediated gene delivery and the apoptotic effect of p21.
Minimum Requirements for Efficient Transduction of Dividing and Nondividing Cells by Feline Immunodeficiency Virus Vectors
Authors: Johnston Julie C. , Gasmi Mehdi , Lim Leland E. , Elder John H. , Yee Jiing-Kuan , Jolly Douglas J. , Campbell Kevin P. , Davidson Beverly L. , Sauter Sybille L.
Published: December 2019

The development of gene delivery vectors based on feline immunodeficiency virus (FIV) is an attractive alternative to vectors based on primate sources for the delivery of genes into humans. To investigate the requirements for efficient transduction of dividing and nondividing cells by vector particles based on FIV, a series of packaging and vector constructs was generated for which viral gene expression was minimized and from which unnecessary
cis
-acting sequences were deleted. Pseudotyped vector particles produced in 293T cells were used to transduce various target cells, including contact-inhibited human skin fibroblasts and growth-arrested HT1080 cells. FIV vectors in which the U3 promoter was replaced with the cytomegalovirus promoter gave rise to over 50-fold-higher titers than FIV vectors containing the complete FIV 5′ long terminal repeat (LTR). Comparison of the transduction efficiencies of vectors containing different portions of the FIV Gag coding region indicates that at least a functional part of the FIV packaging signal (Ψ) is located within an area which includes the 5′ LTR and the first 350 bp of
gag
. Transduction efficiencies of vectors prepared without FIV
vif
and
orf2
accessory gene expression did not differ substantially from those of vectors prepared with accessory gene expression in either dividing or nondividing cells. The requirement for FIV
rev
-RRE was, however, demonstrated by the inefficient production of vector particles in the absence of
rev
expression. Together, these results demonstrate the efficient transduction of nondividing cells in vitro by a multiply attenuated FIV vector and contribute to an understanding of the minimum requirements for efficient vector production and infectivity. In addition, we describe the ability of an FIV vector to deliver genes in vivo into hamster muscle tissue.
The Carboxy-Terminal Acidic Domain of Rift Valley Fever Virus NSs Protein Is Essential for the Formation of Filamentous Structures but Not for the Nuclear Localization of the Protein
Authors: Yadani Fatima-Zahra , Kohl Alain , Préhaud Christophe , Billecocq Agnès , Bouloy Michèle
Published: December 2019

The ambisense S segment of Rift Valley fever (RVF) virus (a phlebovirus in the
Bunyaviridae
family) codes for two proteins: the viral complementary-sense RNA for the N nucleoprotein and the genomic-sense RNA for the nonstructural protein NSs. Except for the fact that the NSs protein is phosphorylated and forms filamentous structures in the nuclei of infected cells (R. Swanepoel and N. K. Blackburn, J. Gen. Virol. 34:557–561, 1977), its role is poorly understood, especially since the replication cycle of all these viruses takes place in the cytoplasm. To investigate the mechanisms involved in filament formation, we expressed NSs in mammalian cells via a recombinant Semliki Forest virus and demonstrated that the protein alone was able to form structures similar to those observed in RVF virus-infected cells, indicating that the presence of other RVF virus proteins is not required for filament formation. The yeast two-hybrid system was used to show that the protein interacts with itself and to map the interacting domains. Various deletion and substitution mutants were constructed, and the mutant proteins were analyzed by immunoprecipitation, Western blotting and immunofluorescence. These experiments indicated that the 10 to 17 amino acids of the carboxy-terminal domain were involved in self-association of the protein and that deletion of this acidic carboxy-terminal domain prevents the protein from forming filaments but does not affect its nuclear localization. The role of two phosphorylation sites present in this domain was also investigated, but they were not found to have a major influence on the formation of the nuclear filament.
Broad-Spectrum Protection against Tombusviruses Elicited by Defective Interfering RNAs in Transgenic Plants
Authors: Rubio Teresa , Borja Marisé , Scholthof Herman B. , Feldstein Paul A. , Morris T. Jack , Jackson Andrew O.
Published: December 2019

We have designed a DNA cassette to transcribe defective interfering (DI) RNAs of tomato bushy stunt virus (TBSV) and have investigated their potential to protect transgenicNicotiana benthamianaplants from tombusvirus infections. To produce RNAs with authentic 5′ and 3′ termini identical to those of the native B10 DI RNA, the DI RNA sequences were flanked by ribozymes (RzDI). When RzDI RNAs transcribed in vitro were mixed with parental TBSV transcripts and inoculated into protoplasts or plants, they became amplified, reduced the accumulation of the parental RNA, and mediated attenuation of the lethal syndrome characteristic of TBSV infections. Analysis of F1and F2RzDI transformants indicated that uninfected plants expressed the DI RNAs in low abundance, but these RNAs were amplified to very high levels during TBSV infection. By two weeks postinoculation with TBSV, all untransformedN. benthamianaplants and transformed negative controls died. Although infection of transgenic RzDI plants initially induced moderate to severe symptoms, these plants subsequently recovered, flowered, and set seed. Plants from the same transgenic lines also exhibited broad-spectrum protection against related tombusviruses but remained susceptible to a distantly related tombus-like virus and to unrelated viruses.
The Murine Gammaherpesvirus 68 v-Cyclin Gene Is an Oncogene That Promotes Cell Cycle Progression in Primary Lymphocytes
Authors: van Dyk Linda F. , Hess Jay L. , Katz Jonathan D. , Jacoby Meagan , Speck Samuel H. , Virgin Herbert W.
Published: December 2019

Several gammaherpesviruses contain open reading frames encoding proteins homologous to mammalian D-type cyclins. In this study, we analyzed the expression and function of the murine gammaherpesvirus 68 (γHV68) viral cyclin (v-cyclin). The γHV68 v-cyclin gene was expressed in lytically infected fibroblasts as a leaky-late mRNA of approximately 0.9 kb encoding a protein of approximately 25 kDa. To evaluate the effect of the γHV68 v-cyclin on cell cycle progression in primary lymphocytes and to determine if the γHV68 v-cyclin gene is an oncogene, we generated transgenic mice by using thelckproximal promoter to express the γHV68 v-cyclin in early T cells. Expression of the γHV68 v-cyclin significantly increased the number of thymocytes in cell culture, as determined by measuring both DNA content and incorporation of 5-bromo-2-deoxyuridine following in vivo pulse-labeling. Expression of the γHV68 v-cyclin interfered with normal thymocyte maturation, as shown by increased numbers of CD4+CD8+double-positive thymocytes and decreased numbers of CD4+or CD8+single-positive and T-cell-receptor-bright thymocytes and splenocytes in transgenic mice. Despite increased numbers of cycling thymocytes, γHV68–v-cyclin–transgenic mice did not have proportionately increased thymocyte numbers, and staining by terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling demonstrated increased apoptosis in the thymi of v-cyclin-transgenic mice. Fifteen of 38 γHV68–v-cyclin–transgenic mice developed high-grade lymphoblastic lymphoma between 3 and 12 months of age. We conclude that (i) the γHV68 v-cyclin is expressed as a leaky-late gene in lytically infected cells, (ii) expression of the γHV68 v-cyclin in thymocytes promotes cell cycle progression and inhibits normal T-cell differentiation, and (iii) the γHV68 v-cyclin gene is an oncogene.
Colocalization of the Herpes Simplex Virus 1 U
L
4 Protein with Infected Cell Protein 22 in Small, Dense Nuclear Structures Formed prior to Onset of DNA Synthesis
Authors: Jahedi Sogol , Markovitz Nancy S. , Filatov Felix , Roizman Bernard
Published: December 2019

Herpes simplex virus 1 infected cell protein 22 (ICP22) localizes in small, dense nuclear bodies of primate cells early in infection and in the more diffuse replicative complexes after the onset of DNA synthesis. U
L
4, a γ
2
protein, localizes in cytoplasm and in the small nuclear structures containing ICP22 but not in replicative complexes. In rabbit skin cells, both ICP22 and U
L
4 localize in the dense nuclear bodies late in infection. The results suggest that the small nuclear structures perform a function involving both proteins late in infection.
Alfalfa Mosaic Virus RNAs Serve as Cap Donors for Tomato Spotted Wilt Virus Transcription during Coinfection of
Nicotiana benthamiana
Authors: Duijsings Daniël , Kormelink Richard , Goldbach Rob
Published: December 2019

Tomato spotted wilt virus (TSWV) was shown to use alfalfa mosaic virus (AMV) RNAs as cap donors in vivo during a mixed infection in
Nicotiana benthamiana
. By use of nested reverse transcription-PCR, TSWV N and NSs mRNAs provided with capped leader sequences derived from all four AMV RNAs could be cloned and sequenced. The sequence specificity of the putative TSWV endonuclease involved is discussed.
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

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

The 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

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

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

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

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

Human 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

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

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

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

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.