bluuebunny/crossref_metadata_2025_split huggingface dataset:
Herpes Simplex Virus gD and Virions Accumulate in Endosomes by Mannose 6-Phosphate-Dependent and -Independent Mechanisms
Authors: Brunetti Craig R. , Dingwell Kevin S. , Wale Cathy , Graham Frank L. , Johnson David C. 
Published: December 2019

ABSTRACT
Herpes simplex virus (HSV) glycoprotein D (gD) is modified with mannose 6-phosphate (M6P) and binds to M6P receptors (MPRs). MPRs are involved in the well-characterized pathway by which lysosomal enzymes are directed to lysosomes via a network of endosomal membranes. Based on the impaired ability of HSV to form plaques under conditions in which glycoproteins could not interact with MPRs, we proposed that MPRs may function during HSV egress or cell-to-cell spread (C. R. Brunetti, R. L. Burke, B. Hoflack, T. Ludwig, K. S. Dingwell, and D. C. Johnson, J. Virol. 69:3517–3528, 1995). To further analyze M6P modification and intracellular trafficking of gD in the absence of other HSV proteins, adenovirus (Ad) vectors were used to express soluble and membrane-anchored forms of gD. Both membrane-bound and soluble gD were modified with M6P residues and were localized to endosomes that contained the 275-kDa MPR or the transferrin receptor. Similar results were observed in HSV-infected cells. Cell fractionation experiments showed that gD was not present in lysosomes. However, a mutant form of gD and another HSV glycoprotein, gI, that were not modified with M6P were also found in endosomes in HSV-infected cells. Moreover, a substantial fraction of the HSV nucleocapsid protein VP6 was found in endosomes, consistent with accumulation of virions in an endosomal compartment. Therefore, it appears that HSV glycoproteins and virions are directed to endosomes, by M6P-dependent as well as by M6P-independent mechanisms, either as part of the virus egress pathway or by endocytosis from the cell surface.
Human Immunodeficiency Virus Neurotropism: an Analysis of Viral Replication and Cytopathicity for Divergent Strains in Monocytes and Microglia
Authors: Ghorpade Anuja , Nukuna Adeline , Che MyHanh , Haggerty Sheryl , Persidsky Yuri , Carter Eboni , Carhart Leeroy , Shafer Laura , Gendelman Howard E. 
Published: December 2019

ABSTRACTProductive replication of human immunodeficiency virus type 1 (HIV-1) in brain macrophages and microglia is a critical component of viral neuropathogenesis. However, how virus-macrophage interactions lead to neurological disease remains incompletely understood. Possibly, a differential ability of virus to replicate in brain tissue macrophages versus macrophages in other tissues underlies HIV-1 neurovirulence. To these ends, we established systems for the isolation and propagation of pure populations of human microglia and then analyzed the viral life cycles of divergent HIV-1 strains in these cells and in cultured monocytes by using identical viral inocula and indicator systems. The HIV-1 isolates included those isolated from blood, lung tissue, cerebrospinal fluids (CSF), and brain tissues of infected subjects: HIV-1ADAand HIV-189.6(from peripheral blood mononuclear cells), HIV-1DJVand HIV-1JR-FL(from brain tissue), HIV-1SF162(from CSF), and HIV-1BAL(from lung tissue). The synthesis of viral nucleic acids and viral mRNA, cytopathicity, and release of progeny virions were assessed. A significant heterogeneity among macrophage-tropic isolates for infection of monocytes and microglia was demonstrated. Importantly, a complete analysis of the viral life cycle revealed no preferential differences in the abilities of the HIV-1 strains tested to replicate in microglia and/or monocytes. Macrophage tropism likely dictates the abilities of HIV-1 to invade, replicate, and incite disease within its microglial target cells.
Membrane Organization of Bluetongue Virus Nonstructural Glycoprotein NS3
Authors: Bansal Om B. , Stokes Ann , Bansal Anju , Bishop David , Roy Polly 
Published: December 2019

ABSTRACT

The smallest RNA segment (S10) of bluetongue virus (an orbivirus, family
Reoviridae
) encodes two closely related nonstructural proteins, the 229-amino-acid (aa) NS3 and the 216-aa NS3A. The proteins are found in glycosylated and nonglycosylated forms in infected cells (X. Wu, H. Iwata, S.-Y. Chen, R. W. Compans and P. Roy J. Virol. 66:7104–7112, 1992). The NS3/NS3A proteins have two hydrophobic domains (aa 118 to 141 and 162 to 182) and two potential asparagine-linked glycosylation sites (aa 63 and 150), one of which is located between the hydrophobic domains. To determine whether these features were used in the mature protein forms, we generated a series of mutants of the S10 gene and expressed them by using the vaccinia virus T7 polymerase transient-expression system. Our data indicate that both hydrophobic domains of NS3 span the cell membrane and that only the site at aa 150 is responsible for N-linked glycosylation of the NS3 proteins.
A Cytopathogenic, Apoptosis-Inducing Variant of Hepatitis A Virus
Authors: Brack Kerstin , Frings Werner , Dotzauer Andreas , Vallbracht Angelika 
Published: December 2019

ABSTRACT

A cytopathogenic variant of hepatitis A virus (HAV
cyt/HB1.1
) was isolated from persistently infected BS-C-1 cells by serial passages in FRhK-4 cells. This virus shows a rapid replication pattern and high final titers are obtained, which are main characteristics of cytopathogenic HAVs. Sequencing of the nontranslated regions and the coding regions for 2ABC and 3AB revealed that mutations are distributed all over these regions and that certain mutated sites correspond to those in other cytopathogenic HAV variants. Investigating the mechanisms causing the cytopathic effect in FRhK-4 cells infected with this variant, we found that an apoptotic reaction takes place.
Differentiation of Promonocytic U937 Subclones into Macrophagelike Phenotypes Regulates a Cellular Factor(s) Which Modulates Fusion/Entry of Macrophagetropic Human Immunodeficiency Virus Type 1
Authors: Moriuchi Hiroyuki , Moriuchi Masako , Fauci Anthony S. 
Published: December 2019

ABSTRACTMonocytes/macrophages (M/M) and CD4+T cells are two important targets of human immunodeficiency virus (HIV) infection. Different strains of HIV-1 vary markedly in their abilities to infect cells belonging to the M/M lineage. Macrophagetropic (M-tropic) HIV-1 strains replicate well in primary lymphocytes as well as in primary macrophages; however, they generally infect T-cell lines poorly, if at all. Although promonocytic cell lines such as U937 have been used as in vitro models of HIV-1 infection of M/M, these cell lines are susceptible to certain T-cell-tropic (T-tropic) HIV-1 strains but are resistant to M-tropic HIV-1. In this study, we demonstrate that (i) certain U937 clones (“plus” clones), which are susceptible only to T-tropic HIV-1, become highly susceptible to M-tropic HIV-1 upon differentiation with retinoic acid (RA); (ii) other U937 clones (“minus” clones), which are resistant to both T- and M-tropic HIV-1, remain resistant to both viruses; and (iii) RA treatment induces expression of CCR5, a fusion/entry cofactor for M-tropic HIV-1 in both types of U937 clones, and yet enhances the fusogenicity of the plus clones, but not the minus clones, with M-tropic Env’s. These results indicate that the major restriction of M-tropic HIV-1 infection in promonocytic cells occurs at the fusion/entry level, that differentiation into macrophage-like phenotypes renders some of these cells highly susceptible to infection with M-tropic HIV-1, and that CD4 and CCR5 may not be the only determinants of fusion/entry of M-tropic HIV-1 in these cells.
Interaction of Poliovirus with Its Purified Receptor and Conformational Alteration in the Virion
Authors: Arita Minetaro , Koike Satoshi , Aoki Junken , Horie Hitoshi , Nomoto Akio 
Published: December 2019

ABSTRACT

Polypeptides of amino acids 1 to 241 (PVR241) and 1 to 330 (PVR330) of the human poliovirus receptor (hPVR) were produced in a baculovirus expression system. PVR241 contained extracellular domains 1 and 2 of hPVR, and PVR330 contained extracellular domains 1, 2, and 3. These peptides were purified by immunoaffinity column chromatography with an anti-hPVR monoclonal antibody (MAb). After the purification, PVR241 and PVR330 appeared to retain their native conformation as judged by reactivity with an anti-PVR MAb that recognized domain 1 of hPVR in a conformation-dependent manner. The virulent Mahoney strain of poliovirus type 1 was mixed with the purified PVRs in various concentrations. An average of at least 43 PVR330 molecules were able to bind to one virion particle under the conditions used. The equilibrium dissociation constant between the PVR330 molecule and the PVR binding site (canyon) on the virion was determined to be 4.50 ± (0.86) × 10
−8
M at 4°C. Higher rates of conformational change of the virus (160S) to 135S and 80S particles were observed as the concentration of PVR330 was increased. In this in vitro system, the ratio of the amount of the 135S particle to that of the 80S particle seemed to be always constant. After the disappearance of the 160S particle, the amount of the 80S particle was not increased by further incubation at 37°C. These results suggested that the 80S particle was not derived from the 135S particle under the conditions used in this study.
Monoclonal Antibodies to Distinct Sites on Herpes Simplex Virus (HSV) Glycoprotein D Block HSV Binding to HVEM
Authors: Nicola Anthony V. , Ponce de Leon Manuel , Xu Ruliang , Hou Wangfang , Whitbeck J. Charles , Krummenacher Claude , Montgomery Rebecca I. , Spear Patricia G. , Eisenberg Roselyn J. , Cohen Gary H. 
Published: December 2019

ABSTRACT
HVEM (for herpesvirus entry mediator) is a member of the tumor necrosis factor receptor superfamily and mediates entry of many strains of herpes simplex virus (HSV) into normally nonpermissive Chinese hamster ovary (CHO) cells. We used sucrose density centrifugation to demonstrate that purified HSV-1 KOS virions bind directly to a soluble, truncated form of HVEM (HVEMt) in the absence of any other cell-associated components. Therefore, HVEM mediates HSV entry by serving as a receptor for the virus. We previously showed that soluble, truncated forms of HSV glycoprotein D (gDt) bind to HVEMt in vitro. Here we show that antibodies specific for gD, but not the other entry glycoproteins gB, gC, or the gH/gL complex, completely block HSV binding to HVEM. Thus, virion gD is the principal mediator of HSV binding to HVEM. To map sites on virion gD which are necessary for its interaction with HVEM, we preincubated virions with gD-specific monoclonal antibodies (MAbs). MAbs that recognize antigenic sites Ib and VII of gD were the only MAbs which blocked the HSV-HVEM interaction. MAbs from these two groups failed to coprecipitate HVEMt in the presence of soluble gDt, whereas the other anti-gD MAbs coprecipitated HVEMt and gDt. Previous mapping data indicated that site VII includes amino acids 11 to 19 and site Ib includes 222 to 252. The current experiments indicate that these sites contain residues important for HSV binding to HVEM. Group Ib and VII MAbs also blocked HSV entry into HVEM-expressing CHO cells. These results suggest that the mechanism of neutralization by these MAbs is via interference with the interaction between gD in the virus and HVEM on the cell. Group Ia and II MAbs failed to block HSV binding to HVEM yet still neutralized HVEM-mediated entry, suggesting that these MAbs block entry at a step other than HVEM binding.
Collaboration of Antibody and Inflammation in Clearance of Rabies Virus from the Central Nervous System
Authors: Hooper D. Craig , Morimoto Kinjiro , Bette Michael , Weihe Eberhard , Koprowski Hilary , Dietzschold Bernhard 
Published: December 2019

ABSTRACTTo investigate the involvement of various cellular and humoral aspects of immunity in the clearance of rabies virus from the central nervous system, (CNS), we studied the development of clinical signs and virus clearance from the CNS in knockout mice lacking either B and T cells, CD8+cytotoxic T cells, B cells, alpha/beta interferon (IFN-α/β) receptors, IFN-γ receptors, or complement components C3 and C4. Following intranasal infection with the attenuated rabies virus CVS-F3, normal adult mice of different genetic backgrounds developed a transient disease characterized by loss of body weight and appetite depression which peaked at 13 days postinfection (p.i.). While these animals had completely recovered by day 21 p.i., mice lacking either B and T cells or B cells alone developed a progressive disease and succumbed to infection. Mice lacking either CD8+T cells, IFN receptors, or complement components C3 and C4 showed no significant differences in the development of clinical signs by comparison with intact counterparts having the same genetic background. However, while infectious virus and viral RNA could be detected in normal control mice only until day 8 p.i., in all of the gene knockout mice studied except those lacking C3 and C4, virus infection persisted through day 21 p.i. Analysis of rabies virus-specific antibody production together with histological assessment of brain inflammation in infected animals revealed that clearance of CVS-F3 by 21 days p.i. correlated with both a strong inflammatory response in the CNS early in the infection (day 8 p.i.), and the rapid (day 10 p.i.) production of significant levels of virus-neutralizing antibody (VNA). These studies confirm that rabies VNA is an absolute requirement for clearance of an established rabies virus infection. However, for the latter to occur in a timely fashion, collaboration between VNA and inflammatory mechanisms is necessary.
Temporal Mapping of Transcripts in Herpesvirus 6 Variants
Authors: Mirandola Prisco , Menegazzi Paola , Merighi Stefania , Ravaioli Tullia , Cassai Enzo , Di Luca Dario 
Published: December 2019

ABSTRACTTo define the molecular features characteristic of the early stages of infection of lymphocytes with human herpesvirus 6 (HHV-6) variant A or B, we studied the temporal regulation of expression of selected sets of viral genes. Thus, U42, U94, U89-U90, U73, and U39 are α genes since their transcripts (i) were made in the presence of inhibitors of protein synthesis and (ii) were detected 3 h after infection of untreated cells. U41, U53, U31, and U19 are β genes since their expression is inhibited by cycloheximide but not by phosphonoacetate, an inhibitor of DNA synthesis. U100 is a γ gene since its spliced transcript encoding the structural glycoprotein gp82/105 was first detected 16 h after infection of untreated cells but could not be detected in cells treated with phosphonoacetate. HHV-6 variants differ in the transcription patterns of their genes. U16-U17 originates a splice transcript and is regulated as α in HHV-6B and as β in HHV-6A. U91 generates two transcripts, amplified as 476- and 374-bp PCR fragments. The 476-bp fragment is α in HHV-6A-infected cells but β in HHV-6B-infected cells. Conversely, the 374-bp fragment is β in HHV-6A-infected cells and α in HHV-6B-infected cells. Furthermore, the spliced product of U18-U19-U20 (526 bp) is β in HHV-6A-infected cells, but only a partially spliced form (1.9 kb) was detected at late stages of infection in HHV-6B. HHV-6 transcription was also studied in nonproductive lymphoid cells, and the same transcription pattern detected during lytic infection was observed. Also, HHV-6 variants maintain the differences in U91, U16-17, and U18-U19-U20. We conclude that, as expected from the sequencing data, gene expression is generally similar in HHV-6 variants. However, transcription of selected genes in HHV-6A and HHV-6B differs with respect to temporal regulation and splicing pattern. Furthermore, the identification of viral functions expressed during the different stages of lytic replication suggests that reverse transcription-PCR for HHV-6 genes is a useful diagnostic approach to differentiate between latent and productive HHV-6 infection.
T-Cell-Line-Tropic Human Immunodeficiency Virus Type 1 That Is Made Resistant to Stromal Cell-Derived Factor 1α Contains Mutations in the Envelope gp120 but Does Not Show a Switch in Coreceptor Use
Authors: Schols Dominique , Esté José A. , Cabrera Cecilia , De Clercq Erik 
Published: December 2019

ABSTRACT

The NL4.3 T-cell-line-tropic human immunodeficiency virus type 1 strain is sensitive to the CXC chemokine stromal cell-derived factor 1α (SDF-1α), the natural ligand for CXC chemokine receptor 4 (CXCR4); the 50% inhibitory concentration (IC
50
) in MT-4 cells is 130 ng/ml. We generated resistant virus through passaging of the virus in the presence of increasing concentrations of SDF-1α. After 24 passages, the virus was no longer sensitive to SDF-1α (SDF-1α
res
virus) (IC
50
, >2 μg/ml) and became resistant to SDF-1β (IC
50
, >2 μg/ml) and to a specific CXCR4 monoclonal antibody (IC
50
, >20 μg/ml). The SDF-1α
res
virus was about 10-fold less sensitive than the wild-type virus to the bicyclam AMD3100, a specific CXCR4 antagonist. The SDF-1α
res
virus contained the following mutations in the gp120 molecule: N106K in the V1 loop; S134N and F145L in the V2 loop; F245I in the C2 loop; K269E, Q278H, I288V, and N293D in the V3 loop; a deletion of 5 amino acids (FNSTW) at positions 364 to 368 in the V4 loop; and R378T in the CD4 binding domain. Replication of the NL4.3 wild-type virus and the SDF-1α
res
virus was demonstrated in U87 cells that coexpressed CD4 and CXCR4 (U87.CD4.CXCR4) but not in U87.CD4.CCR5 cells. Thus, the resistant virus was not able to switch to the CC chemokine receptor 5 (CCR5) coreceptor (the main coreceptor for macrophage-tropic viruses). The SDF-1α
res
virus replicated in HOS.CD4 cells expressing CCR1, CCR2b, CCR3, CCR4, CCR5, and CXCR4 but also in HOS.CD4.pBABE cells. However, all HOS transfectant cells expressed a low level of CXCR4. Neither of the two virus strains was able to infect HOS.CXCR4 or HOS.CCR5 transfectants, demonstrating the necessity of the CD4 receptor. The T-cell-line-tropic SDF-1α
res
virus was thus able to overcome the inhibitory effect of SDF-1α through mutations in gp120 but still needed CXCR4 to enter the cells.
A Broad Range of Chemokine Receptors Are Used by Primary Isolates of Human Immunodeficiency Virus Type 2 as Coreceptors with CD4
Authors: McKnight Áine , Dittmar Matthias T. , Moniz-Periera José , Ariyoshi Koya , Reeves Jacqueline D. , Hibbitts Sam , Whitby Denise , Aarons Emma , Proudfoot Amanda E. I. , Whittle Hilton , Clapham Paul R. 
Published: December 2019

ABSTRACT
Like human immunodeficiency virus type 1 (HIV-1) and simian immunodeficiency virus (SIV), HIV-2 requires a coreceptor in addition to CD4 for entry into cells. HIV and SIV coreceptor molecules belong to a family of seven-transmembrane-domain G-protein-coupled receptors. Here we show that primary HIV-2 isolates can use a broad range of coreceptor molecules, including CCR1, CCR2b, CCR3, CCR4, CCR5, and CXCR4. Despite broad coreceptor use, the chemokine ligand SDF-1 substantially blocked HIV-2 infectivity of peripheral blood mononuclear cells, indicating that its receptor, CXCR4, was the predominant coreceptor for infection of these cells. However, expression of CXCR4 together with CD4 on some cell types did not confer susceptibility to infection by all CXCR4-using virus isolates. These data therefore indicate that another factor(s) influences the ability of HIV-2 to replicate in human cell types that express the appropriate receptors for virus entry.
Secondary Structures in the Capsid Protein Coding Sequence and 3′ Nontranslated Region Involved in Amplification of the Tobacco Etch Virus Genome
Authors: Haldeman-Cahill Ruth , Daròs José-Antonio , Carrington James C. 
Published: December 2019

ABSTRACT

The 3′-terminal 350 nucleotides of the tobacco etch potyvirus (TEV) genome span the end of the capsid protein (CP)-coding sequence and the 3′ nontranslated region (NTR). The CP-coding sequence within this region contains a 105-nucleotide
cis
-active element required for genome replication (S. Mahajan, V. V. Dolja, and J. C. Carrington, J. Virol. 70:4370–4379, 1996). To investigate the sequence and secondary structure requirements within the CP
cis
-active region and the 3′ NTR, a systematic linker-scanning mutagenesis analysis was done. Forty-six mutations, each with two to six nucleotide substitutions, were introduced at consecutive hexanucleotide positions in the genome of a recombinant TEV strain expressing a reporter protein (β-glucuronidase). Genome amplification activity of each mutant in the protoplast cell culture system was measured. Mutations that severely debilitated genome amplification were identified throughout the CP-coding
cis
-active sequence and at several distinct locations within the 3′ NTR. However, based on a computer model of RNA folding, mutations that had the most severe effects mapped to regions that were predicted to form base-paired secondary structures. Linker-scanning mutations predicted to affect either strand of a base-paired structure within the CP-coding
cis
-active sequence, a base-paired structure between two segments of the CP-coding
cis
-active sequence and a contiguous 14-nucleotide segment of the 3′ NTR, and a base-paired structure near the 3′ terminus of the 3′ NTR inactivated genome amplification. Compensatory mutations that restored base pair interactions in each of these regions restored amplification activity, although to differing levels depending on the structure restored. These data reveal that the 3′ terminus of the TEV genome consists of a series of functionally discrete sequences and secondary structures and that the CP-coding sequence and 3′ NTR are coadapted for genome amplification function through a requirement for base pair interactions.
A Proline-Rich Motif (PPPY) in the Gag Polyprotein of Mason-Pfizer Monkey Virus Plays a Maturation-Independent Role in Virion Release
Authors: Yasuda Jiro , Hunter Eric 
Published: December 2019

ABSTRACT

Virus assembly represents one of the last steps in the retrovirus life cycle. During this process, Gag polyproteins assemble at specific sites within the cell to form viral capsids and induce membrane extrusion (viral budding) either as assembly progresses (type C virus) or following formation of a complete capsid (type B and type D viruses). Finally, the membrane must undergo a fusion event to pinch off the particle in order to release a complete enveloped virion. Structural elements within the MA region of the Gag polyprotein define the route taken to the plasma membrane and direct the process of virus budding. Results presented here suggest that a distinct region of Gag is necessary for virus release. The pp24 and pp16 proteins of the type D retrovirus Mason-Pfizer monkey virus (M-PMV) are phosphoproteins that are encoded in the
gag
gene of the virus. The pp16 protein is a C-terminally located cleavage product of pp24 and contains a proline-rich motif (PPPY) that is conserved among the Gag proteins of a wide variety of retroviruses. By performing a functional analysis of this coding region with deletion mutants, we have shown that the pp16 protein is dispensable for capsid assembly but essential for virion release. Moreover, additional experiments indicated that the virus release function of pp16 was abolished by the deletion of only the PPPY motif and could be restored when this motif alone was reinserted into a Gag polyprotein lacking the entire pp16 domain. Single-amino-acid substitutions for any of the residues within this motif confer a similar virion release-defective phenotype. It is unlikely that the function of the proline-rich motif is simply to inhibit premature activation of protease, since the PPPY deletion blocked virion release in the context of a protease-defective provirus. These results demonstrate that in type D retroviruses a PPPY motif plays a key role in a late stage of virus budding that is independent of and occurs prior to virion maturation.