bluuebunny/crossref_metadata_2025 huggingface dataset:
Nonstructural C Protein Is Required for Efficient Measles Virus Replication in Human Peripheral Blood Cells
Authors: Escoffier Carine , Manié Serge , Vincent Séverine , Muller Claude P. , Billeter Martin , Gerlier Denis
Published: December 2019

The P gene of measles virus (MV) encodes the phosphoprotein, a component of the virus ribonucleoprotein complex, and two nonstructural proteins, C and V, with unknown functions. Growth of recombinant MV, defective in C or V expression, was explored in human peripheral blood mononuclear cells (PBMC). The production of infectious recombinant MV V−was comparable to that of parental MV tag in simian Vero fibroblasts and in PBMC. In contrast, MV C−progeny was strongly reduced in PBMC but not in Vero cells. Consistently, the expression of both hemagglutinin and fusion proteins, as well as that of nucleoprotein mRNA, was lower in MV C−-infected PBMC. Thus, efficient replication of MV in natural host cells requires the expression of the nonstructural C protein. The immunosuppression that accompanies MV infection is associated with a decrease in the in vitro lymphoproliferative response to mitogens. MV C−was as potent as MV tag or MV V−in inhibiting the phytohemagglutinin-induced proliferation of PBMC, indicating that neither the C protein nor the V protein is directly involved in this effect.
Polyomavirus Large T Antigen Binds the Transcriptional Coactivator Protein p300
Authors: Nemethova Maria , Wintersberger Erhard
Published: December 2019

Using coimmunoprecipitation and glutathione
S
-transferase pulldown experiments, we found that polyomavirus large T antigen binds to p300 in vivo and in vitro. The N-terminal region of the viral protein, including the pRB binding motif, was dispensable for this interaction, which involved several regions within the C-terminal half of the large T antigen. Interestingly, anti-T antibody coimmunoprecipitated a subspecies of p300 which has high histone acetyltransferase activity.
Human Adeno-Associated Virus Type 5 Is Only Distantly Related to Other Known Primate Helper-Dependent Parvoviruses
Authors: Bantel-Schaal Ursula , Delius Hajo , Schmidt Rainer , zur Hausen Harald
Published: December 2019

We have characterized 95% (4,404 nucleotides) of the genome of adeno-associated virus type 5 (AAV5), including part of the terminal repeats and the terminal resolution site. Our results show that AAV5 is different from all other described AAV serotypes at the nucleotide level and at the amino acid level. The sequence homology to AAV2, AAV3B, AAV4, and AAV6 at the nucleotide level is only between 54 and 56%. The positive strand contains two large open reading frames (ORFs). The left ORF encodes the nonstructural (Rep) proteins, and the right ORF encodes the structural (Cap) proteins. At the amino acid level the identities with the capsid proteins of other AAVs range between 51 and 59%, with a high degree of heterogeneity in regions which are considered to be on the exterior surface of the viral capsid. The overall identity for the nonstructural Rep proteins at the amino acid level is 54.4%. It is lowest at the C-terminal 128 amino acids (10%). There are only two instead of the common three putative Zn fingers in the Rep proteins. The Cap protein data suggest differences in capsid surfaces and raise the possibility of a host range distinct from those of other parvoviruses. This may have important implications for AAV vectors used in gene therapy.
Dual Stem Loops within the Poliovirus Internal Ribosomal Entry Site Control Neurovirulence
Authors: Gromeier Matthias , Bossert Birgit , Arita Mineo , Nomoto Akio , Wimmer Eckard
Published: December 2019

In the human central nervous system, susceptibility to poliovirus (PV) infection is largely confined to a specific subpopulation of neuronal cells. PV tropism is likely to be determined by cell-external components such as the PV receptor CD155, as well as cell-internal constraints such as the availability of a suitable microenvironment for virus propagation. We reported previously that the exchange of the cognate internal ribosomal entry site (IRES) within the 5′ nontranslated region of PV with its counterpart from human rhinovirus type 2 (HRV2) can eliminate the neuropathogenic phenotype in a transgenic mouse model for poliomyelitis without diminishing the growth properties in HeLa cells. We now show that attenuation of neurovirulence of PV/HRV2 chimeras is not confined to CD155 transgenic mice but is evident also after intraspinal inoculation into
Cynomolgus
monkeys. We have dissected the PV and HRV2 IRES elements to determine those structures responsible for neurovirulence (or attenuation) of these chimeric viruses. We report that two adjacent stem loop structures within the IRES cooperatively determine neuropathogenicity.
Epstein-Barr Virus Nuclear Antigen 1 Sequences in Endemic and Sporadic Burkitt’s Lymphoma Reflect Virus Strains Prevalent in Different Geographic Areas
Authors: Habeshaw G. , Yao Q. Y. , Bell A. I. , Morton D. , Rickinson A. B.
Published: December 2019

The Epstein-Barr virus (EBV) nuclear antigen EBNA1 is the only viral protein detectably expressed in virus genome-positive Burkitt’s lymphoma (BL); recent work has suggested that viral strains with particular EBNA1 sequence changes are preferentially associated with this tumor and that, within a patient, the tumor-associated variant may have arisen de novo as a rare mutant of the dominant preexisting EBV strain (K. Bhatia, A. Raj, M. J. Gutierrez, J. G. Judde, G. Spangler, H. Venkatesh, and I. T. Magrath, Oncogene 13:177–181, 1996). In the present work we first study 12 BL patients and show that the virus strain in the tumor is identical in EBNA1 sequence and that it is matched at several other polymorphic loci to the dominant strain rescued in vitro from the patient’s normal circulating B cells. We then analyze BL-associated virus strains from three different geographic areas (East Africa, Europe, and New Guinea) alongside virus isolates from geographically matched control donors by using sequence changes in two separate regions of the EBNA1 gene (N-terminal codons 1 to 60 and C-terminal codons 460 to 510) to identify the EBNA1 subtype of each virus. Different geographic areas displayed different spectra of EBNA1 subtypes, with only limited overlap between them; even type 2 virus strains, which tended to be more homogeneous than their type 1 counterparts, showed geographic differences at the EBNA1 locus. Most importantly, within any one area the EBNA1 subtypes associated with BL were also found to be prevalent in the general population. We therefore find no evidence that Burkitt lymphomagenesis involves a selection for EBV strains with particular EBNA1 sequence changes.
The Hypervariable Domain of the Murine Leukemia Virus Surface Protein Tolerates Large Insertions and Deletions, Enabling Development of a Retroviral Particle Display System
Authors: Kayman Samuel C. , Park Han , Saxon Maya , Pinter Abraham
Published: December 2019

The surface proteins (SU) of murine type-C retroviruses have a central hypervariable domain devoid of cysteine and rich in proline. This 41-amino-acid region of Friend ecotropic murine leukemia virus SU was shown to be highly tolerant of insertions and deletions. Viruses in which either the N-terminal 30 amino acids or the C-terminal 22 amino acids of this region were replaced by the 7-amino-acid sequence ASAVAGA were fully infectious. Insertions of this 7-amino-acid sequence at the N terminus, center, and the C terminus of the hypervariable domain had little effect on envelope protein (Env) function, while this insertion at a position 10 amino acids following the N terminus partially destabilized the association between the SU and transmembrane subunits of Env. Large, complex domains (either a 252-amino-acid single-chain antibody binding domain [scFv] or a 96-amino-acid V1/V2 domain of HIV-1 SU containing eight N-linked glycosylation sites and two disulfides) did not interfere with Env function when inserted in the center or C-terminal portions of the hypervariable domain. The scFv domain inserted into the C-terminal region of the hypervariable domain was shown to mediate binding of antigen to viral particles, demonstrating that it folded into the active conformation and was displayed on the surface of the virion. Both positive and negative enrichment of virions expressing the V1/V2 sequence were achieved by using a monoclonal antibody specific for a conformational epitope presented by the inserted sequence. These results indicated that the hypervariable domain of Friend ecotropic SU does not contain any specific sequence or structure that is essential for Env function and demonstrated that insertions into this domain can be used to extend particle display methodologies to complex protein domains that require expression in eukaryotic cells for glycosylation and proper folding.
Human Immunodeficiency Virus Type 1 Protease Triggers a Myristoyl Switch That Modulates Membrane Binding of Pr55

gag

and p17MA
Authors: Hermida-Matsumoto Luz , Resh Marilyn D.
Published: December 2019

The human immunodeficiency virus type 1 (HIV-1) Pr55

gag

gene product directs the assembly of virions at the inner surface of the cell plasma membrane. The specificity of plasma membrane binding by Pr55

gag

is conferred by a combination of an N-terminal myristoyl moiety and a basic residue-rich domain. Although the myristate plus basic domain is also present in the p17MA proteolytic product formed upon Pr55

gag

maturation, the ability of p17MA to bind to membranes is significantly reduced. It was previously reported that the reduced membrane binding of p17MA was due to sequestration of the myristate moiety by a myristoyl switch (W. Zhou and M. D. Resh, J. Virol. 70:8540–8548, 1996). Here we demonstrate directly that treatment of membrane-bound Pr55

gag

in situ with HIV-1 protease generates p17MA, which is then released from the membrane. Pr55

gag

was synthesized in reticulocyte lysates, bound to membranes, and incubated with purified HIV-1 protease. The p17MA product in the membrane-bound and soluble fractions was analyzed following proteolysis. Newly generated p17MA initially was membrane bound but then displayed a slow, time-dependent dissociation resulting in 65% solubilization. Residual p17MA could be extracted from the membranes with either high pH or high salt. Treatment of membranes from transfected COS-1 cells with protease revealed that Pr55

gag

was present within sealed membrane vesicles and that the release of p17MA occurred only when detergent and salt were added. We present a model proposing that the HIV-1 protease is the “trigger” for a myristoyl switch mechanism that modulates the membrane associations of Pr55

gag

and p17MA in virions and membranes.
Actin Associates with the Nucleocapsid Domain of the Human Immunodeficiency Virus Gag Polyprotein
Authors: Wilk Thomas , Gowen Brent , Fuller Stephen D.
Published: December 2019

Recently, it was shown that actin molecules are present in human immunodeficiency virus type 1 (HIV-1) particles. We have examined the basis for incorporation and the location of actin molecules within HIV-1 and murine retrovirus particles. Our results show that the retroviral Gag polyprotein is sufficient for actin uptake. Immunolabeling studies demonstrate that actin molecules localize to a specific radial position within the immature particle, clearly displaced from the matrix domain underneath the viral membrane but in proximity to the nucleocapsid (NC) domain of the Gag polyprotein. When virus or subviral Gag particles were disrupted with nonionic detergent, actin molecules remained associated with the disrupted particles. Actin molecules remained in a stable complex with the NC cleavage product (or an NC-RNA complex) after treatment of the disrupted HIV-1 particles with recombinant HIV-1 protease. In contrast, matrix and capsid molecules were released. The same result was obtained when mature HIV-1 particles were disrupted with detergent. Taken together, these results indicate that actin molecules are associated with the NC domain of the viral polyprotein.
Structure of Adeno-Associated Virus Vector DNA following Transduction of the Skeletal Muscle
Authors: Vincent-Lacaze Nathalie , Snyder Richard O. , Gluzman Régis , Bohl Delphine , Lagarde Catherine , Danos Olivier
Published: December 2019

The skeletal muscle provides a very permissive physiological environment for adeno-associated virus (AAV) type 2-mediated gene transfer. We have studied the early steps leading to the establishment of permanent transgene expression, after injection of recombinant AAV (rAAV) particles in the quadriceps muscle of mice. The animals received an rAAV encoding a secreted protein, murine erythropoietin (mEpo), under the control of the human cytomegalovirus major immediate-early promoter and were sacrificed between 1 and 60 days after injection. The measurement of plasma Epo levels and of hematocrits indicated a progressive increase of transgene expression over the first 2 weeks, followed by a stabilization at maximal plateau values. The rAAV sequences were analyzed by Southern blotting following neutral or alkaline gel electrophoresis of total DNA from injected muscles. While a high number of rAAV sequences were detected during the first 5 days following the injection, only a few percent of these sequences was retained in the animals analyzed after 2 weeks, in which transgene expression was maximal. Double-stranded DNA molecules resulting from de novo second-strand synthesis were detected as early as day 1, indicating that this crucial step of AAV-mediated gene transfer is readily accomplished in the muscle. The templates driving stable gene expression at later time points are low in copy number and structured as high-molecular-weight concatemers or interlocked circles. The presence of the circular form of the rAAV genomes at early time points suggests that the molecular transformations involved in the formation of stable concatemers may involve a rolling-circle type of DNA replication.
Human Immunodeficiency Virus Type 1 Tat Induces Apoptosis and Increases Sensitivity to Apoptotic Signals by Up-Regulating FLICE/Caspase-8
Authors: Bartz Steven R. , Emerman Michael
Published: December 2019

Apoptosis contributes to the loss of CD4 cells during human immunodeficiency virus type 1 (HIV-1) infection. Although the product of theenvgene, gp160/gp120, is known to play a role in cell death mediated by HIV-1, the role of other HIV-1 genes in the process is unclear. We found that HIV-1 lacking theenvgene (HIVΔenv) still induced apoptosis in T-cell lines and primary CD4 T cells. The ability to induce apoptosis was attributable to Tat, a viral regulatory protein. Tat induction of apoptosis was separate from the transactivation function of Tat, required expression of the second exon of Tat, and was associated with the increased expression and activity of caspase-8 (casp-8), a signaling molecule in apoptotic pathways. Moreover, induction of apoptosis could be prevented by treating cells with an inhibitor of casp-8. In addition, we show that HIV-1Δenvinfection and Tat expression increased the sensitivity of cells to Fas-mediated apoptosis, an apoptotic pathway that signals via casp-8. The up-regulation of casp-8 by HIV-1 Tat expression may contribute to the increased apoptosis and sensitivity to apoptotic signals observed in the cells of HIV-1-infected persons.
Open Reading Frame 1a-Encoded Subunits of the Arterivirus Replicase Induce Endoplasmic Reticulum-Derived Double-Membrane Vesicles Which Carry the Viral Replication Complex
Authors: Pedersen Ketil W. , van der Meer Yvonne , Roos Norbert , Snijder Eric J.
Published: December 2019

The replicase of equine arteritis virus (EAV; family
Arteriviridae
, order
Nidovirales
) is expressed in the form of two polyproteins (the open reading frame 1a [ORF1a] and ORF1ab proteins). Three viral proteases cleave these precursors into 12 nonstructural proteins, which direct both genome replication and subgenomic mRNA transcription. Immunofluorescence assays showed that most EAV replicase subunits localize to membranes in the perinuclear region of the infected cell. Using replicase-specific antibodies and cryoimmunoelectron microscopy, unusual double-membrane vesicles (DMVs) were identified as the probable site of EAV RNA synthesis. These DMVs were previously observed in cells infected with different arteriviruses but were never implicated in viral RNA synthesis. Extensive electron microscopic analysis showed that they appear to be derived from paired endoplasmic reticulum membranes and that they are most likely formed by protrusion and detachment of vesicular structures with a double membrane. Interestingly, very similar membrane rearrangements were observed upon expression of ORF1a-encoded replicase subunits nsp2 to nsp7 from an alphavirus-based expression vector. Apparently, the formation of a membrane-bound scaffold for the replication complex is a distinct step in the arterivirus life cycle, which is directed by the ORF1a protein and does not depend on other viral proteins and/or EAV-specific RNA synthesis.
Identification of Protein Instability Determinants in the Carboxy-Terminal Region of c-Myb Removed as a Result of Retroviral Integration in Murine Monocytic Leukemias
Authors: Bies Juraj , Nazarov Viktor , Wolff Linda
Published: December 2019

The c-
myb
oncogene has been a target of retroviral insertional mutagenesis in murine monocytic leukemias. One mechanism by which c-
myb
can be activated is through the integration of a retroviral provirus into the central portion of the locus, causing premature termination of c-
myb
transcription and translation. We had previously shown that a leukemia-specific c-Myb protein, truncated at the site of proviral integration by 248 amino acids, had approximately a fourfold-increased half-life compared to the normal c-Myb protein, due to its ability to escape rapid degradation by the ubiquitin-26S proteasome pathway. Here we provide evidence for the existence of more than one instability determinant in the carboxy-terminal region of the wild-type protein, which appear to act independently of each other. The data were derived from examination of premature termination mutants and deletion mutants of the normal protein, as well as analysis of another carboxy-terminally truncated protein expressed in leukemia. Evidence is provided that one instability determinant is located in the terminal 87 amino acids of the protein and another is located in the vicinity of the internal region that has leucine zipper homology. In leukemias, different degrees of protein stability are attained following proviral integration depending upon how many determinants are removed. Interestingly, although PEST sequences (rich in proline, glutamine, serine, and threonine), often associated with degradation, are found in c-Myb, deletion of PEST-containing regions had no effect on protein turnover. This study provides further insight into how inappropriate expression of c-Myb may contribute to leukemogenesis. In addition, it will facilitate further studies aimed at characterizing the specific role of individual regions of the normal protein in targeting to the 26S proteasome.
The Replicative Capacities of Large E1B-Null Group A and Group C Adenoviruses Are Independent of Host Cell p53 Status
Authors: Turnell Andrew S. , Grand Roger J. A. , Gallimore Phillip H.
Published: December 2019

Recent reports suggest that an early region 1B (E1B) 55,000-molecular-weight polypeptide (55K)-null adenovirus type 5 (Ad5) mutant (
dl
1520) can replicate to the same extent as wild-type (wt) Ad5 in cells either deficient or mutated in p53, implicating p53 in limiting viral replication in vivo. In contrast, we show here that the replicative capacity of Ad5
dl
1520 is wholly independent of host cell p53 status, as is the replicative capacity of comparable Ad12 E1B 54K-null adenoviruses (Ad12
dl
620 and Ad12
hr
703). Furthermore, we show that there is no requirement for complex formation between p53 and Ad5 E1B 55K or Ad12 E1B 54K for a productive infection, such that wt Ad5 and wt Ad12 will both replicate in cells which are null for p53. In addition, we find that these Ad5 and Ad12 mutant viruses induce S phase irrespective of the p53 status of the cell and that, therefore, S-phase induction does not correlate with the replicative capacity of the virus. Interestingly, the replicative capacities of the large E1B-null adenoviruses correlated positively with the ability to express E1B 19K and were related to the ability to repress premature adenovirus-induced apoptosis. Infection of primary human cells indicated that Ad5
dl
1520, wt Ad5, and wt Ad12 replicated better in cycling normal human skin fibroblasts (HSFs) than in quiescent HSFs. Thus, the cell cycle status of the host cell, upon infection, also influences viral yield.
DNA Vaccine Encoding Hemagglutinin Provides Protective Immunity against H5N1 Influenza Virus Infection in Mice
Authors: Kodihalli Shantha , Goto Hideo , Kobasa Darwyn L. , Krauss Scott , Kawaoka Yoshihiro , Webster Robert G.
Published: December 2019

In Hong Kong in 1997, a highly lethal H5N1 avian influenza virus was apparently transmitted directly from chickens to humans with no intermediate mammalian host and caused 18 confirmed infections and six deaths. Strategies must be developed to deal with this virus if it should reappear, and prospective vaccines must be developed to anticipate a future pandemic. We have determined that unadapted H5N1 viruses are pathogenic in mice, which provides a well-defined mammalian system for immunological studies of lethal avian influenza virus infection. We report that a DNA vaccine encoding hemagglutinin from the index human influenza isolate A/HK/156/97 provides immunity against H5N1 infection of mice. This immunity was induced against both the homologous A/HK/156/97 (H5N1) virus, which has no glycosylation site at residue 154, and chicken isolate A/Ck/HK/258/97 (H5N1), which does have a glycosylation site at residue 154. The mouse model system should allow rapid evaluation of the vaccine’s protective efficacy in a mammalian host. In our previous study using an avian model, DNA encoding hemagglutinin conferred protection against challenge with antigenic variants that differed from the primary antigen by 11 to 13% in the HA1 region. However, in our current study we found that a DNA vaccine encoding the hemagglutinin from A/Ty/Ir/1/83 (H5N8), which differs from A/HK/156/97 (H5N1) by 12% in HA1, prevented death but not H5N1 infection in mice. Therefore, a DNA vaccine made with a heterologous H5 strain did not prevent infection by H5N1 avian influenza viruses in mice but was useful in preventing death.
Extensive Mutagenesis of the Hepatitis B Virus Core Gene and Mapping of Mutations That Allow Capsid Formation
Authors: Koschel Matthias , Thomssen Reiner , Bruss Volker
Published: December 2019

We generated a large number of mutations in the hepatitis B virus (HBV) core gene inserted into a bacterial expression vector. The new mutagenesis procedure generated deletions and insertions (as sequence repeats) of various lengths at random positions between M1 and E145 but not substitutions. The R-rich 30-amino-acid C-terminal domain was not analyzed. A total of 50,000 colonies were tested with a polyclonal human serum for the expression of hepatitis B core or e antigen. A total of 110 mutants randomly chosen from 1,500 positive colonies were genotyped. Deletions and insertions were clustered in four regions: D2 to E14, corresponding to the N-terminal loop in a model for the core protein fold (B. Bottcher, S. A. Wynne, and R. A. Crowther, Nature 386:88–91, 1997); V27 to P50 (second loop); L60 to V86 (upper half of the alpha helix forming the N-terminal part of the spike and the tip of the spike); and V124 to L140 (C-terminal part of the C-terminal helix and downstream loop). Deletions or insertions in the remaining parts of the molecule forming the compact center of the fold seemed to destabilize the protein. Of the 110 mutations, 38 allowed capsid formation in
Escherichia coli
. They mapped exclusively to nonhelical regions of the proposed fold. The mutations form a basis for subsequent analysis of further functions of the HBV core protein in the viral life cycle.
Hypoxia Blocks In Vivo Initiation of Simian Virus 40 Replication at a Stage Preceding Origin Unwinding
Authors: Riedinger Hans-Jörg , van Betteraey Maria , Probst Hans
Published: December 2019

Simian virus 40 (SV40)-infected CV1 cells transiently exposed to hypoxia show a burst of viral replication immediately after reoxygenation. DNA precursor incorporation and analysis of growing daughter strands by alkaline sedimentation demonstrated that SV40 DNA synthesis began with a lag of about 3 to 5 min after reoxygenation followed by a largely synchronous viral replication round. Viral RNA-DNA primers complementary to the SV40 origin region were not detectable before 3 min upon reoxygenation. A distinct form of circular closed, supercoiled SV40 DNA was detectable as soon as 3 min after reoxygenation but not under hypoxia. Sensitivity to the DNA nuclease
Bal
31 and migration behavior in chloroquine-containing agarose gels suggested that this DNA species was highly underwound compared to other SV40 topoisomers and was probably related to the highly underwound form U DNA first described by Dean et al. (F. B. Dean, P. Bullock, Y. Murakami, C. R. Wobbe, L. Weissbach, and J. Hurwitz, Proc. Natl. Acad. Sci. USA 84:16–20, 1987), in vitro. 3′-OH ends of presumed RNA-DNA primers could be detected in form U by 3′ end labeling with T7 polymerase. Addition of aphidicolin to the cells before reoxygenation led to a pronounced accumulation of form U DNA containing RNA-DNA primers. In vivo pulse-chase kinetic studies performed with aphidicolin-treated SV40-infected cells showed that form U is an initial intermediate of SV40 DNA replication which matures into higher-molecular-weight replication intermediates and into SV40 form I DNA after removal of the inhibitor. These results suggest that in vivo initiation of SV40 replication is arrested by hypoxia before origin unwinding and primer synthesis.
Alterations to both the Primary and Predicted Secondary Structure of Stem-Loop IIIc of the Hepatitis C Virus 1b 5′ Untranslated Region (5′UTR) Lead to Mutants Severely Defective in Translation Which Cannot Be Complemented in
trans
by the Wild-Type 5′UTR Sequence
Authors: Tang Shixing , Collier Adam J. , Elliott Richard M.
Published: December 2019

Cap-independent translation of the hepatitis C virus (HCV) genomic RNA is mediated by an internal ribosome entry site (IRES) within the 5′ untranslated region (5′UTR) of the virus RNA. To investigate the effects of alterations to the primary sequence of the 5′UTR on IRES activity, a series of HCV genotype 1b (HCV-1b) variant IRES elements was generated and cloned into a bicistronic reporter construct. Changes from the prototypic HCV-1b 5′UTR sequence were identified at various locations throughout the 5′UTR. The translation efficiencies of these IRES elements were examined by an in vivo transient expression assay in transfected BHK-21 cells and were found to range from 0.4 to 95.8% of the activity of the prototype HCV-1b IRES. Further mutational analysis of the three single-point mutants most severely defective in activity, whose mutations were all located in or near stem-loop IIIc, demonstrated that both the primary sequence and the maintenance of base pairing within this stem structure were critical for HCV IRES function. Complementation studies indicated that defective mutants containing either point mutations or major deletions within the IRES elements could not be complemented in
trans
by a wild-type IRES.
Mutant Influenza Viruses with a Defective NS1 Protein Cannot Block the Activation of PKR in Infected Cells
Authors: Hatada Eriko , Saito Sakura , Fukuda Ryuji
Published: December 2019

A short model genome RNA and also the genome RNA of influenza A virus bearing both 5′- and 3′-terminal common sequences activated the interferon-induced double-stranded-RNA-dependent protein kinase, PKR, by stimulating autophosphorylation in vitro. The activated PKR catalyzed phosphorylation of the alpha subunit of eucaryotic translation initiation factor 2 (eIF2α). The NS1 protein efficiently eliminated the PKR-activating activity of these RNAs by binding to them. Two mutant NS1 proteins, each harboring a single amino acid substitution at different regions, exhibited temperature sensitivity in their RNA binding activity in the mutant virus-infected cell lysates as well as when they were prepared as fusion proteins expressed in bacteria. The virus strains carrying these mutant NS1 proteins exhibited temperature sensitivity in virus protein synthesis at the translational level, as reported previously, and could not repress the autophosphorylation of PKR developing during the virus growth, which is normally suppressed by a viral function(s). As a result, the level of eIF2α phosphorylation was elevated 2.5- to 3-fold. The defect in virus protein synthesis was well correlated with the level of phosphorylation of PKR and eIF2α.
Moloney Murine Leukemia Virus-Induced Preleukemic Thymic Atrophy and Enhanced Thymocyte Apoptosis Correlate with Disease Pathogenicity
Authors: Bonzon Christine , Fan Hung
Published: December 2019

Moloney murine leukemia virus (M-MuLV) is a replication-competent, simple retrovirus that induces T-cell lymphoma with a mean latency of 3 to 4 months. During the preleukemic period (4 to 10 weeks postinoculation) a marked decrease in thymic size is apparent for M-MuLV-inoculated mice in comparison to age-matched uninoculated mice. We were interested in studying whether the thymic regression was due to an increased rate of thymocyte apoptosis in the thymi of M-MuLV-inoculated mice. Neonatal NIH/Swiss mice were inoculated subcutaneously (s.c.) with wild-type M-MuLV (approximately 10
5
XC PFU). Mice were sacrificed at 4 to 11 weeks postinoculation. Thymic single-cell suspensions were prepared and tested for apoptosis by two-parameter flow cytometry. Indications of apoptosis included changes in cell size and staining with 7-aminoactinomycin D or annexin V. The levels of thymocyte apoptosis were significantly higher in M-MuLV-inoculated mice than in uninoculated control animals, and the levels of apoptosis were correlated with thymic atrophy. To test the relevance of enhanced thymocyte apoptosis to leukemogenesis, mice were inoculated with the Mo+PyF101 enhancer variant of M-MuLV. When inoculated intraperitoneally, a route that results in wild-type M-MuLV leukemogenesis, mice displayed levels of enhanced thymocyte apoptosis comparable to those seen with wild-type M-MuLV. However, in mice inoculated s.c., a route that results in attenuated leukemogenesis, significantly lower levels of apoptosis were observed. This supported a role for higher levels of thymocyte apoptosis in M-MuLV leukemogenesis. To examine the possible role of mink cell focus-forming (MCF) recombinant virus in raising levels of thymocyte apoptosis, MCF-specific focal immunofluorescence assays were performed on thymocytes from preleukemic mice inoculated with M-MuLV and Mo+PyF101 M-MuLV. The results indicated that infection of thymocytes by MCF virus recombinants is not required for the increased level of apoptosis and thymic atrophy.
A Conserved Tryptophan-Rich Motif in the Membrane-Proximal Region of the Human Immunodeficiency Virus Type 1 gp41 Ectodomain Is Important for Env-Mediated Fusion and Virus Infectivity
Authors: Salzwedel Karl , West John T. , Hunter Eric
Published: December 2019

Mutations were introduced into the ectodomain of the human immunodeficiency virus type 1 (HIV-1) transmembrane envelope glycoprotein, gp41, within a region immediately adjacent to the membrane-spanning domain. This region, which is predicted to form an α-helix, contains highly conserved hydrophobic residues and is unusually rich in tryptophan residues. In addition, this domain overlaps the epitope of a neutralizing monoclonal antibody, 2F5, as well as the sequence corresponding to a peptide, DP-178, shown to potently neutralize virus. Site-directed mutagenesis was used to create deletions, substitutions, and insertions centered around a stretch of 17 hydrophobic and uncharged amino acids (residues 666 to 682 of the HXB2 strain of HIV-1) in order to determine the role of this region in the maturation and function of the envelope glycoprotein. Deletion of the entire stretch of 17 amino acids abrogated the ability of the envelope glycoprotein to mediate both cell-cell fusion and virus entry without affecting the normal maturation, transport, or CD4-binding ability of the protein. This phenotype was also demonstrated by substituting alanine residues for three of the five tryptophan residues within this sequence. Smaller deletions, as well as multiple amino acid substitutions, were also found to inhibit but not block cell-cell fusion. These results demonstrate the crucial role of a tryptophan-rich motif in gp41 during a post-CD4-binding step of glycoprotein-mediated fusion. The basis for the invariant nature of the tryptophans, however, appears to be at the level of glycoprotein incorporation into virions. Even the substitution of phenylalanine for a single tryptophan residue was sufficient to reduce Env incorporation and drop the efficiency of virus entry approximately 10-fold, despite the fact that the same mutation had no significant effect on syncytium formation.
Productive Infection of Human Peripheral Blood Mononuclear Cells by Feline Immunodeficiency Virus: Implications for Vector Development
Authors: Johnston James , Power Christopher
Published: December 2019

Feline immunodeficiency virus (FIV) is a lentivirus causing immune suppression and neurological disease in cats. Like primate lentiviruses, FIV utilizes the chemokine receptor CXCR4 for infection. In addition, FIV gene expression has been demonstrated in immortalized human cell lines. To investigate the extent and mechanism by which FIV infected primary and immortalized human cell lines, we compared the infectivity of two FIV strains, V
1
CSF and Petaluma, after cell-free infection. FIV genome was detected in infected human peripheral blood mononuclear cells (PBMC) and macrophages at 21 and 14 days postinfection, respectively. Flow cytometry analysis of FIV-infected human PBMC indicated that antibodies to FIV p24 recognized 12% of the cells. Antibodies binding the CCR3 chemokine receptor maximally inhibited infection of human PBMC by both FIV strains compared to antibodies to CXCR4 or CCR5. Reverse transcriptase levels increased in FIV-infected human PBMC, with detection of viral titers of 10
1.3
to 10
2.1
50% tissue culture infective doses/10
6
cells depending on the FIV strain examined. Cell death in human PBMC infected with either FIV strain was significantly elevated relative to uninfected control cultures. These findings indicate that FIV can productively infect primary human cell lines and that viral strain specificity should be considered in the development of an FIV vector for gene therapy.
Residues Critical for Duck Hepatitis B Virus Neutralization Are Involved in Host Cell Interaction
Authors: Sunyach Claire , Rollier Christine , Robaczewska Magdalena , Borel Christelle , Barraud Luc , Kay Alan , Trépo Christian , Will Hans , Cova Lucyna
Published: December 2019

To date, no detailed analysis of the neutralization properties of duck hepatitis B virus (DHBV) has been reported, and it is not clear whether any of the known neutralization epitopes correspond to the viral receptor binding site or to sequences involved in the cell entry pathway. We demonstrate here that antibodies directed against two overlapping peptides (amino acids 83 to 97 and 93 to 107), covering the sequences of most DHBV pre-S neutralizing epitopes, both inhibit virus binding to primary duck hepatocytes and neutralize virus infectivity. An extensive mutagenesis of the motif
88
WTP
90
, which is the shortest sequence of the epitope recognized by the virus-neutralizing monoclonal antibody (MAb) 900 was performed in order to define the amino acids involved in these interactions. Single point mutations within this epitope affected neither virus replication nor infectivity but abolished virus neutralization by MAb 900 completely. Interestingly, mutants with two and three consecutive residue replacements (SIP and SIH) within this epitope retained replication competence but were no longer infectious. The loss of infectivity of SIH and SIP mutant particles was associated with significantly reduced binding to primary duck hepatocytes and could be rescued by
trans
complementation with wild-type pre-S protein. Taken together, these results indicate that each amino acid of the DHBV pre-S sequence
88
WTP
90
is critical for recognition by the neutralizing MAb 900 and that replacement of the first two or all three residues strongly reduces virus interaction with hepatocytes and abrogates infectivity. These data imply that the motif
88
WTP
90
contains key residues which are critical for interaction with both the neutralizing MAb and the host cell.
EBP2, a Human Protein That Interacts with Sequences of the Epstein-Barr Virus Nuclear Antigen 1 Important for Plasmid Maintenance
Authors: Shire Kathy , Ceccarelli Derek F. J. , Avolio-Hunter Tina M. , Frappier Lori
Published: December 2019

The replication and stable maintenance of latent Epstein-Barr virus (EBV) DNA episomes in human cells requires only one viral protein, Epstein-Barr nuclear antigen 1 (EBNA1). To gain insight into the mechanisms by which EBNA1 functions, we used a yeast two-hybrid screen to detect human proteins that interact with EBNA1. We describe here the isolation of a protein, EBP2 (EBNA1 binding protein 2), that specifically interacts with EBNA1. EBP2 was also shown to bind to DNA-bound EBNA1 in a one-hybrid system, and the EBP2-EBNA1 interaction was confirmed by coimmunoprecipitation from insect cells expressing these two proteins. EBP2 is a 35-kDa protein that is conserved in a variety of organisms and is predicted to form coiled-coil interactions. We have mapped the region of EBNA1 that binds EBP2 and generated internal deletion mutants of EBNA1 that are deficient in EBP2 interactions. Functional analyses of these EBNA1 mutants show that the ability to bind EBP2 correlates with the ability of EBNA1 to support the long-term maintenance in human cells of a plasmid containing the EBV origin,
oriP
. An EBNA1 mutant lacking amino acids 325 to 376 was defective for EBP2 binding and long-term
oriP
plasmid maintenance but supported the transient replication of
oriP
plasmids at wild-type levels. Thus, our results suggest that the EBNA1-EBP2 interaction is important for the stable segregation of EBV episomes during cell division but not for the replication of the episomes.
Drastic Fitness Loss in Human Immunodeficiency Virus Type 1 upon Serial Bottleneck Events
Authors: Yuste Eloisa , Sánchez-Palomino Sonsoles , Casado Concha , Domingo Esteban , López-Galíndez Cecilio
Published: December 2019

Muller’s ratchet predicts fitness losses in small populations of asexual organisms because of the irreversible accumulation of deleterious mutations and genetic drift. This effect should be enhanced if population bottlenecks intervene and fixation of mutations is not compensated by recombination. To study whether Muller’s ratchet could operate in a retrovirus, 10 biological clones were derived from a human immunodeficiency virus type 1 (HIV-1) field isolate by MT-4 plaque assay. Each clone was subjected to 15 plaque-to-plaque passages. Surprisingly, genetic deterioration of viral clones was very drastic, and only 4 of the 10 initial clones were able to produce viable progeny after the serial plaque transfers. Two of the initial clones stopped forming plaques at passage 7, two others stopped at passage 13, and only four of the remaining six clones yielded infectious virus. Of these four, three displayed important fitness losses. Thus, despite virions carrying two copies of genomic RNA and the system displaying frequent recombination, HIV-1 manifested a drastic fitness loss as a result of an accentuation of Muller’s ratchet effect.
A Second-Site Mutation That Restores Replication of a Tat-Defective Human Immunodeficiency Virus
Authors: Verhoef Koen , Berkhout Ben
Published: December 2019

We previously constructed a large set of mutants of the human immunodeficiency virus type 1 (HIV-1) regulatory protein Tat with conservative amino acid substitutions in the activation domain. These Tat variants were analyzed in the context of the infectious virus, and several mutants were found to be defective for replication. In an attempt to obtain second-site suppressor mutations that could provide information on the Tat protein structure, some of the replication-impaired viruses were used as a parent for the isolation of revertant viruses with improved replication capacity. Sequence analysis of revertant viruses frequently revealed changes within the
tat
gene, most often first-site reversions either to the wild-type amino acid or to related amino acids that restore, at least partially, the Tat function and virus replication. Of 30 revertant cultures, we identified only one second-site suppressor mutation. The inactive Y26A mutant yielded the second-site suppressor mutation Y47N that partially restored
trans
-activation activity and virus replication. Surprisingly, when the suppressor mutation was introduced in the wild-type Tat background, it also improved the
trans
-activation function of this protein about twofold. We conclude that the gain of function measured for the Y47N change is not specific for the Y26A mutant, arguing against a direct interaction of Tat amino acids 26 and 47 in the three-dimensional fold of this protein. Other revertant viruses did not contain any additional Tat changes, and some viruses revealed putative second-site Tat mutations that did not significantly improve Tat function and virus replication. We reason that these mutations were introduced by chance through founder effects or by linkage to suppressor mutations elsewhere in the virus genome. In conclusion, the forced evolution of mutant HIV-1 genomes, which is an efficient approach for the analysis of RNA regulatory motifs, seems less suited for the analysis of the structure of this small transcription factor, although protein variants with interesting properties can be generated.
Interaction of the Human Immunodeficiency Virus Type 1 Nucleocapsid with Actin
Authors: Liu Bindong , Dai Renke , Tian Chun-Juan , Dawson Liza , Gorelick Robert , Yu Xiao-Fang
Published: December 2019

The nucleocapsid (NC) domain of the retrovirus Gag protein plays several important roles in the viral life cycle, including virus assembly, viral genomic RNA encapsidation, primer tRNA placement, and enhancement of viral reverse transcription. In this study, deletion of NC domain of human immunodeficiency virus type 1 (HIV-1) Gag was found to drastically reduce virus particle production in CD4
+
T cells. Cellular fractionation experiments showed that although most of the uncleaved wild-type HIV-1 Gag, unmyristylated Gag, and p6
Gag
domain-truncated Gag molecules copurified with the host cell cytoskeleton, most of the mutant Gag molecules lacking both the NC and p6
Gag
domains failed to cofractionate with cytoskeleton. In wild-type virus-infected cells, in which the viral protease was active, the cleaved NCp7 copurified with the cytoskeleton, whereas most of the MAp17 and CAp24 did not. Monoclonal antibody against actin coimmunoprecipitated full-length Gag and p6
Gag
domain-truncated Gag molecules from cell lysates but failed to precipitate the truncated mutant Gag molecules lacking NC plus p6
Gag
. Purified recombinant NCp7, but not CAp24, was able to bind F-actin in cosedimentation experiments. Furthermore, wild-type NCp7 and a zinc finger mutant NCp7(F16A), like a cellular actin-binding protein (the villin headpiece), bound F-actin in a dose-dependent fashion in vitro. Taken together, these results suggest that HIV-1 NCp7 can bind F-actin directly and that interaction between HIV-1 Gag and the actin cytoskeleton through the NC domain may play an important role in HIV-1 assembly and/or other steps of the viral life cycle.
RNA-Stimulated ATPase and RNA Helicase Activities and RNA Binding Domain of Hepatitis G Virus Nonstructural Protein 3
Authors: Gwack Yousang , Yoo Hyouna , Song Inyoung , Choe Joonho , Han Jang H.
Published: December 2019

Hepatitis G virus (HGV) nonstructural protein 3 (NS3) contains amino acid sequence motifs typical of ATPase and RNA helicase proteins. In order to examine the RNA helicase activity of the HGV NS3 protein, the NS3 region (amino acids 904 to 1580) was fused with maltose-binding protein (MBP), and the fusion protein was expressed in
Escherichia coli
and purified with amylose resin and anion-exchange chromatography. The purified MBP-HGV/NS3 protein possessed RNA-stimulated ATPase and RNA helicase activities. Characterization of the ATPase and RNA helicase activities of MBP-HGV/NS3 showed that the optimal reaction conditions were similar to those of other
Flaviviridae
viral NS3 proteins. However, the kinetic analysis of NTPase activity showed that the MBP-HGV/NS3 protein had several unique properties compared to the other
Flaviviridae
NS3 proteins. The HGV NS3 helicase unwinds RNA-RNA duplexes in a 3′-to-5′ direction and can unwind RNA-DNA heteroduplexes and DNA-DNA duplexes as well. In a gel retardation assay, the MBP-HGV/NS3 helicase bound to RNA, RNA/DNA, and DNA duplexes with 5′ and 3′ overhangs but not to blunt-ended RNA duplexes. We also found that the conserved motif VI was important for RNA binding. Further deletion mapping showed that the RNA binding domain was located between residues 1383 and 1395, QRRGRTGRGRSGR. Our data showed that the MBP-HCV/NS3 protein also contains the RNA binding domain in the similar domain.
Human Antibody Responses to Mature and Immature Forms of Viral Envelope in Respiratory Syncytial Virus Infection: Significance for Subunit Vaccines
Authors: Sakurai Hiroshi , Williamson R. Anthony , Crowe James E. , Beeler Judy A. , Poignard Pascal , Bastidas Raiza B. , Chanock Robert M. , Burton Dennis R.
Published: December 2019

A number of antibodies generated during human respiratory syncytial virus (RSV) infection have been cloned by the phage library approach. Antibodies reactive with an immunodominant epitope on the F glycoprotein of this virus have a high affinity for affinity-purified F antigen. These antibodies, however, have a much lower affinity for mature F glycoprotein on the surface of infected cells and are nonneutralizing. In contrast, a potent neutralizing antibody has a high affinity for mature F protein but a much lower affinity for purified F protein or F protein in viral lysates. The data indicate that at least two F protein immunogens are produced during natural RSV infection: immature F, found in viral lysates, and mature F, found on infected cells or virions. Binding studies with polyclonal human immunoglobulin G suggest that the antibody responses to the two immunogens are of similar magnitudes. Competitive binding studies suggest that overlap between the responses is relatively limited. A mature envelope with an antigenic configuration different from that of the immature envelope has an evolutionary advantage in that the infecting virus is less subject to neutralization by the humoral response to the immature envelope that inevitably arises following lysis of infected cells. Subunit vaccines may be at a disadvantage because they most often resemble immature envelope molecules and ignore this aspect of viral evasion.