bluuebunny/crossref_metadata_2025 huggingface dataset:
Characterization of and Functional Antigen Presentation by Central Nervous System Mononuclear Cells from Mice Infected with Theiler’s Murine Encephalomyelitis Virus
Authors: Pope Jonathan G. , Vanderlugt Carol L. , Rahbe Sandra M. , Lipton Howard L. , Miller Stephen D.
Published: December 2019

We examined the phenotype and function of cells infiltrating the central nervous system (CNS) of mice persistently infected with Theiler’s murine encephalomyelitis virus (TMEV) for evidence that viral antigens are presented to T cells within the CNS. Expression of major histocompatibility complex (MHC) class II in the spinal cords of mice infected with TMEV was found predominantly on macrophages in demyelinating lesions. The distribution of I-Asstaining overlapped that of the macrophage marker sialoadhesin in frozen sections and coincided with that of another macrophage/microglial cell marker, F4/80, by flow cytometry. In contrast, astrocytes, identified by staining with glial fibrillary acidic protein, rarely expressed detectable MHC class II, although fibrillary gliosis associated with the CNS damage was clearly seen. The costimulatory molecules B7-1 and B7-2 were expressed on the surface of most MHC class II-positive cells in the CNS, at levels exceeding those found in the spleens of the infected mice. Immunohistochemistry revealed that B7-1 and B7-2 colocalized on large F4/80+macrophages/microglia in the spinal cord lesions. In contrast, CD4+T cells in the lesions expressed mainly B7-2, which was found primarily on blastoid CD4+T cells located toward the periphery of the lesions. Most interestingly, plastic-adherent cells freshly isolated from the spinal cords of TMEV-infected mice were able to process and present TMEV and horse myoglobin to antigen-specific T-cell lines. Furthermore, these cells were able to activate a TMEV epitope-specific T-cell line in the absence of added antigen, providing conclusive evidence for the endogenous processing and presentation of virus epitopes within the CNS of persistently infected SJL/J mice.
Encapsidation of Viral DNA Requires the Adenovirus L1 52/55-Kilodalton Protein
Authors: Gustin Kurt E. , Imperiale Michael J.
Published: December 2019

Previous work demonstrated that the adenovirus L1 52/55-kDa protein is required for assembly of viral particles, although its exact role in the assembly process is unclear. The 52/55-kDa protein’s early expression, however, suggests that it might have other roles at earlier times during infection. To uncover any role the 52/55-kDa protein might have at early times and to better characterize its role in assembly, a mutant adenovirus incapable of expressing the 52/55-kDa protein was constructed (H5pm8001). Analysis of the onset and extent of DNA replication and late protein synthesis revealed that H5pm8001-infected 293 cells entered the late stage of infection at the same time as did adenovirus type 5 (Ad5)-infected cells. Interestingly, H5pm8001-infected cells displayed slightly lower levels of replicated viral DNA and late proteins, suggesting that although not required, the 52/55-kDa protein does augment these activities during infection. Analysis of transcripts produced from the major late and IVa2 promoters indicated a slight reduction in H5pm8001-infected compared to Ad5-infected cells at 18 h postinfection that was not apparent at later times. Analysis of particles formed in H5pm8001 cells revealed that empty capsids could form, suggesting that the 52/55-kDa protein does not function as a scaffolding protein. Subsequent characterization of these particles demonstrated that they lacked any associated viral DNA. These findings indicate that the 52/55 kDa-protein is required to mediate stable association between the viral DNA and empty capsid and suggest that it functions in the DNA encapsidation process.
Genetic Variation in a Human Immunodeficiency Virus Type 2 Live-VirusMacaca nemestrinaVaccine Model
Authors: Radaelli Antonia , Kraus Günter , Schmidt Ann , Badel Patricia , McClure Jan , Hu Shiu-Lok , Morton William , De Giuli Morghen Carlo , Wong-Staal Flossie , Looney D.
Published: December 2019

Four pigtailed macaques were inoculated with an infectious, apathogenic human immunodeficiency virus type 2 (HIV-2) molecular clone (HIV-2KR) and subsequently challenged with a highly pathogenic strain, HIV-2287, together with two naive control animals. After challenge, two animals inoculated with a high dose of the immunizing strain were protected from CD4 decline and immunodeficiency. To examine the role of genetic heterogeneity in protection, fragments of theenvgene were amplified from peripheral blood mononuclear cell DNA and plasma RNA of challenged animals by PCR, examined by using a heteroduplex tracking assay (HTA), and sequenced. By HTA, variation was detected principally within the V1 and V2 regions of envelope. Extent of variation in viral DNA clones as assessed by HTA correlated with inoculum size, as did the degree of variation in sequences of clones derived from viral DNA. Conversely, a rapid reduction in the number of plasma viral RNA variants was noted by HTA at 8 weeks postinfection in protected animals; this reduction was not present in naive or unprotected macaques. Sequences derived from plasma viral RNA were found to be more closely related than corresponding viral DNA sequences, and protection correlated with a significant reduction in variation in plasma RNA sequences in animals given the identical inocula of HIV-2287. Nonsynonymous mutations were significantly less prevalent in the protected animals. An additional potential glycosylation site was predicted to be present in the V2 region in all but one clone, and amino acid signatures related to protection were identified in viral DNA and RNA clones within both the V1 and V2 regions. Examination of the role of viral variation in this HIV-2 live-virus vaccine model may provide valuable insights into immunopathogenesis.
Analysis of Constructed E Gene Mutants of Mouse Hepatitis Virus Confirms a Pivotal Role for E Protein in Coronavirus Assembly
Authors: Fischer Françoise , Stegen Carola F. , Masters Paul S. , Samsonoff William A.
Published: December 2019

Expression studies have shown that the coronavirus small envelope protein E and the much more abundant membrane glycoprotein M are both necessary and sufficient for the assembly of virus-like particles in cells. As a step toward understanding the function of the mouse hepatitis virus (MHV) E protein, we carried out clustered charged-to-alanine mutagenesis on the E gene and incorporated the resulting mutations into the MHV genome by targeted recombination. Of the four possible clustered charged-to-alanine E gene mutants, one was apparently lethal and one had a wild-type phenotype. The two other mutants were partially temperature sensitive, forming small plaques at the nonpermissive temperature. Revertant analyses of these two mutants demonstrated that the created mutations were responsible for the temperature-sensitive phenotype of each and provided support for possible interactions among E protein monomers. Both temperature-sensitive mutants were also found to be markedly thermolabile when grown at the permissive temperature, suggesting that there was a flaw in their assembly. Most significantly, when virions of one of the mutants were examined by electron microscopy, they were found to have strikingly aberrant morphology in comparison to the wild type: most mutant virions had pinched and elongated shapes that were rarely seen among wild-type virions. These results demonstrate an important, probably essential, role for the E protein in coronavirus morphogenesis.
Deoxyribonucleoside Triphosphate Pool Imbalances In Vivo Are Associated with an Increased Retroviral Mutation Rate
Authors: Julias John G. , Pathak Vinay K.
Published: December 2019

Deoxyribonucleoside triphosphate (dNTP) pool imbalances are associated with an increase in the rate of misincorporation and hypermutation during in vitro reverse transcription reactions. However, the effects of in vivo dNTP pool imbalances on the accuracy of reverse transcription are unknown. We sought to determine the effects of in vivo dNTP pool imbalances on retroviral mutation rates and to test our hypothesis that 3′-azido-3′-deoxythymidine (AZT) increases the retroviral mutation rates through induction of dNTP pool imbalances. D17 cells were treated with thymidine, hydroxyurea (HU), or AZT, and the effects on in vivo dNTP pools were measured. Thymidine and HU treatments induced significant dNTP pool imbalances. In contrast, AZT treatment had very little effect on the dNTP pools. The effects of in vivo dNTP pool imbalances induced by thymidine and HU treatments on the retroviral mutation rates were also determined. Spleen necrosis virus (SNV)-based and murine leukemia virus (MLV)-based retroviral vectors that expressed the
lacZ
mutant reporter gene were used. The frequencies of inactivating mutations introduced in the
lacZ
gene in a single replication cycle provided a measure of the retroviral mutation rates. Treatment of D17 target cells with 500 μM thymidine increased the SNV and MLV mutant frequencies 4.7- and 4-fold, respectively. Treatment of D17 target cells with 2 mM HU increased the SNV and MLV mutant frequencies 2.1- and 2.7-fold, respectively. These results demonstrate that dNTP pool imbalances are associated with an increase in the in vivo retroviral mutation rates, but AZT treatment results in an increase in the retroviral mutation rates by a mechanism not involving alterations in dNTP pools.
Analysis of Minimal Human Immunodeficiency Virus Type 1
gag
Coding Sequences Capable of Virus-Like Particle Assembly and Release
Authors: Wang Chin-Tien , Lai Hsiu-Yu , Li Jue-Jyh
Published: December 2019

We have constructed a series of human immunodeficiency virus (HIV)
gag
mutants by progressive truncation of the
gag
coding sequence from the C terminus and have combined these mutants with an assembly-competent matrix domain deletion mutation (ΔMA). By using several methods, the particle-producing capabilities of each mutant were examined. Our analysis indicated that truncated Gag precursors lacking most of C-terminal
gag
gene products assembled and were released from 293T cells. Additionally, a mutant with a combined deletion of the MA (ΔMA) and p6 domains even produced particles at levels comparable to that of the wild-type (wt) virus. However, most mutants derived from combination of the ΔMA and the C-terminal truncation mutations did not release particles as well as the wt. Our smallest HIV
gag
gene product capable of virus-like particle formation was a 28-kDa protein which consists of a few MA amino acids and the CA-p2 domain. Sucrose density gradient fractionation analysis indicated that most mutants exhibited a wt retrovirus particle density. Exceptions to this rule were mutants with an intact MA domain but deleted downstream of the p2 domains. These C-terminal truncation mutants possessed particle densities of 1.13 to 1.15 g/ml, lower than that of the wt. The N-terminal portions of the CA domain, which have been shown to be dispensable for core assembly, became critical when most of the MA domain was deleted, suggesting a requirement for an intact CA domain to assemble and release particles.
Carboxypeptidase D (gp180), a Golgi-Resident Protein, Functions in the Attachment and Entry of Avian Hepatitis B Viruses
Authors: Breiner Klaus M. , Urban Stephan , Schaller Heinz
Published: December 2019

Carboxypeptidase D (gp180), one of many candidate receptors proposed for hepatitis B viruses (HBVs), was examined and found to be the actual cellular receptor for avian HBVs. This conclusion was based on the following observations: (i) gp180 was the only host protein that bound with high affinity to the pre-S ectodomain of the large duck hepatitis B virus (DHBV) envelope protein, which is known to be essential for virus infection; (ii) a pre-S subdomain which determines physical binding to gp180 was found to coincide with a domain functionally defined in infection competition experiments as a receptor binding domain; (iii) soluble gp180, lacking the membrane anchor, efficiently inhibited DHBV infection; (iv) efficient interspecies gp180–pre-S interaction was limited to the natural hosts of avian hepadnaviruses; and (v) expression of gp180 in a heterologous hepatoma cell line mediated cellular attachment and subsequent internalization of fluorescently labeled viral particles into vesicular structures. However, gp180 expression did not render transfected heterologous cells permissive for productive infection, suggesting that a species-specific coreceptor is required for fusion to complete viral entry. In contrast to the case for known virus receptors, gp180 was not detected on the hepatocyte cell surface but was found to be concentrated in the Golgi apparatus, from where it functions by cycling to and from the plasma membrane.
Activation of the Epstein-Barr Virus Transcription Factor BZLF1 by 12-
O
-Tetradecanoylphorbol-13-Acetate-Induced Phosphorylation
Authors: Baumann Matthias , Mischak Harald , Dammeier Sascha , Kolch Walter , Gires Olivier , Pich Dagmar , Zeidler Reinhard , Delecluse Henri-Jacques , Hammerschmidt Wolfgang
Published: December 2019

BZLF1 is a member of the extended AP-1 family of transcription factors which binds to specific BZLF1 sequence motifs within early Epstein-Barr virus (EBV) promoters and to closely related AP-1 motifs. BZLF1’s activity is regulated at the transcriptional level as well as through protein interactions and posttranslational modifications. Phorbol esters or immunoglobulin cross-linking both reactivate EBV from latently infected B cells via transactivation of BZLF1. We report here that the phorbol ester 12-
O
-tetradecanoylphorbol-13-acetate (TPA) is capable of inducing BZLF1’s activity even further. The induction occurs at the posttranscriptional level and depends on a single serine residue located in the DNA binding domain of BZLF1. This serine residue (S186) is phosphorylated by protein kinase C in vitro and in vivo after stimulation with TPA. Phosphorylation of S186 per se interferes with the DNA binding affinity of BZLF1 in vitro but is mandatory for TPA-induced increase in DNA binding of BZLF1, as shown in gel retardation assays and reconstruction experiments with cellular extracts. In transcriptional reporter assays, S186 is essential for the activation of BZLF1 by TPA. Presumably, a yet-to-be-identified cellular factor restores the DNA binding affinity and enhances the transcriptional activity of S186-phosphorylated BZLF1, which is required to induce the lytic phase of EBV’s life cycle.
Transactivation by the E2 Protein of Oncogenic Human Papillomavirus Type 31 Is Not Essential for Early and Late Viral Functions
Authors: Stubenrauch Frank , Colbert Angela M. E. , Laimins Laimonis A.
Published: December 2019

The activation of transcription and of DNA replication are, in some cases, mediated by the same proteins. A prime example is the E2 protein of human papillomaviruses (HPVs), which binds ACCN
6
GGT sequences and activates heterologous promoters from multimerized binding sites. The E2 protein also has functions in replication, where it complexes with the virally encoded origin recognition protein, E1. Much of the information on these activities is based on transient-transfection assays as well as biochemical analyses; however, their importance in the productive life cycle of oncogenic HPVs remains unclear. To determine the contributions of these E2 functions to the HPV life cycle, a genetic analysis was performed by using an organotypic tissue culture model. HPV type 31 (HPV31) genomes that contained mutations in the N terminus of E2 (amino acid 73) were constructed; these mutants retained replication activities but were transactivation defective. Following transfection of normal human keratinocytes, these mutant genomes were established as stable episomes and expressed early viral transcripts at levels similar to those of wild-type HPV31. Upon differentiation in organotypic raft cultures, the induction of late gene expression and amplification of viral DNA were detected in cell lines harboring mutant genomes. Interestingly, only a modest reduction in late gene expression was observed in the mutant lines. We conclude that the transactivation function of E2 is not essential for the viral life cycle of oncogenic HPVs, although it may act to moderately augment late expression. Our studies suggest that the primary positive role of E2 in the viral life cycle is as a replication factor.
Human Immunodeficiency Virus Type 1 Induction Mediated by Genistein Is Linked to Cell Cycle Arrest in G2
Authors: Gozlan Joel , Lathey Janet L. , Spector Stephen A.
Published: December 2019

Protein tyrosine kinase (PTK) phosphorylation is involved in cellular proliferation and differentiation processes that are key factors for human immunodeficiency virus type 1 (HIV-1) regulation in infected monocytic cells. Short-term exposure of the chronically infected promyelocytic OM10 cell line with the PTK inhibitor genistein induced a dose-dependent increase in p24 antigen production in culture supernatants. This induction persisted in the presence of the reverse transcriptase inhibitor, zidovudine, and was associated with an increased transcription of HIV-1 multiply spliced and unspliced RNAs, suggesting a transcriptional mechanism targeting the integrated provirus. Genistein induced cell differentiation, apoptosis, and a G2arrest in the OM10 cells. Cell differentiation and apoptosis were not directly involved in the observed increase in HIV-1 replication that was closely linked to genistein-induced G2arrest. Alleviation of the G2arrest by pentoxyfylline resulted in a concomitant reduction of HIV-1 to baseline replication. Additionally, by flow cytometry, a significant increase in the number of p24 antigen-expressing cells was observed in cells arrested in G2compared to those located in G1or S. Tyrosine kinase inhibition was found not to be essential for enhanced viral replication, which seemed to be related to two other properties of genistein, inhibition of topoisomerase II activity and inhibition of phosphotidylinositol turnover. These findings are consistent with the recent observation that HIV-1 Vpr induces viral replication through preventing proliferation of cells by arresting them in G2of the cell cycle and strongly suggest that manipulation of the cell cycle plays an important role in HIV-1 pathogenesis.
The RNA Polymerase of Influenza Virus, Bound to the 5′ End of Virion RNA, Acts in
cis
To Polyadenylate mRNA
Authors: Poon Leo L. M. , Pritlove David C. , Sharps Jane , Brownlee George G.
Published: December 2019

We previously demonstrated, by limited mutagenesis, that conserved sequence elements within the 5′ end of influenza virus virion RNA (vRNA) are required for the polyadenylation of mRNA in vitro. To further characterize the nucleotide residues at the 5′ end of vRNA which might be involved in polyadenylation, a complete set of short and long model vRNA-like templates with mutations at nucleotides 1′ to 13′ (prime notation denotes numbering from the 5′ end) of vRNA were synthesized and transcribed in vitro. The products were assayed for mRNA production with both reverse transcription-PCR and [α-
32
P]ATP incorporation assays. Results from these independent assays showed that vRNA templates with point mutations at positions 2′, 3′, 7′ to 9′, and 11′ to 13′ synthesized polyadenylated transcripts inefficiently compared with those with mutations at positions 1′, 4′ to 6′, and 10′. Positions 2′, 3′, 7′ to 9′, and 11′ are known to be involved in RNA polymerase binding. Furthermore, residues at positions 11′ to 13′ are known to be involved in base pairing between the 3′ and 5′ ends of vRNA. These findings demonstrate that the RNA polymerase has to bind to the 5′ end of the template vRNA, which must then interact with the 3′ end of the same template for polyadenylation to occur. These results support a model in which a
cis
-acting RNA polymerase is required for the polyadenylation of influenza virus.
Naı̈ve and Memory CD4 T Cells Differ in Their Susceptibilities to Human Immunodeficiency Virus Type 1 Infection following CD28 Costimulation: Implications for Transmission and Pathogenesis
Authors: Riley James L. , Levine Bruce L. , Craighead Nancy , Francomano Tara , Kim Daniel , Carroll Richard G. , June Carl H.
Published: December 2019

In vitro evidence suggests that memory CD4+cells are preferentially infected by human immunodeficiency virus type 1 (HIV-1), yet studies of HIV-1-infected individuals have failed to detect preferential memory cell depletion. To explore this paradox, we stimulated CD45RA+CD4+(naı̈ve) and CD45RO+CD4+(memory) cells with antibodies to CD3 and CD28 and infected them with either CCR5-dependent (R5) or CXCR4-dependent (X4) HIV-1 isolates. Naı̈ve CD4+cells supported less X4 HIV replication than their memory counterparts. However, naı̈ve cells were susceptible to R5 viral infection, while memory cells remained resistant to infection and viral replication. As with the unseparated cells, mixing the naı̈ve and memory cells prior to infection resulted in cells resistant to R5 infection and highly susceptible to X4 infection. While both naı̈ve and memory CD4+subsets downregulated CCR5 expression in response to CD28 costimulation, only the memory cells produced high levels of the β-chemokines RANTES, MIP-1α, and MIP-1β upon stimulation. Neutralization of these β-chemokines rendered memory CD4+cells highly sensitive to infection with R5 HIV-1 isolates, indicating that downregulation of CCR5 is not sufficient to mediate complete protection from CCR5 strains of HIV-1. These results indicate that susceptibility to R5 HIV-1 isolates is determined not only by the level of CCR5 expression but also by the balance of CCR5 expression and β-chemokine production. Furthermore, our results suggest a model of HIV-1 transmission and pathogenesis in which naı̈ve rather than memory CD4+T cells serve as the targets for early rounds of HIV-1 replication.
Long-Term CD4 Th1 and Th2 Memory following Acute Lymphocytic Choriomeningitis Virus Infection
Authors: Whitmire Jason K. , Asano Mary S. , Murali-Krishna Kaja , Suresh M. , Ahmed Rafi
Published: December 2019

CD4 T cells play a central role in viral immunity. They provide help for B cells and CD8 T cells and can act as effectors themselves. Despite their importance, relatively little is known about the magnitude and duration of virus-specific CD4 T-cell responses. In particular, it is not known whether both CD4 Th1 memory and CD4 Th2 memory can be induced by viral infections. To address these issues, we quantitated virus-specific CD4 Th1 (interleukin 2 [IL-2] and gamma-interferon) and Th2 (IL-4) responses in mice acutely infected with lymphocytic choriomeningitis virus (LCMV). Using two sensitive assays (enzyme-linked immunospot assay and intracellular stain) to measure cytokine production at the single-cell level, we found that both CD4 Th1 and Th2 responses were induced during primary LCMV infection. At the peak (day 8) of the response, the frequency of LCMV-specific CD4 Th1 cells was 1/35 to 1/160 CD4 T cells, and the frequency of Th2 cells was 1/400. After viral clearance, the numbers of virus-specific CD4 T cells dropped to 1/260 to 1/3,700 and then were maintained at this level indefinitely. Upon rechallenge with LCMV, both CD4 Th1 and Th2 memory cells made an anamnestic response in vivo. These results show that unlike some microbial infections in which only Th1 or Th2 responses are seen, an acute viral infection can induce a mixed CD4 T-cell response with long-term memory.
Establishment and Characterization of a Human Epstein-Barr Virus-Associated Gastric Carcinoma in SCID Mice
Authors: Iwasaki Yoshiaki , Chong Ja-Mun , Hayashi Yukiko , Ikeno Rie , Arai Kuniyoshi , Kitamura Masatsugu , Koike Morio , Hirai Kanji , Fukayama Masashi
Published: December 2019

A transplantable human Epstein-Barr virus-associated gastric carcinoma (EBVaGC), designated KT, was propagated in severe combined immunodeficiency (SCID) mice for 12 passages. Mucin and cytokeratin expression and the
Alu
sequence in tumor DNA confirmed that the KT tumor was derived from human epithelial tissue. The identity of clonal EBV in the original and KT tumors was demonstrated by terminal repeat analysis of EBV DNA. The pattern of latency gene expression of EBV was the same in both tumors. EBER1 was presented similarly in tumor cell nuclei by in situ hybridization. Reverse transcription-PCR analysis also demonstrated Q-promoter-driven EBNA1 expression but not BZLF1, EBNA2, or LMP1 expression. Thus, the transplantable human EBVaGC KT retains the original EBV with the same latency gene expression and can serve as a model for this unique type of gastric carcinoma.
Protection of Mice against Lethal Coxsackievirus B3 Infection by Using DNA Immunization
Authors: Henke Andreas , Wagner Elke , Whitton J. Lindsay , Zell Roland , Stelzner Axel
Published: December 2019

Vaccination with DNA and recombinant vaccinia viruses (rec.VV) has been studied with the coxsackievirus B3 (CVB3) model system. Plasmids encoding all structural proteins of CVB3, when injected intramuscularly, induced only low levels of virus-specific antibodies. However, DNA vaccination with the major structural protein VP1 protected 72.2% of mice from lethal challenge, whereas VP1 expressed by rec.VV was much less efficient.
Location-Specific, Unequal Contribution of the N Glycans in Simian Immunodeficiency Virus gp120 to Viral Infectivity and Removal of Multiple Glycans without Disturbing Infectivity
Authors: Ohgimoto Shinji , Shioda Tatsuo , Mori Kazuyasu , Nakayama Emi E. , Hu Huiling , Nagai Yoshiyuki
Published: December 2019

One of the striking features of human immunodeficiency virus, simian immunodeficiency virus (SIV), and other lentiviruses is extensive N glycosylation of the envelope protein. To assess the requirement of each N glycan for viral infectivity, we individually silenced all 23 N glycosylation sites in the gp120 subunit of SIVmac239 envelope protein by mutagenizing the canonical Asn-Xaa-Thr/Ser N glycosylation motif in an infectious molecular clone, attempted to rescue viruses from the clones, and compared the replication capability of the rescued viruses in MT4 cells. The mutation resulted in either the recovery of a fully infectious virus (category I); recovery of a faster-replicating virus, compared with the parental virus (category II); or no virus recovery (category III). These categorically different sites were not distributed randomly but were clustered. The sites of category I were localized largely in the N-terminal half, whereas the sites of categories II and III were localized in the C-terminal region, including the CD4 binding site, and the central part, including the C loop, respectively. To learn how far SIV can tolerate the removal of glycans, multiplex mutagenesis was also attempted. When they were appreciably distant from one another in the primary sequence, up to five sites could be silenced in combination without disturbing infectivity. On the other hand, it was difficult to silence contiguous sites. Thus, it appeared that a certain degree of sugar chain density over the local region had to be preserved. We discuss the potential utility of these variously deglycosylated mutants for clarifying the role of N glycans in SIV replication in vivo, as well as in the host response, and for designing vaccines and the generation of glycoprotein crystals.
Proviral Structure, Chromosomal Location, and Expression of HERV-K-T47D, a Novel Human Endogenous Retrovirus Derived from T47D Particles
Authors: Seifarth Wolfgang , Baust Corinna , Murr Andreas , Skladny Heyko , Krieg-Schneider Frank , Blusch Jürgen , Werner Thomas , Hehlmann Rüdiger , Leib-Mösch Christine
Published: December 2019

We previously described that type B retrovirus-like particles released from the human mammary carcinoma cell line T47D are pseudotypes and package retroviral RNA of different origins (W. Seifarth, H. Skladny, F. Krieg-Schneider, A. Reichert, R. Hehlmann, and C. Leib-Mösch, J. Virol. 69:6408–6416, 1995). One preferentially packaged retroviral sequence, ERV-MLN, has now been used to isolate the corresponding full-length provirus from a human genomic library. The 9,315-bp proviral genome comprises a complete retroviral structure except for a 3′ long terminal repeat (LTR) truncation. A lysine tRNA primer-binding site and phylogenetic analyses assign this human endogenous retroviral element, now called HERV-K-T47D, to the HML-4 subgroup of the HERV-K superfamily. The
gag
,
prt
,
pol
, and
env
genes exhibit 40 to 60% amino acid identity to HERV-K10. HERV-K-T47D is located on human chromosome 10, with five closely related elements on chromosomes 8, 9, 15, 16, and 19 and several hundred HERV-K-T47D-related solitary LTRs dispersed over the human genome. HERV-K-T47D-related sequences are detected in the genomes of higher primates and Old World monkeys but not in those of New World monkeys. High HERV-K-T47D transcription levels were observed in human placenta tissue, whereas transcription in T47D cells was strictly steroid dependent.
Specific and Independent Recognition of U3 and U5
att
Sites by Human Immunodeficiency Virus Type 1 Integrase In Vivo
Authors: Masuda Takao , Kuroda Marcelo J. , Harada Shinji
Published: December 2019

The retroviral attachment (
att
) sites at viral DNA ends are
cis
-acting regions essential for proviral integration. To investigate the sequence features of
att
important for human immunodeficiency virus type 1 (HIV-1) integration in vivo, we generated a series of 25
att
mutants of HIV-1 by mutagenesis of the U3, U5, or both boundaries of
att
. Our results indicated that the terminal 11 or 12 bp of viral DNA are sufficient for specific recognition by HIV-1 integrase (IN) and suggested that IN might recognize each
att
site independently in vivo.
Characterization of Simian-Human Immunodeficiency Virus Envelope Glycoprotein Epitopes Recognized by Neutralizing Antibodies from Infected Monkeys
Authors: Etemad-Moghadam Bijan , Karlsson Gunilla B. , Halloran Matilda , Sun Ying , Schenten Dominik , Fernandes Mark , Letvin Norman L. , Sodroski Joseph
Published: December 2019

We characterized human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein epitopes recognized by neutralizing antibodies from monkeys recently infected by molecularly cloned simian-human immunodeficiency virus (SHIV) variants. The early neutralizing antibody response in each infected animal was directed mainly against a single epitope. This primary neutralizing epitope, however, differed among individual monkeys infected by identical viruses. Two such neutralization epitopes were determined by sequences in the V2 and V3 loops of the gp120 envelope glycoprotein, while a third neutralization epitope, apparently discontinuous, was determined by both V2 and V3 sequences. These results indicate that the early neutralizing antibody response in SHIV-infected monkeys is monospecific and directed against epitopes composed of the gp120 V2 and V3 variable loops.
Inactivation of p53 but Not p73 by Adenovirus Type 5 E1B 55-Kilodalton and E4 34-Kilodalton Oncoproteins
Authors: Roth Judith , König Claudia , Wienzek Sandra , Weigel Silke , Ristea Susanne , Dobbelstein Matthias
Published: December 2019

The adenovirus E1B 55-kDa and E4 34-kDa oncoproteins bind and inactivate the p53 tumor suppressor gene product, resulting in cell transformation. A recently discovered cellular protein, p73, shows extensive similarities to p53 in structure and function. Here we show that the simultaneous transient expression of E1B 55-kDa and E4 34-kDa proteins is sufficient to drastically shorten the intracellular half-life of p53, leading to strongly reduced steady-state p53 levels. Concomitantly, the E1B 55-kDa and E4 34-kDa proteins act synergistically to inactivate the transcriptional activity of p53. Mutational analysis suggests that physical interactions between the E1B 55-kDa protein and p53 and between the E1B 55-kDa and E4 34-kDa proteins are both required for p53 degradation. In contrast, the ability of p53 to interact with the cellular mdm2 oncoprotein or with its cognate DNA element appears to be dispensable for its destabilization by adenovirus gene products. The adenovirus E1B 55-kDa protein did not detectably interact with p73 and failed to inhibit p73-mediated transcription; also, the E1B 55-kDa and E4 34-kDa proteins did not promote p73 degradation. When five amino acids near the amino termini were exchanged at corresponding positions between p53 and p73, this rendered p53 resistant and p73 susceptible to complex formation and inactivation by the E1B 55-kDa protein. Our results suggest that while p53 inactivation is a central step in virus-induced tumor development, efficient transformation can occur without targeting p73.
Coronavirus Transcription Early in Infection
Authors: An Sungwhan , Maeda Akihiko , Makino Shinji
Published: December 2019

We studied the accumulation kinetics of murine coronavirus mouse hepatitis virus (MHV) RNAs early in infection by using cloned MHV defective interfering (DI) RNA that contained an intergenic sequence from which subgenomic DI RNA is synthesized in MHV-infected cells. Genomic DI RNA and subgenomic DI RNA accumulated at a constant ratio from 3 to 11 h postinfection (p.i.) in the cells infected with MHV-containing DI particles. Earlier, at 1 h p.i., this ratio was not constant; only genomic DI RNA accumulated, indicating that MHV RNA replication, but not MHV RNA transcription, was active during the first hour of MHV infection. Negative-strand genomic DI RNA and negative-strand subgenomic DI RNA were first detectable at 1 and 3 h p.i., respectively, and the amounts of both RNAs increased gradually until 6 h p.i. These data showed that at 2 h p.i., subgenomic DI RNA was undergoing synthesis in the cells in which negative-strand subgenomic DI RNA was undetectable. These data, therefore, signify that negative-strand genomic DI RNA, but not negative-strand subgenomic DI RNA, was an active template for subgenomic DI RNA synthesis early in infection.
Coronavirus Pseudoparticles Formed with Recombinant M and E Proteins Induce Alpha Interferon Synthesis by Leukocytes
Authors: Baudoux Pierre , Carrat Charles , Besnardeau Lydia , Charley Bernard , Laude Hubert
Published: December 2019

Transmissible gastroenteritis virus (TGEV), an enteric coronavirus of swine, is a potent inducer of alpha interferon (IFN-α) both in vivo and in vitro. Incubation of peripheral blood mononuclear cells with noninfectious viral material such as inactivated virions or fixed, infected cells leads to early and strong IFN-α synthesis. Previous studies have shown that antibodies against the virus membrane glycoprotein M blocked the IFN induction and that two viruses with a mutated protein exhibited a decreased interferogenic activity, thus arguing for a direct involvement of M protein in this phenomenon. In this study, the IFN-α-inducing activity of recombinant M protein expressed in the absence or presence of other TGEV structural proteins was examined. Fixed cells coexpressing M together with at least the minor structural protein E were found to induce IFN-α almost as efficiently as TGEV-infected cells. Pseudoparticles resembling authentic virions were released in the culture medium of cells coexpressing M and E proteins. The interferogenic activity of purified pseudoparticles was shown to be comparable to that of TGEV virions, thus establishing that neither ribonucleoprotein nor spikes are required for IFN induction. The replacement of the externally exposed, N-terminal domain of M with that of bovine coronavirus (BCV) led to the production of chimeric particles with no major change in interferogenicity, although the structures of the TGEV and BCV ectodomains markedly differ. Moreover, BCV pseudoparticles also exhibited interferogenic activity. Together these observations suggest that the ability of coronavirus particles to induce IFN-α is more likely to involve a specific, multimeric structure than a definite sequence motif.
Synthetic DNA Replication Bubbles Bound and Unwound with Twofold Symmetry by a Simian Virus 40 T-Antigen Double Hexamer
Authors: Smelkova Natalia V. , Borowiec James A.
Published: December 2019

Dimerization of simian virus 40 T-antigen hexamers (TAgH) into double hexamers (TAgDH) on model DNA replication forks has been found to greatly stimulate T-antigen DNA helicase activity. To explore the interaction of TAgDHwith DNA during unwinding, we examined the binding of TAgDHto synthetic DNA replication bubbles. Tests of replication bubble substrates containing different single-stranded DNA (ssDNA) lengths indicated that efficient formation of a TAgDHrequires ≥40 nucleotides (nt) of ssDNA. DNase I probing of a substrate containing a 60-nt ssDNA bubble complexed with a TAgDHrevealed that T antigen bound the substrate with twofold symmetry. The strongest protection was observed over the 5′ junction on each strand, with 5 bp of duplex DNA and ∼17 nt of adjacent ssDNA protected from nuclease cleavage. Stimulation of the T-antigen DNA helicase activity by an increase in ATP concentration caused the protection to extend in the 5′ direction into the duplex region, while resulting in no significant changes to the 3′ edge of strongest protection. Our data indicate that each TAgHencircles one ssDNA strand, with a different strand bound at each junction. The process of DNA unwinding results in each TAgHinteracting with a greater length of DNA than was initially bound, suggesting the generation of a more highly processive helicase complex.
Proteolytic Processing and Assembly ofgag and gag-pol Proteins of TED, a Baculovirus-Associated Retrotransposon of the Gypsy Family
Authors: Hajek Kathryn L. , Friesen Paul D.
Published: December 2019

TED (transposable element D) is anenv-containing member of the gypsy family of retrotransposons that represents a possible retrovirus of invertebrates. This lepidopteran (moth) retroelement containsgagandpolgenes that encode proteins capable of forming viruslike particles (VLP) with reverse transcriptase. Since VLP are likely intermediates in TED transposition, we investigated the roles ofgagandpolin TED capsid assembly and maturation. By using constructed baculovirus vectors and TED Gag-specific antiserum, we show that the principal translation product ofgag(Pr55gag) is cleaved to produce a single VLP structural protein, p37gag. Replacement of Asp436within the retrovirus-like active site of thepol-encoded protease (PR) abolished Pr55gagcleavage and demonstrated the requirement for PR in capsid processing. As shown by expression of an in-frame fusion of TEDgagandpol, PR is derived from the Gag-Pol polyprotein Pr195gag-pol. The PR cleavage site within Pr55gagwas mapped to a position near the junction of a basic, nucleocapsid-like domain and a C-terminal acidic domain. Once released by cleavage, the C-terminal fragment was not detected. This acidic fragment was dispensable for VLP assembly, as demonstrated by the formation of VLP by C-terminal Pr55gagtruncation proteins and replacement of the acidic domain with a heterologous protein. In contrast, C-terminal deletions that extended into the adjacent nucleocapsid-like domain of Pr55gagabolished VLP recovery and demonstrated that this central region contributes to VLP assembly or stability, or both. Collectively, these data suggest that the single TED protein p37gagprovides both capsid and nucleocapsid functions. TED may therefore use a simple processing strategy for VLP assembly and genome packaging.
Walleye Retroviruses Associated with Skin Tumors and Hyperplasias Encode Cyclin D Homologs
Authors: LaPierre Lorie A. , Casey James W. , Holzschu Donald L.
Published: December 2019

Walleye dermal sarcoma (WDS) and walleye epidermal hyperplasia (WEH) are skin diseases of walleye fish that appear and regress on a seasonal basis. We report here that the complex retroviruses etiologically associated with WDS (WDS virus [WDSV]) and WEH (WEH viruses 1 and 2 [WEHV1 and WEHV2, respectively]) encode D-type cyclin homologs. The retroviral cyclins (rv-cyclins) are distantly related to one another and to known cyclins and are not closely related to any walleye cellular gene based on low-stringency Southern blotting. Since aberrant expression of D-type cyclins occurs in many human tumors, we suggest that expression of the rv-cyclins may contribute to the development of WDS or WEH. In support of this hypothesis, we show that rv-cyclin transcripts are made in developing WDS and WEH and that the rv-cyclin of WDSV induces cell cycle progression in yeast (Saccharomyces cerevisiae). WEHV1, WEHV2, and WDSV are the first examples of retroviruses that encode cyclin homologs. WEH and WDS and their associated retroviruses represent a novel paradigm of retroviral tumor induction and, importantly, tumor regression.
CD4 Promoter Transactivation by Human Herpesvirus 6
Authors: Flamand Louis , Romerio Fabio , Reitz Marvin S. , Gallo Robert C.
Published: December 2019

The observation that human herpesvirus 6 (HHV-6) can induce CD4 gene transcription and expression in CD4−cells was reported several years ago (P. Lusso, A. De Maria, M. Malnati, F. Lori, S. E. DeRocco, M. Baseler, and R. C. Gallo, Nature 349:533–535, 1991) and subsequently confirmed (P. Lusso, M. S. Malnati, A. Garzino-Demo, R. W. Crowley, E. O. Long, and R. C. Gallo, Nature 362:458–462, 1993; G. Furlini, M. Vignoli, E. Ramazzotti, M. C. Re, G. Visani, and M. LaPlaca, Blood 87:4737–4745, 1996). Our objective was to identify the mechanisms underlying such phenomena. Using reporter gene constructs driven by the CD4 promoter, we report that HHV-6 can efficiently transactivate such genetic elements. Activation of the CD4 promoter occurs in the presence of the viral DNA polymerase inhibitor phosphonoformic acid, which limits expression to the immediate-early and early classes of viral genes. Using deletion mutants and specific CD4 promoter mutants, we identified an ATF/CRE binding site located at nucleotides −67 to −60 upstream of the CD4 gene transcription start site that is important for HHV-6 transactivation. The ATF/CRE site is also essential for CD4 promoter activation by forskolin, an activator of adenylate cyclase. Using electrophoretic mobility shift assays and specific antibodies, we showed that CREB-1 binds specifically to the −79 to −52 region of the CD4 promoter. Last, we have identified two open reading frames (ORFs) of HHV-6, U86 and U89 from the immediate-early locus A, that can transactivate the CD4 promoter in HeLa cells. However, transactivation of the CD4 promoter by ORFs U86 and U89 is independent of the CRE element, suggesting that additional HHV-6 ORFs are likely to contribute to CD4 gene activation. Taken together, our results will help to understand the complex interactions occurring between HHV-6 and the CD4 promoter and provide additional information regarding the class of transcription factors involved in the control of CD4 gene expression.
Adenovirus Endocytosis Requires Actin Cytoskeleton Reorganization Mediated by Rho Family GTPases
Authors: Li Erguang , Stupack Dwayne , Bokoch Gary M. , Nemerow Glen R.
Published: December 2019

Adenovirus (Ad) endocytosis via αvintegrins requires activation of the lipid kinase phosphatidylinositol-3-OH kinase (PI3K). Previous studies have linked PI3K activity to both the Ras and Rho signaling cascades, each of which has the capacity to alter the host cell actin cytoskeleton. Ad interaction with cells also stimulates reorganization of cortical actin filaments and the formation of membrane ruffles (lamellipodia). We demonstrate here that members of the Rho family of small GTP binding proteins, Rac and CDC42, act downstream of PI3K to promote Ad endocytosis. Ad internalization was significantly reduced in cells treated withClostridium difficiletoxin B and in cells expressing a dominant-negative Rac or CDC42 but not a H-Ras protein. Viral endocytosis was also inhibited by cytochalasin D as well as by expression of effector domain mutants of Rac or CDC42 that impair cytoskeletal function but not JNK/MAP kinase pathway activation. Thus, Ad endocytosis requires assembly of the actin cytoskeleton, an event initiated by activation of PI3K and, subsequently, Rac and CDC42.
Virion Incorporation of Human Immunodeficiency Virus Type 1 Nef Is Mediated by a Bipartite Membrane-Targeting Signal: Analysis of Its Role in Enhancement of Viral Infectivity
Authors: Welker Reinhold , Harris Mark , Cardel Bettina , Kräusslich Hans-Georg
Published: December 2019

Thenefgene of primate immunodeficiency viruses is essential for high-titer virus replication and AIDS pathogenesis in vivo. In tissue culture, Nef is not required for human immunodeficiency virus (HIV) infection but enhances viral infectivity. We and others have shown that Nef is incorporated into HIV-1 particles and cleaved by the viral proteinase. To determine the signal for Nef incorporation and to analyze whether virion-associated Nef is responsible for enhancement of infectivity, we generated a panel ofnefmutants and analyzed them for virion incorporation of Nef and for their relative infectivities. We report that N-terminal truncations of Nef abolished its incorporation into HIV particles. Incorporation was reconstituted by targeting the respective proteins to the plasma membrane by using a heterologous signal. Mutational analysis revealed that both myristoylation and an N-terminal cluster of basic amino acids were required for virion incorporation and for plasma membrane targeting of Nef. Grafting the N-terminal anchor domain of Nef onto the green fluorescent protein led to membrane targeting and virion incorporation of the resulting fusion protein. These results indicate that Nef incorporation into HIV-1 particles is mediated by plasma membrane targeting via an N-terminal bipartite signal which is reminiscent of a Src homology region 4. Virion incorporation of Nef correlated with enhanced infectivity of the respective viruses in a single-round replication assay. However, the phenotypes of HIV mutants with reduced Nef incorporation only partly correlated with their ability to replicate in primary lymphocytes, indicating that additional or different mechanisms may be involved in this system.