bluuebunny/crossref_metadata_2025_split huggingface dataset:
Analysis of the Assembly Function of the Human Immunodeficiency Virus Type 1 Gag Protein Nucleocapsid Domain
Authors: Zhang Yaqiang , Qian Haoyu , Love Zachary , Barklis Eric 
Published: December 2019

ABSTRACT

Previous studies have shown that in addition to its function in specific RNA encapsidation, the human immunodeficiency virus type 1 (HIV-1) nucleocapsid (NC) is required for efficient virus particle assembly. However, the mechanism by which NC facilitates the assembly process is not clearly established. Formally, NC could act by constraining the Pr55

gag

polyprotein into an assembly-competent conformation or by masking residues which block the assembly process. Alternatively, the capacity of NC to bind RNA or make interprotein contacts might affect particle assembly. To examine its role in the assembly process, we replaced the NC domain in Pr55

gag

with polypeptide domains of known function, and the chimeric proteins were analyzed for their abilities to direct the release of virus-like particles. Our results indicate that NC does not mask inhibitory domains and does not act passively, by simply providing a stable folded monomeric structure. However, replacement of NC by polypeptides which form interprotein contacts permitted efficient virus particle assembly and release, even when RNA was not detected in the particles. These results suggest that formation of interprotein contacts by NC is essential to the normal HIV-1 assembly process.
Characterization of an Adenovirus Vector Containing a Heterologous Peptide Epitope in the HI Loop of the Fiber Knob
Authors: Krasnykh Victor , Dmitriev Igor , Mikheeva Galina , Miller C. Ryan , Belousova Natalya , Curiel David T. 
Published: December 2019

ABSTRACT
The utility of the present generation of recombinant adenovirus vectors for gene therapy applications could potentially be improved by designing targeted vectors capable of gene delivery to selected cell types in vivo. In order to achieve such targeting, we are investigating the possibilities of incorporation of ligands in the adenovirus fiber protein, which mediates primary binding of adenovirus to its cell surface receptor. Based on the proposed structure of the cell-binding domain of the fiber, we hypothesized that the HI loop of the fiber knob can be utilized as a convenient locale for incorporation of heterologous ligands. In this study, we utilized recombinant fiber proteins expressed in baculovirus-infected insect cells to demonstrate that the incorporation of the FLAG octapeptide into the HI loop does not ablate fiber trimerization and does not disturb formation of the cell-binding site localized in the knob. We then generated a recombinant adenovirus containing this modified fiber and showed that the short peptide sequence engineered in the knob is compatible with the biological functions of the fiber. In addition, by using a ligand-specific antibody, we have shown that the peptide incorporated into the knob remains available for binding in the context of mature virions containing modified fibers. These findings suggest that heterologous ligands can be incorporated into the HI loop of the fiber knob and that this locale possesses properties consistent with its employment in adenovirus retargeting strategies.
Lymphocyte Apoptosis during Classical Swine Fever: Implication of Activation-Induced Cell Death
Authors: Summerfield Artur , Knötig Sonja M. , McCullough Kenneth C. 
Published: December 2019

ABSTRACTInfection of pigs with classical swine fever virus (CSFV), a member of theFlaviviridaefamily, causes a severe leukopenia, particularly notable with the lymphocytes. The goal of this study was to analyze mechanisms behind this CSFV-induced lymphopenia. To this end, the kinetics of leukocyte depletion, the appearance of apoptotic cells, and virus infection of leukocytes after infection of pigs with the virulent CSFV strain Brescia were analyzed. Depletion of B and T lymphocytes was noted as early as 1 day postinfection (p.i.). Circulating viable lymphocytes with reduced mitochondrial transmembrane potential—a particular early marker for apoptosis—were also detectable as early as 1 day p.i. When isolated peripheral blood mononuclear cells were cultured for 6 h, significantly more sub-G1cells with reduced DNA content were detected among the lymphocytes from CSFV-infected animals, again as early as 1 to 3 days p.i. The first time virus was first found in the plasma, as well as infection of leukocytes, was 3 days p.i. However, throughout the observation time of 1 week, <3% of the circulating leukocytes and no lymphocytes contained virus or viral antigen. Further analysis of the T lymphocytes from infected animals demonstrated an increase in CD49d, major histocompatibility complex class II, and Fas expression. An increased susceptibility to apoptosis in vitro was also observed, particularly after addition of concanavalin A as well as apoptosis-inducing anti-Fas antibody to the cultures. Taken together, these results imply that activation-induced programmed cell death was the mechanism behind lymphopenia during classical swine fever.
Evidence that Antibody-Mediated Neutralization of Human Immunodeficiency Virus Type 1 by Sera from Infected Individuals Is Independent of Coreceptor Usage
Authors: Montefiori David C. , Collman Ronald G. , Fouts Timothy R. , Zhou Ji Ying , Bilska Miroslawa , Hoxie James A. , Moore John P. , Bolognesi Dani P. 
Published: December 2019

ABSTRACTHuman immunodeficiency virus type 1 (HIV-1) uses a variety of chemokine receptors as coreceptors for virus entry, and the ability of the virus to be neutralized by antibody may depend on which coreceptors are used. In particular, laboratory-adapted variants of the virus that use CXCR4 as a coreceptor are highly sensitive to neutralization by sera from HIV-1-infected individuals, whereas primary isolates that use CCR5 instead of, or in addition to, CXCR4 are neutralized poorly. To determine whether this dichotomy in neutralization sensitivity could be explained by differential coreceptor usage, virus neutralization by serum samples from HIV-1-infected individuals was assessed in MT-2 cells, which express CXCR4 but not CCR5, and in mitogen-stimulated human peripheral blood mononuclear cells (PBMC), where multiple coreceptors including CXCR4 and CCR5 are available for use. Our results showed that three of four primary isolates with a syncytium-inducing (SI) phenotype and that use CXCR4 and CCR5 were neutralized poorly in both MT-2 cells and PBMC. The fourth isolate, designated 89.6, was more sensitive to neutralization in MT-2 cells than in PBMC. We showed that the neutralization of 89.6 in PBMC was not improved when CCR5 was blocked by having RANTES, MIP-1α, and MIP-1β in the culture medium, indicating that CCR5 usage was not responsible for the decreased sensitivity to neutralization in PBMC. Consistent with this finding, a laboratory-adapted strain of virus (IIIB) was significantly more sensitive to neutralization in CCR5-deficient PBMC (homozygous Δ32-CCR5 allele) than were two of two SI primary isolates tested. The results indicate that the ability of HIV-1 to be neutralized by sera from infected individuals depends on factors other than coreceptor usage.
Comparative Analyses of Intracellularly Expressed Antisense RNAs as Inhibitors of Human Immunodeficiency Virus Type 1 Replication
Authors: Veres Gabor , Junker Uwe , Baker Jenny , Barske Carmen , Kalfoglou Creton , Ilves Heini , Escaich Sonia , Kaneshima Hideto , Böhnlein Ernst 
Published: December 2019

ABSTRACT

The antiviral activities of intracellularly expressed antisense RNAs complementary to the human immunodeficiency virus type 1 (HIV-1)
pol
,
vif
, and
env
genes and the 3′ long terminal repeat (LTR) sequence were evaluated in this comparative study. Retroviral vectors expressing the antisense RNAs as part of the Moloney murine leukemia virus LTR promoter-directed retroviral transcript were constructed. The CD4
+
T-cell line CEM-SS was transduced with retroviral constructs, and Northern blot analyses showed high steady-state antisense RNA expression levels. The most efficient inhibition of HIV-1 replication was observed with the
env
antisense RNA, followed by the
pol
complementary sequence, leading to 2- to 3-log
10
reductions in p24 antigen production even at high inoculation doses (4 × 10
4
50% tissue culture infective doses) of the HIV-1 strain HXB3. The strong antiviral effect correlated with a reduction of HIV-1 steady-state RNA levels, and with intracellular Tat protein production, suggesting that antisense transcripts act at an early step of HIV-1 replication. A lower steady-state antisense RNA level was detected in transduced primary CD4
+
lymphocytes than in CEM-SS cells. Nevertheless, replication of the HIV-1 JR-CSF isolate was reduced with both the
pol
and
env
antisense RNA. Intracellularly expressed antisense sequences demonstrated more pronounced antiviral efficacy than the
trans
-dominant RevM10 protein, making these antisense RNAs a promising gene therapy strategy for HIV-1.
Mapping the Interacting Domains between the Rabies Virus Polymerase and Phosphoprotein
Authors: Chenik M. , Schnell M. , Conzelmann K. K. , Blondel D. 
Published: December 2019

ABSTRACT
The RNA polymerase of rabies virus consists of two subunits, the large (L) protein and the phosphoprotein (P), with 2,127 and 297 amino acids, respectively. When these proteins were coexpressed via the vaccinia virus-T7 RNA polymerase recombinant in mammalian cells, they formed a complex as detected by coimmunoprecipitation. Analysis of P and L deletion mutants was performed to identify the regions of both proteins involved in complex formation. The interaction of P with L was not disrupted by large deletions removing the carboxy-terminal half of the P protein. On the contrary, P proteins containing a deletion in the amino terminus were defective in complex formation with L. Moreover, fusion proteins containing the 19 or the 52 first residues of P in frame with green fluorescent protein (GFP) still bound to L. These results indicate that the major L binding site resides within the 19 first residues of the P protein. We also mapped the region of L involved in the interaction with P. Mutant L proteins consisting of the carboxy-terminal 1,656, 956, 690, and 566 amino acids all bound to the P protein, whereas deletion of 789 residues within the terminal region eliminated binding to P protein. This result demonstrates that the carboxy-terminal domain of L is required for the interaction with P.
Mutational Analysis of the Virus and Monoclonal Antibody Binding Sites in MHVR, the Cellular Receptor of the Murine Coronavirus Mouse Hepatitis Virus Strain A59
Authors: Wessner David R. , Shick Paul C. , Lu Jin-Hua , Cardellichio Christine B. , Gagneten Sara E. , Beauchemin Nicole , Holmes Kathryn V. , Dveksler Gabriela S. 
Published: December 2019

ABSTRACTThe primary cellular receptor for mouse hepatitis virus (MHV), a murine coronavirus, is MHVR (also referred to as Bgp1aor C-CAM), a transmembrane glycoprotein with four immunoglobulin-like domains in the murine biliary glycoprotein (Bgp) subfamily of the carcinoembryonic antigen (CEA) family. Other murine glycoproteins in the Bgp subfamily, including Bgp1band Bgp2, also can serve as MHV receptors when transfected into MHV-resistant cells. Previous studies have shown that the 108-amino-acid N-terminal domain of MHVR is essential for virus receptor activity and is the binding site for monoclonal antibody (MAb) CC1, an antireceptor MAb that blocks MHV infection in vivo and in vitro. To further elucidate the regions of MHVR required for virus receptor activity and MAb CC1 binding, we constructed chimeras between MHVR and other members of the CEA family and tested them for MHV strain A59 (MHV-A59) receptor activity and MAb CC1 binding activity. In addition, we used site-directed mutagenesis to introduce selected amino acid changes into the N-terminal domains of MHVR and these chimeras and tested the abilities of these mutant glycoproteins to bind MAb CC1 and to function as MHV receptors. Several recombinant glycoproteins exhibited virus receptor activity but did not bind MAb CC1, indicating that the virus and MAb binding sites on the N-terminal domain of MHVR are not identical. Analysis of the recombinant glycoproteins showed that a short region of MHVR, between amino acids 34 and 52, is critical for MHV-A59 receptor activity. Additional regions of the N-terminal variable domain and the constant domains, however, greatly affected receptor activity. Thus, the molecular context in which the amino acids critical for MHV-A59 receptor activity are found profoundly influences the virus receptor activity of the glycoprotein.
The Cell Tropism of Human Immunodeficiency Virus Type 1 Determines the Kinetics of Plasma Viremia in SCID Mice Reconstituted with Human Peripheral Blood Leukocytes
Authors: Picchio Gastón R. , Gulizia Richard J. , Wehrly Kathy , Chesebro Bruce , Mosier Donald E. 
Published: December 2019

ABSTRACT

Most individuals infected with human immunodeficiency virus type 1 (HIV-1) initially harbor macrophage-tropic, non-syncytium-inducing (M-tropic, NSI) viruses that may evolve into T-cell-tropic, syncytium-inducing viruses (T-tropic, SI) after several years. The reasons for the more efficient transmission of M-tropic, NSI viruses and the slow evolution of T-tropic, SI viruses remain unclear, although they may be linked to expression of appropriate chemokine coreceptors for virus entry. We have examined plasma viral RNA levels and the extent of CD4
+
T-cell depletion in SCID mice reconstituted with human peripheral blood leukocytes following infection with M-tropic, dual-tropic, or T-tropic HIV-1 isolates. The cell tropism was found to determine the course of viremia, with M-tropic viruses producing sustained high viral RNA levels and sparing some CD4
+
T cells, dual-tropic viruses producing a transient and lower viral RNA spike and extremely rapid depletion of CD4
+
T cells, and T-tropic viruses causing similarly lower viral RNA levels and rapid-intermediate rates of CD4
+
T-cell depletion. A single amino acid change in the V3 region of gp120 was sufficient to cause one isolate to switch from M-tropic to dual-tropic and acquire the ability to rapidly deplete all CD4
+
T cells.
Construction and Characterization of a Temperature-Sensitive Human Immunodeficiency Virus Type 1 Reverse Transcriptase Mutant
Authors: Huang Mingjun , Zensen Ralf , Cho Michael , Martin Malcolm A. 
Published: December 2019

ABSTRACT

A temperature-sensitive (
ts
) human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) mutant was generated by charged-cluster-to-alanine mutagenesis. The mutant virus, containing three charged residues within the RT finger domain changed to alanine (K64A, K66A, and D67A), replicated normally at 34.5 but not 39.5°C. Quantitating virus particle production by p24 antigen capture or virion-associated RT activity and virus infectivity by the MAGI cell assay, we found that (i) mutant virions produced at the permissive temperature were indistinguishable from wild-type virus in assays performed at the nonpermissive temperature, suggesting that the
ts
mutation did not impair early steps in the virus replication cycle and that the mutant RT enzyme was not
ts
; and (ii) virus particle production in cells transfected with the
ts
mutant at the nonpermissive temperature was comparable to that of wild-type virus. However, the particle-associated RT activity and infectivity of mutant virions produced at the nonpermissive temperature were greatly reduced when assays were conducted at the permissive temperature. These results are consistent with an irreversible
ts
event affecting RT that occurs during virus particle production. Radioimmunoprecipitation analyses revealed that both p66 and p51 RT subunits were absent from mutant virions generated at 39.5°C. The presence of normal levels of HIV-1 integrase in mutant particles produced at the nonpermissive temperature was inconsistent with defective Gag-Pol synthesis or Gag-Pol incorporation into progeny virions. Furthermore, wild-type levels of the mutant Pr160

gag-pol

were detected in virions produced at the nonpermissive temperature when the HIV-1 protease was inactivated by site-specific mutagenesis. Taken together, these results are most consistent with a
ts
defect affecting the degradation or aberrant processing of the mutated RT during its processing/maturation within nascent particles.
A Putative α-Helical Structure Which Overlaps the Capsid-p2 Boundary in the Human Immunodeficiency Virus Type 1 Gag Precursor Is Crucial for Viral Particle Assembly
Authors: Accola Molly A. , Höglund Stefan , Göttlinger Heinrich G. 
Published: December 2019

ABSTRACT
The capsid (CA) and nucleocapsid domains of the human immunodeficiency virus type 1 Gag polyprotein are separated by the p2 spacer peptide, which is essential for virus replication. Previous studies have revealed that p2 has an important role in virus morphogenesis. In this paper, we show that a crucial assembly determinant maps to the highly conserved N terminus of p2, which is predicted to form part of an α-helix that begins in CA. A mutational analysis indicates that the ability of the N terminus of p2 to adopt an α-helical structure is essential for its function during virus assembly. To prevent CA-p2 processing, it was necessary to mutate both the CA-p2 cleavage site and an internal cleavage site within p2. Virions produced by the double mutant lacked a conical core shell and instead contained a thin electron-dense shell about 10 nm underneath the virion membrane. These results suggest that p2 is transiently required for proper assembly, but needs to be removed from the C terminus of CA to weaken CA-CA interactions and allow the rearrangement of the virion core shell during virus maturation.
Bovine Papillomavirus Type 1 Genomes and the E2 Transactivator Protein Are Closely Associated with Mitotic Chromatin
Authors: Skiadopoulos Mario H. , McBride Alison A. 
Published: December 2019

ABSTRACT
The bovine papillomavirus type 1 E2 transactivator protein is required for viral transcriptional regulation and DNA replication and may be important for long-term episomal maintenance of viral genomes within replicating cells (M. Piirsoo, E. Ustav, T. Mandel, A. Stenlund, and M. Ustav, EMBO J. 15:1–11, 1996). We have evidence that, in contrast to most other transcriptional transactivators, the E2 transactivator protein is associated with mitotic chromosomes in dividing cells. The shorter E2-TR and E8/E2 repressor proteins do not bind to mitotic chromatin, and the N-terminal transactivation domain of the E2 protein is necessary for the association. However, the DNA binding function of E2 is not required. We have found that bovine papillomavirus type 1 genomes are also associated with mitotic chromosomes, and we propose a model in which E2-bound viral genomes are transiently associated with cellular chromosomes during mitosis to ensure that viral genomes are segregated to daughter cells in approximately equal numbers.
Cytosolic Gag p24 as an Index of Productive Entry of Human Immunodeficiency Virus Type 1
Authors: Maréchal Valérie , Clavel François , Heard Jean Michel , Schwartz Olivier 
Published: December 2019

ABSTRACT
We have investigated the cellular uptake of Gag p24 shortly after exposure of cells to human immunodeficiency virus (HIV) particles. In the absence of envelope glycoprotein on virions or of viral receptors or coreceptors at the cell surface, p24 was incorporated in intracellular vesicles but not detected in the cytosolic subcellular fraction. When appropriate envelope-receptor interactions could occur, the nonspecific vesicular uptake was still intense and cytosolic p24 represented 10 to 40% of total intracellular p24. The measurement of cytosolic p24 early after exposure to HIV type 1 is a reliable assay for investigating virus entry and early events leading to authentic cell infection.
Insect Virus Proteins (FALPE and p10) Self-Associate To Form Filaments in Infected Cells
Authors: Alaoui-Ismaili Moulay Hicham , Richardson Christopher D. 
Published: December 2019

ABSTRACTEntomopoxviruses and baculoviruses are pathogens of insects which replicate in the cytoplasm and nuclei of their host cells, respectively. During the late stages of infection, both groups of viruses produce occlusion bodies which serve to protect virions from the external environment. Immunofluorescence and electron microscopy studies have shown that large bundles of filaments are associated with these occlusion bodies. Entomopoxviruses produce cytoplasmic fibrils which appear to be composed of the filament-associated late protein of entomopoxviruses (FALPE). Baculoviruses, on the other hand, yield filaments in the nuclei and cytoplasm of the infected cell which are composed of a protein called p10. Despite significant differences in their sequences, FALPE and p10 have similar hydrophilicity profiles, and each has a proline-rich stretch of amino acids at its carboxyl terminus. Evidence that FALPE and p10 could produce filaments in the absence of other viral proteins is presented. When FALPE was expressed in insect cells from a recombinant baculovirus, filaments similar to those produced by the wild-typeAmsacta mooreientomopoxvirus were observed. In addition, when expression plasmids containing FALPE or p10 genes were transfected into Vero monkey kidney cells, filament structures similar to those found in infected insect cells were produced. The manner in which FALPE and p10 subunits interact to form polymers was investigated through deletion and site-specific mutagenesis in conjunction with immunofluorescence microscopy, yeast two-hybrid protein interaction analysis, and chemical cross-linking of adjacent molecules. These studies indicated that the amino termini of FALPE and p10 were essential for subunit interaction. Although deletion of the carboxy termini did not affect this interaction, it did inhibit filament formation. In addition, modification of several potential sites for phosphorylation also abolished filament assembly. We concluded that although the sequences of FALPE and p10 were different, the structural and functional properties of the two polypeptides appeared to be similar.
Enhanced Virus Clearance by Early Inducible Lymphocytic Choriomeningitis Virus-Neutralizing Antibodies in Immunoglobulin-Transgenic Mice
Authors: Seiler Peter , Kalinke Ulrich , Rülicke Thomas , Bucher Etienne M. , Böse Christian , Zinkernagel Rolf M. , Hengartner Hans 
Published: December 2019

ABSTRACTFollowing infection of mice with lymphocytic choriomeningitis virus (LCMV), virus-neutralizing antibodies appear late, after 30 to 60 days. Such neutralizing antibodies play an important role in protection against reinfection. To analyze whether a neutralizing antibody response which developed earlier could contribute to LCMV clearance during the acute phase of infection, we generated transgenic mice expressing LCMV-neutralizing antibodies. Transgenic mice expressing the immunoglobulin μ heavy chain of the LCMV-neutralizing monoclonal antibody KL25 (H25 transgenic mice) mounted LCMV-neutralizing immunoglobulin M (IgM) serum titers within 8 days after infection. This early inducible LCMV-neutralizing antibody response significantly improved the host’s capacity to clear the infection and did not cause an enhancement of disease after intracerebral (i.c.) LCMV infection. In contrast, mice which had been passively administered LCMV-neutralizing antibodies and transgenic mice exhibiting spontaneous LCMV-neutralizing IgM serum titers (HL25 transgenic mice expressing the immunoglobulin μ heavy and the κ light chain) showed an enhancement of disease after i.c. LCMV infection. Thus, early-inducible LCMV-neutralizing antibodies can contribute to viral clearance in the acute phase of the infection and do not cause antibody-dependent enhancement of disease.