bluuebunny/crossref_metadata_2025 huggingface dataset:
Polarized Budding of Measles Virus Is Not Determined by Viral Surface Glycoproteins
Authors: Maisner A. , Klenk H.-D. , Herrler G.
Published: December 2019

For viruses that mature by a budding process, the envelope glycoproteins are considered the major determinants for the site of virus release from polarized epithelial cells. Viruses are usually released from that membrane domain where the viral surface glycoproteins are transported to. We here report that measles virus has developed a different maturation strategy. Measles virus was found to be released from the apical membrane domain of polarized epithelial cells, though the surface glycoproteins H and F were transported in a nonpolarized fashion and to the basolateral membrane domain, respectively.
Cleavage of Rhesus Rotavirus VP4 after Arginine 247 Is Essential for Rotavirus-Like Particle-Induced Fusion from Without
Authors: Gilbert Joanna M. , Greenberg Harry B.
Published: December 2019

We recently described our finding that recombinant baculovirus-produced virus-like particles (VLPs) can induce cell-cell fusion similar to that induced by intact rotavirus in our assay for viral entry into tissue culture cells (J. M. Gilbert and H. B. Greenberg, J. Virol. 71:4555–4563, 1997). The conditions required for syncytium formation are similar to those for viral penetration of the plasma membrane during the course of viral infection. This VLP-mediated fusion activity was dependent on the presence of the outer-layer proteins, viral protein 4 (VP4) and VP7, and on the trypsinization of VP4. Fusion activity occurred only with cells that are permissive for rotavirus infection. Here we begin to dissect the role of VP4 in rotavirus entry by examining the importance of the precise trypsin cleavage of VP4 and the activation of VP4 function related to viral entry. We present evidence that the elimination of the three trypsin-susceptible arginine residues of VP4 by specific site-directed mutagenesis prevents syncytium formation. Two of the three arginine residues in VP4 are dispensable for syncytium formation, and only the arginine residue at site 247 appears to be required for activation of VP4 functions and cell-cell fusion. Using the recombinant VLPs in our syncytium assay will aid in understanding the conformational changes that occur in VP4 involved in rotavirus penetration into host cells.
In Vivo Immune Evasion Mediated by the Herpes Simplex Virus Type 1 Immunoglobulin G Fc Receptor
Authors: Nagashunmugam Thandavarayan , Lubinski John , Wang Liyang , Goldstein Lester T. , Weeks Benjamin S. , Sundaresan Periasamy , Kang Eugene H. , Dubin Gary , Friedman Harvey M.
Published: December 2019

Herpes simplex virus (HSV) glycoproteins gE and gI form an immunoglobulin G (IgG) Fc receptor (FcγR) that binds the Fc domain of human anti-HSV IgG and inhibits Fc-mediated immune functions in vitro. gE or gI deletion mutant viruses are avirulent, probably because gE and gI are also involved in cell-to-cell spread. In an effort to modify FcγR activity without affecting other gE functions, we constructed a mutant virus, NS-gE339, that has four amino acids inserted into gE within the domain homologous to mammalian IgG FcγRs. NS-gE339expresses gE and gI, is FcγR−, and does not participate in antibody bipolar bridging since it does not block activities mediated by the Fc domain of anti-HSV IgG. In vivo studies were performed with mice because the HSV-1 FcγR does not bind murine IgG; therefore, the absence of an FcγR should not affect virulence in mice. NS-gE339causes disease at the skin inoculation site comparably to wild-type and rescued viruses, indicating that the FcγR−mutant virus is pathogenic in animals. Mice were passively immunized with human anti-HSV IgG and then infected with mutant or wild-type virus. We postulated that the HSV-1 FcγR should protect wild-type virus from antibody attack. Human anti-HSV IgG greatly reduced viral titers and disease severity in NS-gE339-infected animals while having little effect on wild-type or rescued virus. We conclude that the HSV-1 FcγR enables the virus to evade antibody attack in vivo, which likely explains why antibodies are relatively ineffective against HSV infection.
Characterization of the Transcriptional Repressive Element of the Human Cytomegalovirus Immediate-Early US3 Gene
Authors: Biegalke Bonita J.
Published: December 2019

Transcriptional repression is utilized by human cytomegalovirus to regulate expression of the immediate-early US3 gene. Sequences located 3′ of the US3 TATA box are required for down regulation of expression. Mutagenesis of US3 sequences identified a 10-nucleotide region that is essential for transcriptional repression. In addition to the 10-nucleotide element, an additional region, which includes the US3 initiator element, was needed to confer repression on a heterologous promoter. Thus, a 19-nucleotide element (−18 to +1 relative to the transcription start site) functioned as a transcriptional repressive element (
tre
). The
tre
repressed transcription in a position-dependent but orientation-independent manner. In vivo footprinting experiments demonstrated that transcriptional repression is associated with a decrease in protein interactions with the US3 promoter and surrounding sequences. The data presented here suggest that the association of an as yet unidentified repressor protein with the
tre
represses transcription by inhibiting assembly of the transcription initiation complex on the US3 promoter.
Derivation and Functional Characterization of a Consensus DNA Binding Sequence for the Tas Transcriptional Activator of Simian Foamy Virus Type 1
Authors: Kang Yibin , Cullen Bryan R.
Published: December 2019

Although DNA binding sites specific for the Bel-1 and Tas transcriptional activators, encoded, respectively, by the human and simian foamy viruses, have been mutationally defined, they show little evident sequence identity. As a result, the sequence determinants for DNA binding by both Bel-1 and Tas have remained unclear. Here, we report the use of a novel in vivo randomization and selection strategy to identify a Tas DNA binding site consensus. This approach takes advantage of the fact that Tas can effectively activate gene expression in yeast cells via a Tas DNA binding site derived from the simian foamy virus type 1 (SFV-1) internal promoter. The defined Tas DNA binding site consensus extends over approximately 25 bp and contains a critical core sequence of ∼5 bp. Positions adjacent to this core sequence, while clearly also subject to selection, show a significantly higher level of sequence variation. Surprisingly, the wild-type SFV-1 internal promoter Tas DNA binding site fails to conform to the consensus at several positions. Further analysis demonstrated that the consensus sequence bound Tas more effectively than did the wild-type sequence in vitro and could mediate an enhanced Tas response in vivo when substituted into the SFV-1 internal promoter context. These findings explain the limited sequence identity observed for mutationally defined Tas or Bel-1 response elements and should facilitate the identification of Tas DNA target sites located elsewhere in the SFV-1 genome.
An Interferon Regulatory Factor Binding Site in the U5 Region of the Bovine Leukemia Virus Long Terminal Repeat Stimulates Tax-Independent Gene Expression
Authors: Kiermer Véronique , Van Lint Carine , Briclet Delphine , Vanhulle Caroline , Kettmann Richard , Verdin Eric , Burny Arsène , Droogmans Louis
Published: December 2019

Bovine leukemia virus (BLV) replication is controlled by bothcis- andtrans-acting elements. The virus-encoded transactivator, Tax, is necessary for efficient transcription from the BLV promoter, although it is not present during the early stages of infection. Therefore, sequences that control Tax-independent transcription must play an important role in the initiation of viral gene expression. This study demonstrates that the R-U5 sequence of BLV stimulates Tax-independent reporter gene expression directed by the BLV promoter. R-U5 was also stimulatory when inserted immediately downstream from the transcription initiation site of a heterologous promoter. Progressive deletion analysis of this region revealed that a 46-bp element corresponding to the 5′ half of U5 is principally responsible for the stimulation. This element exhibited enhancer activity when inserted upstream or downstream from the herpes simplex virus thymidine kinase promoter. This enhancer contains a binding site for the interferon regulatory factors IRF-1 and IRF-2. A 3-bp mutation that destroys the IRF recognition site caused a twofold decrease in Tax-independent BLV long terminal repeat-driven gene expression. These observations suggest that the IRF binding site in the U5 region of BLV plays a role in the initiation of virus replication.
The Neuroinvasiveness of a Murine Retrovirus Is Influenced by a Dileucine-Containing Sequence in the Cytoplasmic Tail of Glycosylated Gag
Authors: Fujisawa Ryuichi , McAtee Frank J. , Wehrly Kathy , Portis John L.
Published: December 2019

The tempo and intensity of retroviral neuropathogenesis are dependent on the capacity of the virus to invade the central nervous system. For murine leukemia viruses, an important determinant of neuroinvasiveness is the virus-encoded protein glycosylated Gag, the function of which in the virus life cycle is not known. While this protein is dispensable for virus replication, mutations which prevent its expression slow the spread of virus in vivo and restrict virus dissemination to the brain. To further explore the function of this protein, we compared two viruses, CasFr
KP
(KP) and CasFr
KP41
(KP41), which differ dramatically in neurovirulence. KP expresses high early viremia titers, is neuroinvasive, and induces clinical neurologic disease in 100% of neonatally inoculated mice, with an incubation period of 18 to 23 days. In contrast, KP41 expresses early viremia titers 100- fold lower than those of KP, exhibits attenuated neuroinvasiveness, and induces clinical neurologic disease infrequently, with a relatively long incubation period. The genomes of these two viruses differ by only 10 nucleotides, resulting in differences at five residues, all located within the N-terminal cytoplasmic tail of glycosylated Gag. In this study, using KP as the parental virus, we systematically mutated each of the five amino acid residues to those of KP41 and found that substitution mutation of two membrane-proximal residues, E
53
and L
56
, to K and P, respectively produced the greatest effect on early viremia kinetics and neurovirulence. These mutations disrupted the KP sequence E
53
FLL
56
, the leucine dipeptide of which suggests the possibility that it may represent a sorting signal for glycosylated Gag. Supporting this idea was the finding that alteration of this sequence motif increased the level of cell surface expression of the protein, which suggests that analysis of the intracellular trafficking of glycosylated Gag may provide further clues to its function.
Human Cytomegalovirus Persistently Infects Aortic Endothelial Cells
Authors: Fish Kenneth N. , Soderberg-Naucler Cecilia , Mills Lisa K. , Stenglein Stephan , Nelson Jay A.
Published: December 2019

Endothelial cells (EC) have been implicated as constituting an important cell type in the pathogenesis of human cytomegalovirus (HCMV). Microvascular and macrovascular EC exhibit different biochemical and functional properties depending on the organ of origin. Phenotypic differences between microvascular and macrovascular EC may alter the ability of these cells to support HCMV replication. In this study, we compared the replication of HCMV in primary macrovascular aortic EC (AEC) with that in brain microvascular EC (BMVEC). An examination of IE72, pp65, and gB viral antigen expression in BMVEC and AEC by immunoflourescence revealed similar frequencies of infected cells. Intracellular production of virus was 3 log units greater in BMVEC than in AEC, while equal quantities of extracellular virus were produced in both cell types. HCMV infection of BMVEC resulted in rapid cellular lysis, while the virus was nonlytic and continuously released from HCMV-infected AEC for the life span of the culture. An examination of infected cells by electron microscopy revealed the formation of abundant nucleocapsids in both AEC and BMVEC. However, significant amounts of mature viral particles were only detected in the cytoplasm of BMVEC. These observations indicate that levels of HCMV replication in EC obtained from different organs are distinct and suggest that persistently infected AEC may serve as a reservoir of virus.
Rabbit Cells Expressing Human CD4 and Human CCR5 Are Highly Permissive for Human Immunodeficiency Virus Type 1 Infection
Authors: Speck Roberto F. , Penn Michael L. , Wimmer Jörg , Esser Ursula , Hague Bishop F. , Kindt Thomas J. , Atchison Robert E. , Goldsmith Mark A.
Published: December 2019

To evaluate the feasibility of using transgenic rabbits expressing CCR5 and CD4 as a small-animal model of human immunodeficiency virus type 1 (HIV) disease, we examined whether the expression of the human chemokine receptor (CCR5) and human CD4 would render a rabbit cell line (SIRC) permissive to HIV replication. Histologically, SIRC cells expressing CD4 and CCR5 formed multinucleated cells (syncytia) upon exposure to BaL, a macrophagetropic strain of HIV that uses CCR5 for cell entry. Intracellular viral capsid p24 staining showed abundant viral gene expression in BaL-infected SIRC cells expressing CD4 and CCR5. In contrast, neither SIRC cells expressing CD4 alone nor murine 3T3 cells expressing CCR5 and CD4 exhibited significant expression of p24. These stably transfected rabbit cells were also highly permissive for the production of virions upon infection by two other CCR5-dependent strains (JR-CSF and YU-2) but not by a CXCR4-dependent strain (NL4-3). The functional integrity of these virions was demonstrated by the successful infection of human peripheral blood mononuclear cells (PBMC) with viral stocks prepared from these transfected rabbit cells. Furthermore, primary rabbit PBMC were found to be permissive for production of infectious virions after circumventing the cellular entry step. These results suggest that a transgenic rabbit model for the study of HIV disease may be feasible.
Human Immunodeficiency Virus Type 1 Vectors Efficiently Transduce Human Hematopoietic Stem Cells
Authors: Sutton Richard E. , Wu Henry T. M. , Rigg Richard , Böhnlein Ernst , Brown Patrick O.
Published: December 2019

Lentiviruses are potentially advantageous compared to oncoretroviruses as gene transfer agents because they can infect nondividing cells. We demonstrate here that human immunodeficiency virus type 1 (HIV-1)-based vectors were highly efficient in transducing purified human hematopoietic stem cells. Transduction rates, measured by marker gene expression or by PCR of the integrated provirus, exceeded 50%, and transduction appeared to be independent of mitosis. Derivatives of HIV-1 were constructed to optimize the vector, and a deletion of most of Vif and Vpr was required to ensure the long-term persistence of transduced cells with relatively stable expression of the marker gene product. These results extend the utility of this lentivirus vector system.
Genetically Related Human Immunodeficiency Virus Type 1 in Three Adults of a Family with No Identified Risk Factor for Intrafamilial Transmission
Authors: Bélec Laurent , Mohamed Ali Si , Müller-Trutwin Michaela C. , Gilquin Jacques , Gutmann Laurent , Safar Michel , Barré-Sinoussi Françoise , Kazatchkine Michel D.
Published: December 2019

A small number of cases of human immunodeficiency virus (HIV) infection have been reported in individuals with no identified risk factors for transmission. We report on the seroconversion of the 61-year-old mother and the subsequent finding of HIV seropositivity in the 66-year-old father of a 31-year-old AIDS patient. Extensive investigation failed to identify any risk factor for intrafamilial transmission. We conducted a genetic analysis and determined the amino acid signature patterns of the V3, V4, and V5 hypervariable domains and flanking regions in the HIV-1 gp120
env
gene of 26 clones derived from proviral DNA in peripheral blood mononuclear cells of the members of the family.
env
sequences of the viruses isolated from the patients were compared with sequences of HIV-1 subtype B viruses from Europe and local field isolates. Phylogenetic analysis revealed that the sequences of the viruses isolated from the patients were genetically related and formed an intrafamilial cluster of HIV-1 distinct from other subtype B viruses. Interindividual nucleotide variability in the C2-V3 and V4-C4-V5 domains ranged between 1.2 and 5.0% and between 2.2 and 7.5%, respectively, whereas divergence between HIV strains from the patients and control viral strains ranged from 6.6 to 29.3%. The amino acid signature patterns of viral clones from the three patients were closely related. In the C2-V3 region, two minor clones derived from the son’s virus showed less nucleotide divergence (mean, 3.5 and 3.9%) than did the clones derived from the viruses of both parents or the seven other predominant clones derived from the virus from the son (mean, 5.4%). The top of the V3 loop of the last two clones and of all viral clones from the parents exhibited an unusual GPGG sequence. This is the first report of genotypic relatedness of HIV-1 in three adults of the same family in the absence of identified risk factor for transmission between the members of the family. Our findings suggest that atypical transmission of HIV may occur.
Contact with Thymic Epithelial Cells as a Prerequisite for Cytokine-Enhanced Human Immunodeficiency Virus Type 1 Replication in Thymocytes
Authors: Rothe Maurice , Chêne Laurent , Nugeyre Marie-Thérèse , Braun Joséphine , Barré-Sinoussi Françoise , Israël Nicole
Published: December 2019

We report here that human immunodeficiency virus type 1 (HIV-1)-infected human thymocytes, in the absence of any exogenous stimulus but cocultivated with autologous thymic epithelial cells (TEC), obtained shortly (3 days) after thymus excision produce a high and sustained level of HIV-1 particles. The levels and kinetics of HIV-1 replication were similar for seven distinct viral strains irrespective of their phenotypes and genotypes. Contact of thymocytes with TEC is a critical requirement for optimal viral replication. Rather than an inductive signal resulting from the contact itself, soluble factors produced in the mixed culture are responsible for this effect. Specifically, the synergistic effects of tumor necrosis factor, interleukin-1 (IL-1), IL-6, and granulocyte-macrophage colony-stimulating factor may account by themselves for the high level of HIV-1 replication in thymocytes observed in mixed cultures. In conclusion, the microenvironment generated by TEC-thymocyte interaction might greatly favor optimal HIV-1 replication in the thymus.
Functional Analysis of the Core Human Immunodeficiency Virus Type 1 Packaging Signal in a Permissive Cell Line
Authors: Harrison Geoffrey P. , Miele Gino , Hunter Eric , Lever Andrew M. L.
Published: December 2019

Packaging of type C retrovirus genomic RNAs into budding virions requires a highly specific interaction between the viral Gag precursor and unique
cis
-acting packaging signals on the full-length RNA genome, allowing the selection of this RNA species from among a pool of spliced viral RNAs and similar cellular RNAs. This process is thought to involve RNA secondary and tertiary structural motifs since there is little conservation of the primary sequence of this region between retroviruses. To confirm RNA secondary structures, which we and others have predicted for this region, disruptive, compensatory, and deletion mutations were introduced into proviral constructs, which were then assayed in a permissive cell line. Disruption of either of two predicted stem-loops was found to greatly reduce RNA encapsidation and replication, whereas compensatory mutations restoring base pairing to these stem-loops had a wild-type phenotype. A GGNGR motif was identified in the loops of three hairpins in this region. Results were consistent with the hypothesis that the process of efficient RNA encapsidation is linked to dimerization. Replication and encapsidation were shown to occur at a reduced rate in the absence of the previously described kissing hairpin motif.
Antiapoptotic Activity of the Herpesvirus Saimiri-Encoded Bcl-2 Homolog: Stabilization of Mitochondria and Inhibition of Caspase-3-Like Activity
Authors: Derfuss Tobias , Fickenscher Helmut , Kraft Michael S. , Henning Golo , Lengenfelder Doris , Fleckenstein Bernhard , Meinl Edgar
Published: December 2019

Viruses have evolved different strategies to interfere with host cell apoptosis. Herpesvirus saimiri (HVS) and other lymphotropic herpesviruses code for proteins that are homologous to the cellular antiapoptotic Bcl-2. In this study HVS-Bcl-2 was stably expressed in the human leukemia cell line Jurkat and in the murine T-cell hybridoma DO to assess its antiapoptotic spectrum and to gain further insight into its mode of action. HVS- Bcl-2 prevented apoptosis that occurs as a result of a disturbance of intracellular homeostasis by, for example, DNA damage or menadione, which gives rise to oxygen radicals. In Jurkat cells, HVS-Bcl-2 also inhibited apoptosis mediated by the death receptor CD95. In DO cells, HVS-Bcl-2 did not interfere with CD95-mediated apoptosis but blocked dexamethasone-induced cell death. Mitochondrial damage is a central coordinating event in apoptosis induced by different stimuli. To assess the integrity of mitochondria, we used rhodamine 123, which is released upon disturbance of the mitochondrial membrane potential, and determined the release of cytochromecinto the cytosol. Both signs of mitochondrial damage were prevented by HVS-Bcl-2. This viral protein also inhibited the generation of caspase-3-like DEVDase activity and blocked the cleavage of poly(ADP-ribose) polymerase, a natural substrate of caspase-3-like proteases. In conclusion, HVS-Bcl-2 protects against a great variety of apoptotic stimuli, stabilizes mitochondria, and acts upstream of the generation of caspase-3-like activity.
AR1 Is an Integral Part of the Adenovirus Type 2 E1A-CR3 Transactivation Domain
Authors: Ström Anne-Christine , Ohlsson Petra , Akusjärvi Göran
Published: December 2019

We have previously shown that the nonconserved carboxy-terminal exon of the adenovirus type 2 E1A-289R protein contains two interchangeable sequence elements, auxiliary region (AR) 1 and AR2, that are required for efficient CR3-mediated transcriptional activation of the viral E4 promoter (M. Bondesson, C. Svensson, S. Linder, and G. Akusjärvi, EMBO J. 11:3347–3354, 1992). Here we show that CR3-mediated transactivation of all adenovirus early promoters and the HSP70 promoter requires the AR1 element. We further show that AR2 can substitute for AR1 only when artificially juxtaposed to CR3. AR1 consists of six tandem glutamic acid-proline (EP) repeats and is positioned immediately downstream of CR3. Genetic dissection of AR1 showed that the number of EP repeats in AR1 is critical for CR3 function. Thus, reducing or increasing the number of EP repeats reduces the CR3 transactivation capacity. Furthermore, the introduction of amino acid substitutions into AR1 suggested that the net negative charge in AR1 is of critical importance for its function as an enhancer of CR3-mediated transcriptional activation. Using an in vitro binding approach, we showed that the AR1 element is not part of the CR3 promoter localization signal mediating contact with the Sp1, ATF-2, or c-Jun upstream-binding transcription factors. Previous studies have suggested that the 49-amino-acid sequence constituting CR3 represents the minimal domain required for E1A-induced activation of viral early promoters. Since AR1 was required for efficient CR3-mediated transcriptional activation of all tested promoters, we suggest that the carboxy-terminal boundary for the CR3 transactivation domain should be extended to include the AR1 element.
Interaction of the Human Immunodeficiency Virus Type 1 Vpr Protein with the Nuclear Pore Complex
Authors: Fouchier Ron A. M. , Meyer Barbara E. , Simon James H. M. , Fischer Utz , Albright Andrew V. , González-Scarano Francisco , Malim Michael H.
Published: December 2019

The Vpr protein of human immunodeficiency virus type 1 (HIV-1) performs a number of functions that are associated with the nucleus. Vpr enhances the nuclear import of postentry viral nucleoprotein complexes, arrests proliferating cells in the G
2
phase of the cell cycle, and acts as a modest transcriptional activator. For this paper, we have investigated the nuclear import of Vpr. Although Vpr does not encode a sequence that is recognizable as a nuclear localization signal (NLS), Vpr functions as a transferable NLS both in somatic cells and in
Xenopus laevis
oocytes. In certain contexts, Vpr also mediates substantial accumulation at the nuclear envelope and, in particular, at nuclear pore complexes (NPCs). Consistent with this, Vpr is shown to interact specifically with nucleoporin phenylalanine-glycine (FG)-repeat regions. These findings not only demonstrate that Vpr harbors a bona fide NLS but also raise the possibility that one (or more) of Vpr’s functions may take place at the NPC.
Exclusive and Persistent Use of the Entry Coreceptor CXCR4 by Human Immunodeficiency Virus Type 1 from a Subject Homozygous for
CCR5
Δ32
Authors: Michael Nelson L. , Nelson Julie A. E. , KewalRamani Vineet N. , Chang George , O’Brien Stephen J. , Mascola John R. , Volsky Barbara , Louder Mark , White Gilbert C. , Littman Dan R. , Swanstrom Ronald , O’Brien Thomas R.
Published: December 2019

Individuals who are homozygous for the 32-bp deletion in the gene coding for the chemokine receptor and major human immunodeficiency virus type 1 (HIV-1) coreceptor CCR5 (
CCR5
−/−) lack functional cell surface CCR5 molecules and are relatively resistant to HIV-1 infection. HIV-1 infection in
CCR5
−/− individuals, although rare, has been increasingly documented. We now report that the viral quasispecies from one such individual throughout disease is homogenous, T cell line tropic, and phenotypically syncytium inducing (SI); exclusively uses CXCR4; and replicates well in
CCR5
−/− primary T cells. The recently discovered coreceptors BOB and Bonzo are not used. Although early and persistent SI variants have been described in longitudinal studies, this is the first demonstration of exclusive and persistent CXCR4 usage. With the caveat that the earliest viruses available from this subject were from approximately 4 years following primary infection, these data suggest that HIV-1 infection can be mediated and persistently maintained by viruses which exclusively utilize CXCR4. The lack of evolution toward the available minor coreceptors in this subject underscores the dominant biological roles of the major coreceptors CCR5 and CXCR4. This and two similar subjects (R. Biti, R. Ffrench, J. Young, B. Bennetts, G. Stewart, and T. Liang, Nat. Med. 3:252–253, 1997; I. Theodoreu, L. Meyer, M. Magierowska, C. Katlama, and C. Rouzioux, Lancet 349:1219–1220, 1997) showed relatively rapid CD4
+
T-cell declines despite average or low initial viral RNA load. Since viruses which use CXCR4 exclusively cannot infect macrophages, these data have implications for the relative infection of the T-cell compartment versus the macrophage compartment in vivo and for the development of CCR5-based therapeutics.
Role of Individual T-Cell Epitopes of Theiler’s Virus in the Pathogenesis of Demyelination Correlates with the Ability To Induce a Th1 Response
Authors: Yauch Robert L. , Palma JoAnn P. , Yahikozawa Hiroyuki , Koh Chang-Sung , Kim Byung S.
Published: December 2019

Intracerebral inoculation of susceptible strains of mice with Theiler’s murine encephalomyelitis virus (TMEV) results in immune-mediated demyelination. Three major T-cell epitopes have previously been identified within the VP1 (VP1233–250), VP2 (VP274–86), and VP3 (VP324–37) capsid proteins in virus-infected SJL/J mice. These epitopes appear to account for the majority (∼90%) of major histocompatibility complex class II-restricted T-cell responses to TMEV. Interestingly, the effect of immunization with synthetic peptides bearing the predominant T-cell epitopes on the course of TMEV-induced demyelination indicates that T cells reactive to the VP1 and VP2 epitopes, but not VP3, accelerate the pathogenesis of demyelination. The predominant pathogenic role of the T cells is verified by similar immunization with the fusion proteins containing the entire individual capsid proteins. The order of appearance and level of T cells specific for the individual epitopes during the course of demyelination are similar to each other. However, cytokine profiles of T cells from virus-infected mice indicate that T cells specific for the VP1 (and perhaps the VP2) epitope are Th1, whereas T cells reactive to VP3 are primarily Th2. These results suggest that Th1-type cells specific for VP1 and VP2 are involved in the pathogenesis of viral demyelination induced by TMEV. Thus, a predominance of Th1-inducing viral epitopes is likely critical for the pathogenesis of demyelination.
Woodchuck Hepatitis Virus Enhancer I and Enhancer II Are Both Involved in N-
myc2
Activation in Woodchuck Liver Tumors
Authors: Flajolet Marc , Tiollais Pierre , Buendia Marie-Annick , Fourel Geneviève
Published: December 2019

Direct activation of the N-
myc2
oncogene by insertion of woodchuck hepatitis virus (WHV) DNA is a major oncogenic step in woodchuck hepatocarcinogenesis. We previously reported that WHV enhancer II (We2), which controls expression of the core/pregenome RNA, can also activate the N-
myc2
promoter in hepatoma cell lines. To better define the integrated WHV regulatory sequences responsible for N-
myc2
promoter activation in woodchuck liver tumors, we analyzed the structure and enhancer activity of a single viral integrant found at the
win
locus in tumor 2260T1 and mapping approximately 175 kb 3′ of N-
myc2
. This viral insert was made of 11 concatemerized WHV fragments, 5 of which overlapped with We2 sequences and 1 with WHV sequence homologous to that of hepatitis B virus enhancer I (We1). In transient transfection assays in hepatoma-derived cells, the We2 activator was found to be fully effective only when inserted in close proximity to the N-
myc2
promoter whereas the We1 element by itself was apparently devoid of activity. In contrast, the 2260T1 viral insert exhibited a potent enhancer capacity that depended both on multimerized We2 and on We1 sequences. In a survey of different woodchuck hepatomas, both elements were commonly found within integrated viral sequences involved in long-range N-
myc2
activation.
The NS5A/NS5 Proteins of Viruses from Three Genera of the Family
Flaviviridae
Are Phosphorylated by Associated Serine/Threonine Kinases
Authors: Reed Karen E. , Gorbalenya Alexander E. , Rice Charles M.
Published: December 2019

Phosphorylation of the expressed NS5A protein of hepatitis C virus (HCV), a member of the
Hepacivirus
genus of the family
Flaviviridae
, has been demonstrated in mammalian cells and in a cell-free assay by an associated kinase activity. In this report, phosphorylation is also shown for the NS5A and NS5 proteins, respectively, of bovine viral diarrhea virus (BVDV) and yellow fever virus (YF), members of the other two established genera in this family. Phosphorylation of BVDV NS5A and YF NS5 was observed in infected cells, transient expression experiments, and a cell-free assay similar to the one developed for HCV NS5A. Phosphoamino acid analyses indicated that all three proteins were phosphorylated by serine/threonine kinases. Similarities in the properties of BVDV NS5A, YF NS5, and HCV NS5A phosphorylation in vitro further suggested that closely related kinases or the same kinase may phosphorylate these viral proteins. Conservation of this trait among three quite distantly related viruses representing three separate genera suggests that phosphorylation of the NS5A/NS5 proteins or their association with cellular kinases may play an important role in the flavivirus life cycle.
Effects of Substituting Granulin or a Granulin-Polyhedrin Chimera for Polyhedrin on Virion Occlusion and Polyhedral Morphology in
Autographa californica
Multinucleocapsid Nuclear Polyhedrosis Virus
Authors: Eason Jane E. , Hice Robert H. , Johnson Jeffrey J. , Federici Brian A.
Published: December 2019

Substitution of granulin from the
Trichoplusia ni
granulosis virus (TnGV) for polyhedrin of the
Autographa californica
multinucleocapsid nuclear polyhedrosis virus (Ac
M
NPV) yielded a few very large (2 to 5 μm) cuboidal inclusions in the cytoplasm and nucleus of infected cells. These polyhedra lacked the beveled edges characteristic of wild-type Ac
M
NPV polyhedra, contained fractures, and occluded few virions. Placing a nuclear localization signal (KRKK) in granulin directed more granulin to the nucleus and resulted in more structurally uniform cuboidal inclusions in which no virions were observed. A granulin-polyhedrin chimera produced tetrahedral occlusions with more virions than granulin inclusions but many fewer than wild-type polyhedra. Despite the unusual structure of the granulin and granulin-polyhedrin inclusions, they interacted with Ac
M
NPV p10 fibrillar structures and electron-dense spacers that are precursors of the polyhedral calyx. The change in inclusion shape obtained with the granulin-polyhedrin chimera demonstrates that the primary amino acid sequence affects occlusion body shape, but the large cuboidal inclusions formed by granulin indicate that the amino acid sequence is not the only determinant. The failure of granulin or the granulin-polyhedrin chimera to properly occlude Ac
M
NPV virions suggests that specific interactions occur between polyhedrin and other viral proteins which facilitate normal virion occlusion and occlusion body assembly and shape in baculoviruses.
The Herpesvirus Transactivator VP16 Mimics a Human Basic Domain Leucine Zipper Protein, Luman, in Its Interaction with HCF
Authors: Lu Rui , Yang Ping , Padmakumar Sharmila , Misra Vikram
Published: December 2019

In human cells infected with herpes simplex virus (HSV), viral gene expression is initiated by the virion protein VP16. VP16 does not bind DNA directly but forms a multiprotein complex on the viral immediate-early gene promoters with two cellular proteins: the POU domain protein Oct-1 and host cell factor (HCF; also called C1, VCAF, and CFF). Despite its apparent role in stabilizing the VP16-induced transcription complex, the natural biological role of HCF is unclear. Only recently HCF has been implicated in control of the cell cycle. To determine the role of HCF in cells and answer why HSV has evolved an HCF-dependent mechanism for the initiation of the lytic cycle, we identified the first human ligand for HCF (R. Lu et al., Mol. Cell. Biol. 17:5117–5126, 1997). This protein, Luman, is a member of the CREB/ATF family of transcription factors that can activate transcription from promoters containing cyclic AMP response elements (CRE). Here we provide evidence that Luman and VP16 share two important structural features: an acidic activation domain and a common mechanism for binding HCF. We found that Luman, its homolog in
Drosophila
, dCREB-A (also known as BBF-2), and VP16 bind to HCF by a motif, (D/E)HXY(S/A), present in all three proteins. In addition, a mutation (P134S) in HCF that prevents VP16 binding also abolishes its binding to Luman and dCREB-A. We also show that while interaction with HCF is not required for the ability of Luman to activate transcription when tethered to the GAL4 promoter, it appears to be essential for Luman to activate transcription through CRE sites. These data suggest that the HCF-Luman interaction may represent a conserved mechanism for transcriptional regulation in metazoans, and HSV mimics this interaction with HCF to monitor the physiological state of the host cell.
Cellular Components Interact with Adenovirus Type 5 Minimal DNA Packaging Domains
Authors: Schmid Susanne I. , Hearing Patrick
Published: December 2019

Adenovirus type 5 DNA packaging is initiated from the left end of the viral genome and depends on the presence of a
cis
-acting packaging domain located between nucleotides 194 and 380. Multiple redundant packaging elements (termed A repeats I through VII [AI through AVII]) are contained within this domain and display differential abilities to support DNA packaging in vivo. The functionally most important repeats, AI, AII, AV, and AVI, follow a bipartite consensus motif exhibiting AT-rich and CG-rich core sequences. Results from previous mutational analyses defined a fragment containing AV, AVI, and AVII as a minimal packaging domain in vivo, which supports a functional independence of the respective
cis
-acting sequences. Here we describe multimeric versions of individual packaging elements as minimal packaging domains that can confer viability and packaging activity to viruses carrying gross truncations within their left end. These mutant viruses directly rate the functional role that different packaging elements play relative to each other. The A repeats are likely to be binding sites for limiting,
trans
-acting packaging factors of cellular and/or viral origin. We report here the characterization of two cellular binding activities interacting with all of the minimal packaging domains in vitro, an unknown binding activity termed P-complex, and the transcription factor chicken ovalbumin upstream promoter transcription factor. The binding of both activities is dependent on the integrity of the AT-rich, but not the CG-rich, consensus half site. In the case of P-complex, binding affinity for different minimal packaging domains in vitro correlates well with their abilities to support DNA packaging in vivo. Interestingly, P-complex interacts not only with packaging elements but also with the left terminus of the viral genome, the core origin of replication. Our data implicate cellular factors as components of the viral packaging machinery. The dual binding specificity of P-complex for packaging and replication sequences may further suggest a direct involvement of left-end replication sequences in viral DNA encapsidation.
Binding of Human Immunodeficiency Virus Type 1 to CD4 and CXCR4 Receptors Differentially Regulates Expression of Inflammatory Genes and Activates the MEK/ERK Signaling Pathway
Authors: Popik Waldemar , Hesselgesser Joseph E. , Pitha Paula M.
Published: December 2019

We have previously shown that binding of human immunodeficiency virus type 1 (HIV-1) virions to CD4 receptors stimulates association of Lck with Raf-1 and results in the activation of Raf-1 kinase in a Ras-independent manner. In the present study, we demonstrate that HIV-1 envelope glycoproteins of both T-cell-tropic and macrophagetropic strains rapidly activate the ERK/mitogen-activated protein (MAP) kinase pathway and the binding of nuclear transcription factors (AP-1, NF-κB, and C/EBP) and stimulate expression of cytokine and chemokine genes. The activation of this signaling pathway requires functional CD4 receptors and is independent of binding to CXCR4. Binding of the natural ligand stromal cell-derived factor 1 (SDF-1) to CXCR4, which inhibits entry of T-cell-tropic HIV-1, activates also the ERK/MAP kinase pathway. However, SDF-1 did not affect the CD4-mediated expression of cytokine and chemokine genes. These results provide firm molecular evidence that binding of HIV-1 envelope glycoproteins to CD4 receptor initiates a signaling pathway(s) independent of the binding to the chemokine receptor that leads to the aberrant expression of inflammatory genes and may contribute significantly to HIV-1 replication as well as to deregulation of the immune system.
Naturally Occurring Human Immunodeficiency Virus Type 1 Long Terminal Repeats Have a Frequently Observed Duplication That Binds RBF-2 and Represses Transcription
Authors: Estable Mario Clemente , Bell Brendan , Hirst Martin , Sadowski Ivan
Published: December 2019

Approximately 38% of human immunodeficiency virus type 1 (HIV-1)-infected patients within the Vancouver Lymphadenopathy-AIDS Study have proviruses bearing partial 15- to 34-nucleotide duplications upstream of the NF-κB binding sites within the 5′ long terminal repeat (LTR). This most frequent naturally occurring length polymorphism (MFNLP) of the HIV-1 5′ LTR encompasses potential binding sites for several candidate transcription factors, including TCF-1α/hLEF, c-Ets, AP-4, and Ras-responsive binding factor 2 (RBF-2) (M. C. Estable et al., J. Virol. 70:4053–4062, 1996). RBF-2 and an apparently related factor, RBF-1, bind to at least four
cis
elements within the LTR which are required for full transcriptional responsiveness to protein-tyrosine kinases and v-Ras (B. Bell and I. Sadowski, Oncogene 13:2687–2697, 1996). Here we demonstrate that representative MFNLPs from two patients specifically bind RBF-2. In both cases, deletion of the MFNLP caused elevated LTR-directed transcription in cells expressing RBF-2 but not in cells with undetectable RBF-2. RBF-1, but not RBF-2, appears to contain the Ets transcription factor family member GABPα/GABPβ1. Taken together with the fact that every MFNLP from a comparative study of over 500 LTR sequences from 42 patients contains a predicted binding site for RBF-2, our data suggest that the MFNLP is selected in vivo because it provides a duplicated RBF-2
cis
element, which may limit transcription in monocytes and activated T cells.
Involvement of Aminopeptidase N (CD13) in Infection of Human Neural Cells by Human Coronavirus 229E
Authors: Lachance Claude , Arbour Nathalie , Cashman Neil R. , Talbot Pierre J.
Published: December 2019

Attachment to a cell surface receptor can be a major determinant of virus tropism. Previous studies have shown that human respiratory coronavirus HCV-229E uses human aminopeptidase N (hAPN [CD13]) as its cellular receptor for infection of lung fibroblasts. Although human coronaviruses are recognized respiratory pathogens, occasional reports have suggested their possible neurotropism. We have previously shown that human neural cells, including glial cells in primary cultures, are susceptible to human coronavirus infection in vitro (A. Bonavia, N. Arbour, V. W. Yong, and P. J. Talbot, J. Virol. 71:800–806, 1997). However, the only reported expression of hAPN in the nervous system is at the level of nerve synapses. Therefore, we asked whether hAPN is utilized as a cellular receptor for infection of these human neural cell lines. Using flow cytometry, we were able to show the expression of hAPN on the surfaces of various human neuronal and glial cell lines that are susceptible to HCV-229E infection. An hAPN-specific monoclonal antibody (WM15), but not control antibody, inhibited the attachment of radiolabeled HCV-229E to astrocytic, neuronal, and oligodendrocytic cell lines. A correlation between the apparent amount of cell surface hAPN and the level of virus attachment was observed. Furthermore, the presence of WM15 inhibited virus infection of these cell lines, as detected by indirect immunofluorescence. These results indicate that hAPN (CD13) is expressed on neuronal and glial cell lines in vitro and serves as the receptor for infection by HCV-229E. This further strengthens the neurotropic potential of this human respiratory virus.
Changing the Site of Initiation of Plus-Strand DNA Synthesis Inhibits the Subsequent Template Switch during Replication of a Hepadnavirus
Authors: Loeb Daniel D. , Tian Ru , Gulya Karolyn J. , Qualey Amy E.
Published: December 2019

Unique to hepadnavirus reverse transcription is the process of primer translocation, in which the RNA primer for the initiation of plus-strand DNA synthesis is generated at one site on its template, DR1, and is moved to a new site, DR2. For duck hepatitis B virus (DHBV), DR2 is located within 50 nucleotides of the 5′ end of the minus-strand DNA template. When the synthesis of plus-strand DNA proceeds to the 5′ terminus of the minus strand, the 3′ end of the minus strand becomes the template for DNA synthesis. This switch in templates circularizes the nascent genome and is required for the genesis of the relaxed circular form of the DNA and the mature capsid. Maturation of the capsid is a prerequisite for virus egress. We have analyzed a series of DHBV variants in which plus-strand DNA synthesis was initiated from a new position relative to the 5′ end of the template. For these variants, the subsequent circularization was inhibited. We found that when the number of nucleotides between the site of initiation of plus-strand DNA synthesis and the 5′ end of its template was restored to 54 nucleotides, circularization was substantially restored. These results mean that the process of circularization is influenced by the earlier steps in DNA replication. This sensitivity is consistent with the notion that this region of the nascent genome is in a dynamic structure that is crucial for successful DNA replication.
Involvement of Human CRM1 (Exportin 1) in the Export and Multimerization of the Rex Protein of Human T-Cell Leukemia Virus Type 1
Authors: Hakata Yoshiyuki , Umemoto Tomoe , Matsushita Shuzo , Shida Hisatoshi
Published: December 2019

We investigated the role of human CRM1 (hCRM1) (exportin 1) in the function of Rex protein encoded by human T-cell leukemia virus type 1. hCRM1 promoted the export of Rex protein from the nucleus to the cytoplasm. A Rex protein with a mutation in the activation domain, RexM90, lost both the ability to bind to hCRM1 and the ability to multimerize. The overexpression of hCRM1 complemented the functional defects of RexM64, which contains a mutation in the multimerization domain of Rex. A dominant-negative mutant of Rex which sequesters cofactors of Rex abrogated multimerization as well as the activity of the wild-type Rex protein. These two functions were simultaneously restored by the overexpression of hCRM1. Taken together, these results suggest that hCRM1 plays important roles in the multimerization and export of Rex protein.
Protective Role of Gamma Interferon during the Recall Response to Influenza Virus
Authors: Bot Adrian , Bot Simona , Bona Constantin A.
Published: December 2019

During secondary immune responses to influenza virus, virus-specific T memory cells are a major source of gamma interferon (IFN-γ). We assessed the contribution of IFN-γ to heterologous protection against the A/WSN/33 (H1N1) virus of wild-type and IFN-γ−/− mice previously immunized with the A/HK/68 (H3N2) virus. The IFN-γ−/− mice displayed significantly reduced survival rates subsequent to a challenge with various doses of the A/WSN/33 virus. This was associated with an impaired ability of the IFN-γ−/− mice to completely clear the pulmonary virus by day 7 after the challenge, although significant reduction of the virus titers was noted. However, the IFN-γ−/− mice developed type A influenza virus cross-reactive cytotoxic T lymphocytes (CTLs) similar to the wild-type mice, as demonstrated by both cytotoxicity and a limiting-dilution assay for the estimation of CTL precursor frequency. The pulmonary recruitment of T cells in IFN-γ−/− mice was not dramatically affected, and the percentage of CD4+and CD8+T cells was similar to that of wild-type mice. The T cells from IFN-γ−/− mice did not display a significant switch toward a Th2 profile. Furthermore, the IFN-γ−/− mice retained the ability to mount significant titers of WSN and HK virus-specific hemagglutination-inhibiting antibodies. Together, these results are consistent with a protective role of IFN-γ during the heterologous response against influenza virus independently of the generation and local recruitment of cross-reactive CTLs.