bluuebunny/crossref_metadata_2025 huggingface dataset:
Genome-Wide Screening, Cloning, Chromosomal Assignment, and Expression of Full-Length Human Endogenous Retrovirus Type K
Authors: Tönjes Ralf R. , Czauderna Frank , Kurth Reinhard
Published: December 2019

The human genome harbors 25 to 50 proviral copies of the endogenous retrovirus type K (HERV-K), some of which code for the characteristic retroviral proteins Gag, Pol, and Env. For a genome-wide cloning approach of full-length and intact HERV-K proviruses, a human P1 gene library was screened with a
gag
-specific probe. Both HERV-K type 1 and 2 clones were isolated. Sixteen HERV-K type 2 proviral genomes were characterized by direct coupled in vitro transcription-in vitro translation assays to analyze the coding potential of isolated
gag
,
pol
, and
env
amplicons from individual P1 clones. After determination of long terminal repeat (LTR) sequences and adjacent chromosomal integration sites by inverse PCR techniques, two HERV-K type 2 proviruses displaying long retroviral open reading frames (ORFs) were assigned to chromosomes 7 (C7) and 19 (C19) by using a human-rodent monochromosomal cell hybrid mapping panel. HERV-K(C7) shows an altered (YIDD-to-CIDD) motif in the reverse transcriptase domain. HERV-K(C19) is truncated in the 5′ LTR and harbors a defective protease gene due to a point mutation. Direct amplification of proviral structures from single chromosomes by using chromosomal flanking primers was performed by long PCR for HERV-K(C7) and HERV-K(C19) and for type 1 proviruses HERV-K10 and HERV-K18 from chromosomes 5 and 1, respectively. HERV-K18, in contrast to HERV-K10, bears no intact
gag
ORF and shows close homology to HERV-K/IDDMK
1,2
22. In transfection experiments, HERV-K(C7) and HERV-K cDNA-based expression vectors yielded the proteins Gag and cORF whereas HERV-K10 vectors yielded Gag alone. The data suggest that the human genome does not contain an entire, intact proviral copy of HERV-K.
Assignment of the Multifunctional NS3 Protein of Bovine Viral Diarrhea Virus during RNA Replication: an In Vivo and In Vitro Study
Authors: Grassmann Claus W. , Isken Olaf , Behrens Sven-Erik
Published: December 2019

Studies on the replication of the pestivirus bovine viral diarrhea virus (BVDV) were considerably facilitated by the recent discovery of an autonomous subgenomic BVDV RNA replicon (DI9c). DI9c comprises mainly the untranslated regions of the viral genome and the coding region of the nonstructural proteins NS3, NS4A, NS4B, NS5A, and NS5B. To assess the significance of the NS3-associated nucleoside triphosphatase/helicase activity during RNA replication and to explore other functional features of NS3, we generated a repertoire of DI9c derivatives bearing in-frame mutations in different parts of the NS3 coding unit. Most alterations resulted in deficient replicons, several of which encoded an NS3 protein with an inhibited protease function. Three lesions permitted replication, though at a lower level than that of the wild-type RNA, i.e., replacement of the third position of the DEYH helicase motif II by either T or F and an insertion of four amino acid residues in the C-terminal part of NS3. While polyprotein proteolysis was found to be almost unaffected in these latter replicons, in vitro studies with the purified mutant NS3 proteins revealed a significantly impaired helicase activity for the motif II substitutions. NS3 with a DEFH motif, moreover, showed a significantly lower ATPase activity. In contrast, the C-terminal insertion had no negative impact on the ATPase/RNA helicase activity of NS3. All three mutations affected the synthesis of both replication products—negative-strand intermediate and progeny positive-strand RNA—in a symmetric manner. Unexpectedly, various attempts to rescue or enhance the replication capability of nonfunctional or less functional DI9c NS3 derivatives, respectively, by providing intact NS3 intransfailed. Our experimental data thus demonstrate that the diverse enzymatic activities of the NS3 protein—in particular the ATPase/RNA helicase—play a pivotal role even during early steps of the viral replication pathway. They may further indicate the C-terminal part of NS3 to be an important functional determinant of the RNA replication process.
Genetic Stability of Foamy Viruses: Long-Term Study in an African Green Monkey Population
Authors: Schweizer Matthias , Schleer Hubertus , Pietrek Michael , Liegibel Jürgen , Falcone Valeria , Neumann-Haefelin Dieter
Published: December 2019

The genetic variability of the envelope surface domain (SU) of simian foamy virus (FV) of African green monkeys was studied. To assess the interindividual diversity of FV, isolates were obtained from 19 animals living together in a monkey house. The monkeys had been imported from Kenya prior to being placed in long-term housing in the research institute. In addition, a simian FV isolate and proviral DNA were obtained from an animal caretaker infected in this setting. DNA of the complete SU (1779 to 1793 bp) was analyzed by PCR and sequencing. The sequences revealed four clusters with high homologies (>95%). Between the clusters, divergencies ranged from 3 to 25%. Obviously, the clusters reflect four different strains or subtypes of simian FV type 3 that were prevalent in the colony. In contrast to lentiviruses, hypervariable regions could not be detected in the FV SU. Furthermore, to analyze the intraindividual diversity of FV, we investigated the virus population within an individual monkey at a given time point and its evolution over 13 years. For this purpose, 22 proviral SU clones generated by PCR from one oral swab and seven isolates obtained from the same animal between 1982 and 1995 were examined. These sequences revealed exceptionally high homology rates (99.5 to 100%), and only a minimal genetic drift was recognized within the series of isolates. In conclusion, the low in vivo divergency of FV SU suggests that genetic variability is not important for the maintenance of FV persistence.
Binding of Polyomavirus Small T Antigen to Protein Phosphatase 2A Is Required for Elimination of p27 and Support of S-Phase Induction in Concert with Large T Antigen
Authors: Schüchner Stefan , Wintersberger Erhard
Published: December 2019

Although polyomavirus large T antigen readily transactivates S-phase-specific enzymes in serum-starved Swiss 3T3 mouse fibroblasts, it is incapable by itself to efficiently drive such cells into S phase. We describe here that this inability correlates with a weak proficiency of the viral protein to induce the synthesis of cyclin A and cyclin E and to stimulate the respective cyclin/cdk activities. Polyomavirus small T antigen, which together with the large T protein supports S-phase induction, strongly contributes to the synthesis of cyclin A. In addition, small T antigen causes a dramatic induction of cyclin A- and, together with large T antigen, of cyclin E-specific protein kinase activity. This latter function of polyomavirus small T antigen correlates with its competence to provoke the elimination of the kinase inhibitor p27
Kip1
. An interaction of the small T antigen with the protein phosphatase 2A is essential for this activity. Hence, the ability to drive quiescent Swiss 3T3 cells into S phase results from the capacity of large T antigen to transactivate DNA synthesis enzymes by its interaction with retinoblastoma-type proteins and from the potential of the large and the small T antigens together to stimulate cyclin A synthesis and cyclin A- and cyclin E-dependent protein kinase activity.
Evolution of Envelope Sequences of Human Immunodeficiency Virus Type 1 in Cellular Reservoirs in the Setting of Potent Antiviral Therapy
Authors: Günthard Huldrych F. , Frost Simon D. W. , Leigh-Brown Andrew J. , Ignacio Caroline C. , Kee Kristin , Perelson Alan S. , Spina Celsa A. , Havlir Diane V. , Hezareh Marjan , Looney David J. , Richman Douglas D. , Wong Joseph K.
Published: December 2019

In human immunodeficiency virus (HIV)-infected patients treated with potent antiretroviral therapy, the persistence of latently infected cells may reflect the long decay half-life of this cellular reservoir or ongoing viral replication at low levels with continuous replenishment of the population or both. To address these possibilities, sequences encompassing the C2 and V3 domains of HIV-1
env
were analyzed from virus present in baseline plasma and from viral isolates obtained after 2 years of suppressive therapy in six patients. The presence of sequence changes consistent with evolution was demonstrated for three subjects and correlated with less complete suppression of viral replication, as indicated by the rapidity of the initial virus load decline or the intermittent reappearance of even low levels of detectable viremia. Together, these results provide evidence for ongoing replication. In the remaining three patients, virus recovered after 2 years of therapy was either genotypically contemporary with or ancestral to virus present in plasma 2 years before, indicating that virus recovery had indeed resulted from activation of latently infected cells.
cORF and RcRE, the Rev/Rex and RRE/RxRE Homologues of the Human Endogenous Retrovirus Family HTDV/HERV-K
Authors: Magin Christine , Löwer Roswitha , Löwer Johannes
Published: December 2019

cORF, a protein encoded by the human endogenous retrovirus family HTDV/HERV-K, contains amino acid motifs which resemble the nuclear import and export signals of the viral regulatory proteins Rev (human immunodeficiency virus) and Rex (human T-cell leukemia virus [HTLV]). In this study, we demonstrated that cORF indeed has a Rev-like function and mapped the cORF-responsive RNA element to a sequence in the 3′ long terminal repeat, a localization similar to RxRE, the responsive element in HTLV type 1. Accordingly, we have given the element the designation RcRE. cORF and RcRE stabilize unspliced and incompletely spliced viral transcripts and enhance their nuclear export via the CRM1 export pathway. So far, HTDV/HERV-K is the only endogenous retrovirus family with a complex regulation at the posttranscriptional level. It may be regarded as an intermediate in the evolution from simple to complex retroviruses.
Mutant Cells Selected during Persistent Reovirus Infection Do Not Express Mature Cathepsin L and Do Not Support Reovirus Disassembly
Authors: Baer Geoffrey S. , Ebert Daniel H. , Chung Chia J. , Erickson Ann H. , Dermody Terence S.
Published: December 2019

Persistent reovirus infections of murine L929 cells select cellular mutations that inhibit viral disassembly within the endocytic pathway. Mutant cells support reovirus growth when infection is initiated with infectious subvirion particles (ISVPs), which are intermediates in reovirus disassembly formed following proteolysis of viral outer-capsid proteins. However, mutant cells do not support growth of virions, indicating that these cells have a defect in virion-to-ISVP processing. To better understand mechanisms by which viruses use the endocytic pathway to enter cells, we defined steps in reovirus replication blocked in mutant cells selected during persistent infection. Subcellular localization of reovirus after adsorption to parental and mutant cells was assessed using confocal microscopy and virions conjugated to a fluorescent probe. Parental and mutant cells did not differ in the capacity to internalize virions or distribute them to perinuclear compartments. Using pH-sensitive probes, the intravesicular pH was determined and found to be equivalent in parental and mutant cells. In both cell types, virions localized to acidified intracellular organelles. The capacity of parental and mutant cells to support proteolysis of reovirus virions was assessed by monitoring the appearance of disassembly intermediates following adsorption of radiolabeled viral particles. Within 2 h after adsorption to parental cells, proteolysis of viral outer-capsid proteins was observed, consistent with formation of ISVPs. However, in mutant cells, no proteolysis of viral proteins was detected up to 8 h postadsorption. Since treatment of cells with E64, an inhibitor of cysteine-containing proteases, blocks reovirus disassembly, we used immunoblot analysis to assess the expression of cathepsin L, a lysosomal cysteine protease. In contrast to parental cells, mutant cells did not express the mature, proteolytically active form of the enzyme. The defect in cathepsin L maturation was not associated with mutations in procathepsin L mRNA, was not complemented by procathepsin L overexpression, and did not affect the maturation of cathepsin B, another lysosomal cysteine protease. These findings indicate that persistent reovirus infections select cellular mutations that affect the maturation of cathepsin L and suggest that alterations in the expression of lysosomal proteases can modulate viral cytopathicity.
Proliferation Response to Interleukin-2 and Jak/Stat Activation of T Cells Immortalized by Human T-Cell Lymphotropic Virus Type 1 Is Independent of Open Reading Frame I Expression
Authors: Collins Nathaniel D. , D’Souza Celine , Albrecht Björn , Robek Michael D. , Ratner Lee , Ding Wei , Green Patrick L. , Lairmore Michael D.
Published: December 2019

Human T-cell lymphotropic virus type 1 (HTLV-1), a complex retrovirus, encodes a hydrophobic 12-kD protein from pX open reading frame (ORF) I that localizes to cellular endomembranes and contains four minimal SH3 binding motifs (PXXP). We have demonstrated the importance of ORF I expression in the establishment of infection and hypothesize that p12Ihas a role in T-cell activation. In this study, we tested interleukin-2 (IL-2) receptor expression, IL-2-mediated proliferation, and Jak/Stat activation in T-cell lines immortalized with either wild-type or ORF I mutant clones of HTLV-1. All cell lines exhibited typical patterns of T-cell markers and maintained mutation fidelity. No significant differences between cell lines were observed in IL-2 receptor chain (α, β, or γc) expression, in IL-2-mediated proliferation, or in IL-2-induced phosphorylated forms of Stat3, Stat5, Jak1, or Jak3. The expression of ORF I is more likely to play a role in early HTLV-1 infection, such as in the activation of quiescent T cells in vivo.
Multiple Effects of an Anti-Human Immunodeficiency Virus Nucleocapsid Inhibitor on Virus Morphology and Replication
Authors: Berthoux Lionel , Péchoux Christine , Darlix Jean-Luc
Published: December 2019

Human immunodeficiency virus type 1 nucleocapsid protein is a major structural component of the virion core and a key factor involved in proviral DNA synthesis and virus formation. 2,2′-Dithiobenzamides (DIBA-1) and related compounds that are inhibitors of NCp7 are thought to eject zinc ions from NCp7 zinc fingers, inhibiting the maturation of virion proteins. Here, we show that the presence of DIBA-1 at the time of virus formation causes morphological malformations of the virus and reduces proviral DNA synthesis. Thus, it seems that DIBA-1 is responsible for a “core-freezing effect,” as shown by electron microscopy analyses. DIBA-1 can also directly interfere with the fate of the newly made proviral DNA in a manner independent of its effects on virion core formation. These data strongly suggest that nucleocapsid protein is a prime target for new compounds aimed at inhibiting human immunodeficiency virus and other retroviruses.
Molecular Mechanisms of Coxsackievirus Persistence in Chronic Inflammatory Myopathy: Viral RNA Persists through Formation of a Double-Stranded Complex without Associated Genomic Mutations or Evolution
Authors: Tam Patricia E. , Messner Ronald P.
Published: December 2019

Enterovirus infection and persistence have been implicated in the pathogenesis of certain chronic muscle diseases. In vitro studies suggest that persistent enteroviruses mutate, evolving into forms that are less lytic and display altered tropism, but it is less clear whether these mechanisms operate in vivo. In this study, persistent coxsackievirus RNA from the muscle of mice afflicted with chronic inflammatory myopathy (CIM) was characterized and compared with RNA from a virus that had established a persistent infection of G8 mouse myoblasts for 30 passages in vitro. Competitive strand-specific reverse transcription-PCR and susceptibility to RNase I treatment revealed that plus- and minus-strand viral RNAs were present at nearly equivalent levels in muscle and that they persisted in a double-stranded conformation. All regions of the viral genome persisted and were amplified as a series of seven overlapping fragments. Restriction endonuclease fingerprinting coupled with sequencing indicated that there was no evolution of the viral genome associated with its persistence in muscle. This contrasted with the productive persistent infection that was established in myoblast cultures, where plus-strand RNA predominated and persistent virus developed distinct mutations. In vitro persistence proceeded by a carrier culture mechanism and was completely dependent on production of infectious virus, since persistent viral RNA was not detected in cultures subjected to antibody-mediated curing. These experiments demonstrate that persistence of coxsackievirus RNA in muscle is not facilitated by distinct genetic changes in the virus that give rise to replication-defective forms but occurs primarily through production of stable double-stranded RNA that is produced as the acute viral infection resolves. The data suggest a mechanism for coxsackievirus persistence in myofibers and perhaps other nondividing cells whereby cells that survive infection could harbor persistent viral RNA for extended times without producing detectable levels of infectious virus.
Caspase-Dependent N-Terminal Cleavage of Influenza Virus Nucleocapsid Protein in Infected Cells
Authors: Zhirnov O. P. , Konakova T. E. , Garten W. , Klenk H.-D.
Published: December 2019

The nucleocapsid protein (NP) (56 kDa) of human influenza A viruses is cleaved in infected cells into a 53-kDa form. Likewise, influenza B virus NP (64 kDa) is cleaved into a 55-kDa protein with a 62-kDa intermediate (O. P. Zhirnov and A. G. Bukrinskaya, Virology 109:174–179, 1981). We show now that an antibody specific for the N terminus of influenza A virus NP reacted with the uncleaved 56-kDa form but not with the truncated NP53 form, indicating the removal of a 3-kDa peptide from the N terminus. Amino acid sequencing revealed the cleavage sites ETD16*G for A/Aichi/68 NP and sites DID7*G and EAD61*V for B/Hong Kong/72 NP. With D at position −1, acidic amino acids at position −3, and aliphatic ones at positions −2 and +1, the NP cleavage sites show a recognition motif typical for caspases, key enzymes of apoptosis. These caspase cleavage sites demonstrated evolutionary stability and were retained in NPs of all human influenza A and B viruses. NP of avian influenza viruses, which is not cleaved in infected cells, contains G instead of D at position 16. Oligopeptide DEVD derivatives, specific caspase inhibitors, were shown to prevent the intracellular cleavage of NP. All three events, the NP cleavage, the increase of caspase activity, and the development of apoptosis, coincide in cells infected with human influenza A and B viruses. The data suggest that intracellular cleavage of NP is exerted by host caspases and is associated with the development of apoptosis at the late stages of infection.
Induction of Endogenous Genes following Infection of Human Endothelial Cells with an E1−E4+Adenovirus Gene Transfer Vector
Authors: Ramalingam Ramachandran , Rafii Shahin , Worgall Stefan , Hackett Neil R. , Crystal Ronald G.
Published: December 2019

Recombinant adenovirus (Ad) gene transfer vectors are effective at transferring exogenous genes to a variety of cells and tissue types both in vitro and in vivo. However, in the process of gene transfer, the Ad vectors induce the expression of target cell genes, some of which may modify the function of the target cell and/or alter the local milieu. To develop a broader understanding of Ad vector-mediated induction of endogenous gene expression, genes induced by first-generation E1−E4+Ad vectors in primary human umbilical vein endothelial cells were identified by cDNA subtraction cloning. The identified cDNAs included signaling molecules (lymphoid blast crisis [LBC], guanine nucleotide binding protein α type S [Gα-S], and mitogen kinase [MEK5]), calcium-regulated/cytoskeletal proteins (calpactin p11 and p36 subunits, vinculin, and spinocerebellar ataxia [SCA1]), growth factors (insulin-like growth factor binding protein 4 and transforming growth factor β2), glyceraldehyde-6-phosphate dehydrogenase, an expressed sequence tag, and a novel cDNA showing homology to a LIM domain sequence. Two- to sevenfold induction of the endogenous gene expression was observed at 24 h postinfection, and induction continued up to 72 h, although the timing of gene expression varied among the identified genes. In contrast to that observed in endothelial cells, the Ad vector-mediated induction of gene expression was not found following Ad vector infection of primary human dermal fibroblasts or human alveolar macrophages. Empty Ad capsids did not induce endogenous gene expression in endothelial cells. Interestingly, additional deletion of the E4 gene obviated the upregulation of genes in endothelial cells by the E1−E3−Ad vector, suggesting that genes carried by the E4 region play a central role in modifying target cell gene expression. These findings are consistent with the notion that efficient transfer of exogenous genes to endothelial cells by first-generation Ad vectors comes with the price that these vectors also induce the expression of a variety of cellular genes.
Altering the Cellular Location of an Antigen Expressed by a DNA-Based Vaccine Modulates the Immune Response
Authors: Lewis P. J. , van Drunen Littel-van den Hurk S. , Babiuk L. A.
Published: December 2019

The potential for DNA vaccines encoding mutated versions of the same antigen to modulate immune responses in C3H/HeN mice was investigated. We created expression plasmids that encoded several versions of glycoprotein D (gD) from bovine herpesvirus 1, including authentic membrane-anchored glycoprotein (pSLRSV.AgD), a secreted glycoprotein (pSLRSV.SgD), and an intracellular protein (pSLRSV.CgD). Immunization of an inbred strain of mice with these plasmids resulted in highly efficacious and long-lasting humoral and cell-mediated immunity. We also demonstrated that the cell compartment in which plasmid-encoded gD was expressed caused a deviation in the serum immunoglobulin (Ig) isotype profile as well as the predominant cytokines secreted from the draining lymph node. Immunization of C3H/HeN mice with DNA vaccines encoding cell-associated forms of gD resulted in a predominance of serum IgG2a and gamma interferon-secreting cells within the spleens and draining lymph nodes. In contrast, mice immunized with a secreted form of this same antigen displayed immune responses characterized by greater levels of interleukin 4 in the draining lymph node and IgG1 as the predominant serum isotype. We also showed evidence of compartmentalization of distinct immune responses within different lymphoid organs.
Dependence of Echovirus 9 on the Enterovirus RNA Replication Inhibitor 2-(α-Hydroxybenzyl)-Benzimidazole Maps to Nonstructural Protein 2C
Authors: Hadaschik Dirk , Klein Marcus , Zimmermann Holger , Eggers Hans J. , Nelsen-Salz Birgit
Published: December 2019

HBB [2-(α-hydroxybenzyl)-benzimidazole] selectively inhibits RNA synthesis of most enteroviruses. However, isolation of HBB-dependent variants is possible. Sequence analysis and characterization of recombinant viruses revealed that HBB dependence maps to the nonstructural protein 2C. A single point mutation at position C
4782
U is sufficient to establish the HBB-dependent phenotype in our echovirus 9 model.
Persistent CCR5 Utilization and Enhanced Macrophage Tropism by Primary Blood Human Immunodeficiency Virus Type 1 Isolates from Advanced Stages of Disease and Comparison to Tissue-Derived Isolates
Authors: Li Shan , Juarez Julius , Alali Mohammed , Dwyer Dominic , Collman Ronald , Cunningham Anthony , Naif Hassan M.
Published: December 2019

Viral phenotype, tropism, coreceptor usage, and envelope gene diversity were examined in blood isolates collected from 27 individuals at different stages of human immunodeficiency virus type 1 (HIV-1) disease and tissue derived isolates from 10 individuals with AIDS. The majority (89%) of blood and all tissue HIV-1 isolates from all stages of infection were non-syncytium inducing and macrophage (M) tropic. Tropism and productive infection by HIV isolates in both monocytes and monocyte-derived macrophages (MDM) increased in advanced disease (HIV tropism for monocytes, 1 of 6 from categories I and II versus 11 of 21 [P= 0.05] from category IV and II [CD4 < 250]; and high-level replication in MDM, 1 of 6 from categories I and II versus 16 of 21 from categories IV and II [P= 0.015]). There was a high level of replication of blood and tissue isolates in T lymphocytes without restriction at any stage. Overall, the level of replication in MDM was 5- to 10-fold greater than in monocytes, with restriction in the latter occurring mainly at entry and later stages of replication. Only three blood isolates were identified as syncytium inducing, and all had a dualtropic phenotype. There was a significant increase of HIV envelope gene diversity, as shown by a heteroduplex mobility assay, in advanced disease; this may partly underlie the increase of HIV replication in MDM. Unlike blood isolates (even those from patients with advanced disease), tissue isolates displayed greater similarities (90%) in productive infection between MDM and monocytes. The majority (87%) of all isolates, including those from patients with advanced disease, used CCR5, and only 5 of 37 isolates showed expanded coreceptor usage. These results indicate that in the late stage of disease with increasing viral load and diversity, CCR5 utilization and M-tropism persist in blood and tissue and the replicative ability in macrophages increases. This suggests that these characteristics are advantageous to HIV and are important to disease progression.
Similar Interactions of the Poliovirus and Rhinovirus 3D Polymerases with the 3′ Untranslated Region of Rhinovirus 14
Authors: Meredith Janet M. , Rohll Jonathan B. , Almond Jeffrey W. , Evans David J.
Published: December 2019

We showed previously that a human rhinovirus 14 (HRV14) 3′ untranslated region (3′ UTR) on a poliovirus genome was able to replicate with nearly wild-type kinetics (J. B. Rohll, D. H. Moon, D. J. Evans, and J. W. Almond, J. Virol 69:7835–7844, 1995). This enabled the HRV14 single 3′ UTR stem-loop structure to be studied in combination with a sensitive reporter system, poliovirus FLC/REP, in which the capsid coding region is replaced by an in-frame chloramphemicol acetyltransferase (CAT) gene. Using such a construct, we identified a mutant (designated mut4), in which the structure and stability of the stem were predicted to be maintained, that replicated very poorly as determined by its level of CAT activity. The effect of this mutant 3′ UTR on replication has been further investigated by transferring it onto the full-length cDNAs of both poliovirus type 3 (PV3) and HRV14. Virus was recovered with a parental plaque phenotype at a low frequency, indicating the acquisition of compensating changes, which sequence analysis revealed were, in both poliovirus- and rhinovirus-derived viruses, located in the active-site cleft of 3D polymerase and involved the substitution of Asn18 for Tyr. These results provide further evidence of a specific interaction between the 3′ UTR of picornaviruses and the viral polymerase and also indicate similar interactions of the 3′ UTR of rhinovirus with both poliovirus and rhinovirus polymerases.
Germ Cell Expression of an Isolated Human Endogenous Retroviral Long Terminal Repeat of the HERV-K/HTDV Family in Transgenic Mice
Authors: Casau Armelle E. , Vaughan Joe E. , Lozano Guillermina , Levine Arnold J.
Published: December 2019

In contrast to most other human endogenous retroviral families, various HERV-K members have open reading frames that code for functional viral proteins which can form noninfectious particles in some germ cell tumors. The HERV-K viral genes are highly transcribed in germ cell tumors but are transcribed to lower or undetectable levels in most other tissue and tumor types. To further analyze the expression patterns of these proviruses, long terminal repeats (LTRs) were isolated from the human genome and used in reporter gene assays. Expression of some HERV-K LTRs was found to be high in human and murine germ cell tumors (testicular teratocarcinomas) and low in non-germ-cell tumors. Furthermore, upon differentiation of a teratocarcinoma cell line, the expression of an active LTR dropped dramatically, suggesting developmental regulation of these proviral LTRs. Transgenic mice harboring an active LTR driving
lacZ
expression were generated and analyzed. Adult mouse testes showed the highest levels of expression, and the transgene staining appeared to be restricted primarily to the more undifferentiated spermatocytes. Most other tissues analyzed revealed very low or undetectable levels of expression both by reverse transcription-PCR and by Northern blot analysis. Whether the restricted expression of HERV-K in germ cells and in germ cell-derived tumors is of significant importance during development or tumorigenesis remains to be elucidated. Germ line expression of these viruses would allow for their expansion and movement, while somatic repression would ensure limited insertional mutagenesis and misexpression in an individual.
A Replication-Incompetent Adenovirus Vector with the Preterminal Protein Gene Deleted Efficiently Transduces Mouse Ears
Authors: Moorhead John W. , Clayton Gerald H. , Smith Roderic L. , Schaack Jerome
Published: December 2019

Adenoviruses offer great potential as gene therapy agents but are limited by the strong inflammatory response that occurs in response to the recombinant virus. Since the degree of inflammation correlates in part with the potential of the viral vector for replication, we constructed a preterminal protein (pTP) deletion mutant adenovirus type 5 vector, Ad5
dl
308
ΔpTP
β-gal, that is replication incompetent due to deletion of the pTP gene and that has the E1 genes replaced by the
Escherichia coli lacZ
reporter gene under the control of the cytomegalovirus major immediate-early promoter. This virus was compared with a first-generation, replication-defective adenovirus vector, Ad5
dl
308β-gal, that is isogenic except that it contains a wild-type pTP gene. To examine transduction efficiency and induction of inflammation, we developed a novel system involving intradermal injection of BALB/c mouse ears. Mouse ears can be accurately measured to determine the degree of edema as an indirect measurement of inflammation. Edema and inflammation were induced in a dose- and time-dependent manner by both viruses and correlated well. LacZ activity correlated inversely with edema and inflammation. The pTP-defective vector Ad5
dl
308
ΔpTP
β-gal transduced mouse ears much more efficiently and induced edema and inflammatory cell infiltration approximately 10-fold less efficiently than the first-generation vector Ad5
dl
308β-gal. The diminished inflammatory response and increased efficiency of transduction observed with Ad5
dl
308
ΔpTP
β-gal indicate its promise as a gene therapy agent for other tissues. The results also demonstrate that the mouse ear model offers potential for the study of adenovirus-induced inflammation because of the ready access of the ears, the relative ease of continuous measurement, and the sensitivity to adenovirus transducing vectors.
Conserved Cysteines of the Human Immunodeficiency Virus Type 1 Protease Are Involved in Regulation of Polyprotein Processing and Viral Maturation of Immature Virions
Authors: Davis David A. , Yusa Keisuke , Gillim Laura A. , Newcomb Fonda M. , Mitsuya Hiroaki , Yarchoan Robert
Published: December 2019

We investigated the role of the two highly conserved cysteine residues, cysteines 67 and 95, of the human immunodeficiency virus type 1 (HIV-1) protease in regulating the activity of that protease during viral maturation. To this end, we generated four HIV-1 molecular clones: the wild type, containing both cysteine residues; a protease mutant in which the cysteine at position 67 was replaced by an alanine (C67A); a C95A protease mutant; and a double mutant (C67A C95A). When immature virions were produced in the presence of an HIV-1 protease inhibitor, KNI-272, and the inhibitor was later removed, limited polyprotein processing was observed for wild-type virion preparations over a 20-h period. Treatment of immature wild-type virions with the reducing agent dithiothreitol considerably improved the rate and extent of Gag processing, suggesting that the protease is, in part, reversibly inactivated by oxidation of the cysteine residues. In support of this, C67A C95A virions processed Gag up to fivefold faster than wild-type virions in the absence of a reducing agent. Furthermore, oxidizing agents, such as H
2
O
2
and diamide, inhibited Gag processing of wild-type virions, and this effect was dependent on the presence of cysteine 95. Electron microscopy revealed that a greater percentage of double-mutant virions than wild-type virions developed a mature-like morphology on removal of the inhibitor. These studies provide evidence that under normal culture conditions the cysteines of the HIV-1 protease are susceptible to oxidation during viral maturation, thus preventing immature virions from undergoing complete processing following their release. This is consistent with the cysteines being involved in the regulation of viral maturation in cells under oxidative stress.
Mutational Analysis of Influenza A Virus Nucleoprotein: Identification of Mutations That Affect RNA Replication
Authors: Mena Ignacio , Jambrina Enrique , Albo Carmen , Perales Beatriz , Ortín Juan , Arrese Marta , Vallejo Dolores , Portela Agustín
Published: December 2019

The influenza A virus nucleoprotein (NP) is a multifunctional polypeptide which plays a pivotal role in virus replication. To get information on the domains and specific residues involved in the different NP activities, we describe here the preparation and characterization of 20 influenza A virus mutant NPs. The mutations, mostly single-amino-acid substitutions, were introduced in a cDNA copy of the A/Victoria/3/75 NP gene and, in most cases, affected residues located in regions that were highly conserved across the NPs of influenza A, B, and C viruses. The mutant NPs were characterized (i) in vivo (cell culture) by analyzing their intracellular localization and their functionality in replication, transcription, and expression of model RNA templates; and (ii) in vitro by analyzing their RNA-binding and sedimentation properties. The results obtained allowed us to identify both a mutant protein that accumulated in the cytoplasm and mutations that altered the functionality and/or the oligomerization state of the NP polypeptide. Among the mutations that reduced the NP capability to express chloramphenicol acetyltransferase protein from a model viral RNA (vRNA) template, some displayed a temperature-sensitive phenotype. Interestingly, four mutant NPs, which showed a reduced functionality in synthesizing cRNA molecules from a vRNA template, were fully competent to reconstitute complementary ribonucleoproteins (cRNPs) capable of synthesizing vRNAs, which in turn yielded mRNA molecules. Based on the phenotype of these mutants and on previously published observations, it is proposed that these mutant NPs have a reduced capability to interact with the polymerase complex and that this NP-polymerase interaction is responsible for making vRNPs switch from mRNA to cRNA synthesis.
Mechanism of Suppression of Hepatitis B Virus Precore RNA Transcription by a Frequent Double Mutation
Authors: Li Jie , Buckwold Victor E. , Hon Man-wai , Ou Jing-hsiung
Published: December 2019

A double mutation which converts nucleotide 1765 from A to T and nucleotide 1767 from G to A is frequently found in the hepatitis B virus (HBV) genome isolated from HBV patients with chronic hepatitis symptoms. This double mutation is located in the core promoter that controls the transcription of the precore RNA and the core RNA. In addition, this double mutation also resides in the X protein coding sequence, converting codon 130 from Lys to Met and codon 131 from Val to Ile. Previous studies indicate that this double mutation removes a nuclear receptor binding site in the core promoter, suppresses specifically precore RNA transcription, and enhances viral replication. In this study, we further investigated how this double mutation suppresses precore RNA transcription. We found that this double mutation not only removed the nuclear receptor binding site but also created an HNF1 transcription factor binding site. Further transfection studies using Huh7 hepatoma cells indicate that the removal of the nuclear receptor binding site has no effect on the transcription of HBV RNAs, the two-codon change in the X protein sequence suppresses the transcription of both precore and core RNAs, and the creation of the HNF1 binding site restores the core RNA level. Hence, the specific suppression of precore RNA transcription by this frequent double-nucleotide mutation is the combined result of multiple factors.
Human T-Cell Leukemia Virus Type 1 Tax Protein Abrogates Interleukin-2 Dependence in a Mouse T-Cell Line
Authors: Iwanaga Youichi , Tsukahara Tomonori , Ohashi Takashi , Tanaka Yuetsu , Arai Masaaki , Nakamura Masataka , Ohtani Kiyoshi , Koya Yoshihiro , Kannagi Mari , Yamamoto Naoki , Fujii Masahiro
Published: December 2019

Human T-cell leukemia virus type 1 (HTLV-1) is the etiologic agent of adult T-cell leukemia. Tax, the viral protein, is thought to be crucial in the development of the disease, since it transforms healthy T cells in vitro and induces tumors in transgenic animals. We examined the effect of Tax activity on the growth of the interleukin-2 (IL-2)-dependent T-cell line CTLL-2. Stable expression of Tax in CTLL-2 transformed cell growth from being IL-2 dependent to IL-2 independent. Tax stimulated transcription through NF-κB and the cyclic AMP-responsive element-like sequence in the HTLV-1 promoter. The finding of Tax mutants segregating these two pathways suggested that the NF-κB pathway was essential for IL-2-independent growth of CTLL-2 cells while the CRE pathway was unnecessary. However, both pathways were necessary for another transformation-related activity (colony formation in soft agar) of CTLL-2/Tax. Our results show that Tax has at least two distinct activities on T cells, and suggest that Tax plays a crucial role in IL-2-independent T-cell transformation induced by HTLV-1, in addition to its well-known IL-2-dependent cell transformation.
Identification of Mutations Contributing to the Temperature-Sensitive, Cold-Adapted, and Attenuation Phenotypes of the Live-Attenuated Cold-Passage 45 (
cp
45) Human Parainfluenza Virus 3 Candidate Vaccine
Authors: Skiadopoulos Mario H. , Surman Sonja , Tatem Joanne M. , Paschalis Maribel , Wu Shin-Lu , Udem Stephen A. , Durbin Anna P. , Collins Peter L. , Murphy Brian R.
Published: December 2019

The live-attenuated human parainfluenza virus 3 (PIV3) cold-passage 45 (
cp
45) candidate vaccine was shown previously to be safe, immunogenic, and phenotypically stable in seronegative human infants. Previous findings indicated that each of the three amino acid substitutions in the L polymerase protein of
cp
45 independently confers the temperature-sensitive (
ts
) and attenuation (
att
) phenotypes but not the cold-adaptation (
ca
) phenotype (29).
cp
45 contains 12 additional potentially important point mutations in other proteins (N, C, M, F, and hemagglutinin-neuraminidase [HN]) or in
cis
-acting sequences (the leader region and the transcription gene start [GS] signal of the N gene), and their contribution to these phenotypes was undefined. To further characterize the genetic basis for the
ts
,
ca
, and
att
phenotypes of this promising vaccine candidate, we constructed, using a reverse genetics system, a recombinant
cp
45 virus that contained all 15
cp
45-specific mutations mentioned above, and found that it was essentially indistinguishable from the biologically derived
cp
45 on the basis of plaque size, level of temperature sensitivity, cold adaptation, level of replication in the upper and lower respiratory tract of hamsters, and ability to protect hamsters from subsequent wild-type PIV3 challenge. We then constructed recombinant viruses containing the
cp
45 mutations in individual proteins as well as several combinations of mutations. Analysis of these recombinant viruses revealed that multiple
cp
45 mutations distributed throughout the genome contribute to the
ts
,
ca
, and
att
phenotypes. In addition to the mutations in the L gene, at least one other mutation in the 3′ N region (i.e., including the leader, N GS, and N coding changes) contributes to the
ts
phenotype. A recombinant virus containing all the
cp
45 mutations except those in L was more
ts
than
cp
45, illustrating the complex nature of this phenotype. The
ca
phenotype of
cp
45 also is a complex composite phenotype, reflecting contributions of at least three separate genetic elements, namely, mutations within the 3′ N region, the L protein, and the C-M-F-HN region. The
att
phenotype is a composite of both
ts
and non-
ts
mutations. Attenuating
ts
mutations are located in the L protein, and non-
ts
attenuating mutations are located in the C and F proteins. The presence of multiple
ts
and non-
ts
attenuating mutations in
cp
45 likely contributes to the high level of attenuation and phenotypic stability of this promising vaccine candidate.
Mutational Analysis of the Avian Adenovirus CELO, Which Provides a Basis for Gene Delivery Vectors
Authors: Michou Anne-Isabelle , Lehrmann Heike , Saltik Mediyha , Cotten Matt
Published: December 2019

The avian adenovirus CELO is being developed as a gene transfer tool. Using homologous recombination in
Escherichia coli
, the CELO genome was screened for regions that could be deleted and would tolerate the insertion of a marker gene (luciferase or enhanced green fluorescent protein). For each mutant genome, the production of viable virus able to deliver the transgene to target cells was monitored. A series of mutants in the genome identified a set of open reading frames that could be deleted but which must be supplied in
trans
for virus replication. A region of the genome which is dispensable for viral replication and allows the insertion of an expression cassette was identified and a vector based on this mutation was evaluated as a gene delivery reagent. Transduction of avian cells occurs at 10- to 100-fold greater efficiency (per virus particle) than with an adenovirus type 5 (Ad5)-based vector carrying the same expression cassette. Most important for gene transfer applications, the CELO vector transduced mammalian cells as efficiently as an Ad5 vector. The CELO vector is exceptionally stable, can be grown inexpensively in chicken embryos, and provides a useful alternative to Ad5-based vectors.
Endoproteolytic Processing of the Ebola Virus Envelope Glycoprotein: Cleavage Is Not Required for Function
Authors: Wool-Lewis Rouven J. , Bates Paul
Published: December 2019

Proteolytic processing is required for the activation of numerous viral glycoproteins. Here we show that the envelope glycoprotein from the Zaire strain of Ebola virus (Ebo-GP) is proteolytically processed into two subunits, GP
1
and GP
2
, that are likely covalently associated through a disulfide linkage. Murine leukemia virions pseudotyped with Ebo-GP contain almost exclusively processed glycoprotein, indicating that this is the mature form of Ebo-GP. Mutational analysis identified a dibasic motif, reminiscent of furin-like protease processing sites, as the Ebo-GP cleavage site. However, analysis of Ebo-GP processing in LoVo cells that lack the proprotein convertase furin demonstrated that furin is not required for processing of Ebo-GP. In sharp contrast to other viral systems, we found that an uncleaved mutant of Ebo-GP was able to mediate infection of various cell lines as efficiently as the wild-type, proteolytically cleaved glycoprotein, indicating that cleavage is not required for the activation of Ebo-GP despite the conservation of a dibasic cleavage site in all filoviral envelope glycoproteins.
Differential Effects of Human Immunodeficiency Virus Isolates on β-Chemokine and Gamma Interferon Production and on Cell Proliferation
Authors: Greco Giampaolo , Fujimura Sue H. , Mourich Dan V. , Levy Jay A.
Published: December 2019

All human immunodeficiency virus (HIV) isolates can grow readily in primary CD4+T cells, but they can be distinguished by their ability to replicate in macrophages and established T-cell lines. The macrophage-tropic viruses are generally non-syncytium inducing (NSI), whereas the T-cell-line-tropic viruses are syncytium inducing (SI) in cultured cells. We now demonstrate that infection of CD4+T cells by NSI and SI viruses shows a differential effect on production of β-chemokines and gamma interferon. Infection by NSI viruses increased production of MIP-1α, MIP-1β, and gamma interferon, whereas infection by SI viruses had no effect or decreased production of these cytokines. Production of RANTES was slightly increased during infection by both virus phenotypes. This differential effect of NSI and SI viruses was observed at the level of β-chemokine mRNA as well as at the level of protein expression. Infection by NSI viruses also increased CD4+cell proliferation. These results may have relevance for a differential role of HIV strains in AIDS pathogenesis.
Induction of B-Cell Lymphoma in BALB/c Nude Mice with an Ecotropic, B-Tropic Helper Virus Present in the Murine AIDS Virus Stock
Authors: Tayar Lamin , Higo Kyoko , Kubo Yoshinao , Wang Yun , Lu Ling-Min , Zhang Fengmin , Iwatani Yasumasa , Wang Ling , Ono Takeshi , Maeda Michiyuki , Sakai Hiroyuki , Ishimoto Akinori
Published: December 2019

The pathogenicities of the murine AIDS (MAIDS) virus complex (LP-BM5) and ecotropic helper virus (BM5eco) isolated from the complex to BALB/c nude mice were studied to elucidate the possible role of replication-competent helper virus in inducing the monoclonal outgrowth of lymphoid cells. Neither LP-BM5 nor BM5eco was pathogenic in adult BALB/c nude mice. However, B-cell lymphoma developed with a very high frequency when either virus was inoculated into newborn BALB/c nude (nu/nu) mice. The cells from the B-cell lymphoma were easily transplanted into nude mice. These results suggested that ecotropic helper virus in the MAIDS virus complex plays an important role in inducing the monoclonal outgrowth of lymphoid cells under immunodeficient conditions caused by defective virus.
Exponential Fitness Gains of RNA Virus Populations Are Limited by Bottleneck Effects
Authors: Novella Isabel S. , Quer Josep , Domingo Esteban , Holland John J.
Published: December 2019

Fitness is a parameter that quantitatively measures adaptation of a virus to a given environment. We have previously reported exponential fitness gains of large populations of vesicular stomatitis virus replicating in a constant environment (I. S. Novella et al., Proc. Natl. Acad. Sci. USA 92:5841–5844, 1995). In this paper, we report that during long-term passage of such large viral populations, fitness values reached a high-fitness plateau during which stochastic fitness variations were observed. This effect appears likely to be due to bottleneck effects on very high fitness populations.