bluuebunny/crossref_metadata_2025_split huggingface dataset:
Intracellular Interaction of Simian Immunodeficiency Virus Gag and Env Proteins
Authors: Vincent Martin J. , Melsen Lawrence R. , Martin Annelet S. , Compans Richard W. 
Published: December 2019

ABSTRACTIn polarized epithelial cells, the assembly and release of human immunodeficiency virus type 1 (HIV-1) occur at the basolateral side of the plasma membrane, and the site of assembly is determined by the site of expression of the Env protein. In order to investigate whether the expression of the Env proteins exclusively in the endoplasmic reticulum (ER) can alter the site of virus assembly, we coexpressed the simian immunodeficiency virus (SIV) Gag protein and mutant SIV Env proteins having an ER retrieval signal (KKXX motif). In cells expressing the wild-type (wt) Env protein or coexpressing Env and Gag proteins, the Env protein was processed into the surface (SU) and transmembrane (TM) proteins. In contrast, in cells expressing the mutant Env proteins alone or in combination with Gag, the Env proteins were retrieved to the ER and were not proteolytically processed. Coexpression of the Gag and ER-retained mutant Env proteins resulted in a transient decrease in the release of the Gag protein into the medium, suggesting an interaction between the Gag and ER-retrieved Env proteins. Using saponin-permeabilized cells coexpressing Gag and Env proteins, we obtained further evidence for Env-Gag interaction. A monoclonal antibody specific to the SIV Gag protein was found to coimmunoprecipitate both the Gag and Env proteins. The interaction was specific, as coexpressed SIV Env proteins without the cytoplasmic tail or a chimeric HIV-1 Env proteins with the CD4 cytoplasmic tail were not coimmunoprecipitated by the Gag-specific antibody. Electron microscopic analyses indicated that assembly of virus particles occurred only at the surfaces of cells in which the Gag protein was coexpressed with either the wt or ER-retrieved mutant Env protein. These data indicate that although the Env and Gag proteins interact intracellularly, the site of assembly of SIV is not redirected to an intracellular organelle by the retrieval of the Env protein to the ER.
Bovine Leukemia Virus Structural Gene Vectors Are Immunogenic and Lack Pathogenicity in a Rabbit Model
Authors: Kucerova Lucia , Altanerova Veronika , Altaner Cestmir , Boris-Lawrie Kathleen 
Published: December 2019

ABSTRACT

Infection with a replication-competent bovine leukemia virus structural gene vector (BLV SGV) is an innovative vaccination approach to prevent disease by complex retroviruses. Previously we developed BLV SGV that constitutively expresses BLV
gag
,
pol
, and
env
and related
cis
-acting sequences but lacks
tax
,
rex
,
RIII
, and
GIV
and most of the BLV long terminal repeat sequences, including the
cis
-acting Tax and Rex response elements. The novel SGV virus is replication competent and replicates a selectable vector to a titer similar to that of the parental BLV in cell culture. The overall goal of this study was to test the hypothesis that infection with BLV SGV is nonpathogenic in rabbits. BLV infection of rabbits by inoculation of cell-free BLV or cell-associated BLV typically causes an immunodeficiency-like syndrome and death by 1 year postinfection. We sought to evaluate whether in vivo transfection of BLV provirus recapitulates pathogenic BLV infection and to compare BLV and BLV SGV with respect to infection, immunogenicity, and clinical outcome. Three groups of rabbits were subjected to in vivo transfection with BLV, BLV SGV, or negative control DNA. The results of our 20-month study indicate that in vivo transfection of rabbits with BLV recapitulates the fatal BLV infection produced by cell-free or cell-associated BLV. The BLV-infected rabbits exhibited sudden onset of clinical decline and immunodeficiency-like symptoms that culminated in death. BLV and BLV SGV infected peripheral blood mononuclear cells and induced similar levels of seroconversion to BLV structural proteins. However, BLV SGV exhibited a reduced proviral load and did not trigger the immunodeficiency-like syndrome. These results are consistent with the hypothesis that BLV SGV is infectious and immunogenic and lacks BLV pathogenicity in rabbits, and they support the use of this modified proviral vector delivery system for vaccines against complex retroviruses like BLV.
Factors Affecting the Terminal Resolution Site Endonuclease, Helicase, and ATPase Activities of Adeno-Associated Virus Type 2 Rep Proteins
Authors: Wu Jianwen , Davis Michael D. , Owens Roland A. 
Published: December 2019

ABSTRACT

The Rep68 and Rep78 proteins (Rep68/78) of adeno-associated virus type 2 (AAV) are critical for AAV replication and site-specific integration. They bind specifically to the AAV inverted terminal repeats (ITRs) and possess ATPase, helicase, and strand-specific/site-specific endonuclease activities. In the present study, we further characterized the AAV Rep68/78 helicase, ATPase, and endonuclease activities by using a maltose binding protein-Rep68 fusion (MBP-Rep68Δ) produced in
Escherichia coli
cells and Rep78 produced in vitro in a rabbit reticulocyte lysate system. We found that the minimal length of single-stranded DNA capable of stimulating the ATPase activity of MBP-Rep68Δ is 100 to 200 bases. The degree of stimulation correlated positively with the length of single-stranded DNA added to the reaction mixture. We then determined the ATP concentration needed for optimal MBP-Rep68Δ helicase activity and showed that the helicase is active over a wide range of ATP concentrations. We determined the directionality of MBP-Rep68Δ helicase activity and found that it appears to move in a 3′ to 5′ direction, which is consistent with a model in which AAV Rep68/78 participates in AAV DNA replication by unwinding DNA ahead of a cellular DNA polymerase. In this report, we also demonstrate that single-stranded DNA is capable of inhibiting the MBP-Rep68Δ or Rep78 endonuclease activity greater than 10-fold. In addition, we show that removal of the secondary Rep68/78 binding site, which is found only in the hairpin form of the AAV ITR, causes a three- to eightfold reduction in the ability of the ITR to be used as a substrate for the Rep78 or MBP-Rep68Δ endonuclease activity. This suggests that contact between Rep68/78 and this secondary element may play an important role in the Rep-mediated endonuclease activity.
Avirulent Avian Influenza Virus as a Vaccine Strain against a Potential Human Pandemic
Authors: Takada Ayato , Kuboki Noritaka , Okazaki Katsunori , Ninomiya Ai , Tanaka Hiroko , Ozaki Hiroichi , Itamura Shigeyuki , Nishimura Hidekazu , Enami Masayoshi , Tashiro Masato , Shortridge Kennedy F. , Kida Hiroshi 
Published: December 2019

ABSTRACTIn the influenza H5N1 virus incident in Hong Kong in 1997, viruses that are closely related to H5N1 viruses initially isolated in a severe outbreak of avian influenza in chickens were isolated from humans, signaling the possibility of an incipient pandemic. However, it was not possible to prepare a vaccine against the virus in the conventional embryonated egg system because of the lethality of the virus for chicken embryos and the high level of biosafety therefore required for vaccine production. Alternative approaches, including an avirulent H5N4 virus isolated from a migratory duck as a surrogate virus, H5N1 virus as a reassortant with avian virus H3N1 and an avirulent recombinant H5N1 virus generated by reverse genetics, have been explored. All vaccines were formalin inactivated. Intraperitoneal immunization of mice with each of vaccines elicited the production of hemagglutination-inhibiting and virus-neutralizing antibodies, while intranasal vaccination without adjuvant induced both mucosal and systemic antibody responses that protected the mice from lethal H5N1 virus challenge. Surveillance of birds and animals, particularly aquatic birds, for viruses to provide vaccine strains, especially surrogate viruses, for a future pandemic is stressed.
Proteolytic Cleavage of the Amino Terminus of the U
L
15 Gene Product of Herpes Simplex Virus Type 1 Is Coupled with Maturation of Viral DNA into Unit-Length Genomes
Authors: Salmon Brandy , Nalwanga Dorothy , Fan Ying , Baines Joel D. 
Published: December 2019

ABSTRACT

The U
L
15 gene of herpes simplex virus type 1 (HSV-1), like U
L
6, U
L
17, U
L
28, U
L
32, and U
L
33, is required for cleavage of concatameric DNA into genomic lengths and for packaging of cleaved genomes into preformed capsids. A previous study indicated that the U
L
15 gene encodes minor capsid proteins. In the present study, we have shown that the amino-terminal 509 amino acids of the U
L
15-encoded protein are sufficient to confer capsid association inasmuch as a carboxyl-terminally truncated form of the U
L
15-encoded protein with an
M
r
of approximately 55,000 readily associated with capsids. This and previous studies have shown that, whereas three U
L
15-encoded proteins with apparent
M
r
s of 83,000, 80,000, and 79,000 associated with wild-type B capsids, only the full-length 83,000-
M
r
protein associated with B capsids purified from cells infected with viruses lacking functional U
L
6, U
L
17, U
L
28, U
L
32, and U
L
33 genes (B. Salmon and J. D. Baines, J. Virol. 72:3045–3050, 1998). Thus, all viral mutants that fail to cleave viral DNA into genomic-length molecules also fail to produce capsid-associated U
L
15 80,000- and 79,000-
M
r
proteins. In contrast, the 80,000- and 79,000-
M
r
proteins were readily detected in capsids purified from cells infected with a U
L
25 null virus that cleaves, but does not package, DNA. The conclusion that the amino terminus of the 83,000-
M
r
protein is truncated to produce the 80,000- and/or 79,000-
M
r
protein was supported by the following observations. (i) Whereas the C termini of the 83,000-, 80,000-, and 79,000-
M
r
proteins are identical, immunoreactivity dependent on the first 35 amino acids of the U
L
15 83,000-
M
r
protein was absent from the 80,000- and 79,000-
M
r
proteins. (ii) The 79,000- and 80,000-
M
r
proteins were detected in capsids from cells infected with HSV-1(U
L
15M36V), an engineered virus encoding valine rather than methionine at codon 36. Thus, initiation at codon 36 is unlikely to account for production of the 80,000- and/or 79,000-
M
r
protein. Taken together, these data strongly suggest that capsid-associated U
L
15-encoded protein is proteolytically cleaved near the N terminus and indicate that this modification is tightly linked to maturation of genomic DNA.
A Phylogenetically Conserved Hairpin-Type 3′ Untranslated Region Pseudoknot Functions in Coronavirus RNA Replication
Authors: Williams Gwyn D. , Chang Ruey-Yi , Brian David A. 
Published: December 2019

ABSTRACT
Secondary and tertiary structures in the 3′ untranslated region (UTR) of plus-strand RNA viruses have been postulated to function as control elements in RNA replication, transcription, and translation. Here we describe a 54-nucleotide (nt) hairpin-type pseudoknot within the 288-nt 3′ UTR of the bovine coronavirus genome and show by mutational analysis of both stems that the pseudoknotted structure is required for the replication of a defective interfering RNA genome. The pseudoknot is phylogenetically conserved among coronaviruses both in location and in shape but only partially in nucleotide sequence, and evolutionary covariation of bases to maintain G · U pairings indicates that it functions in the plus strand. RNase probing of synthetic transcripts provided additional evidence of its tertiary structure and also identified the possible existence of two conformational states. These results indicate that the 3′ UTR pseudoknot is involved in coronavirus RNA replication and lead us to postulate that it functions as a regulatory control element.
The Human Immunodeficiency Virus Type 1nefGene Can to a Large Extent Replace Simian Immunodeficiency VirusnefIn Vivo
Authors: Kirchhoff Frank , Münch Jan , Carl Silke , Stolte Nicole , Mätz-Rensing Kerstin , Fuchs Dietmar , Ten Haaft Peter , Heeney Jonathan L. , Swigut Tomek , Skowronski Jacek , Stahl-Hennig Christiane 
Published: December 2019

ABSTRACTThenefgene of the pathogenic simian immunodeficiency virus (SIV) 239 clone was replaced with primary human immunodeficiency virus type 1 (HIV-1)nefalleles to investigate whether HIV-1 Nef can substitute for SIV Nef in vivo. Initially, two rhesus macaques were infected with the chimeric viruses (Nef-SHIVs). Most of thenefalleles obtained from both animals predicted intact open reading frames. Furthermore, forms containing upstream nucleotide substitutions that enhanced expression of the inserted gene became predominant. One animal maintained high viral loads and slowly progressed to immunodeficiency.neflong terminal repeat sequences amplified from this animal were used to generate a second generation of Nef-SHIVs. Two macaques, which were subsequently infected with a mixture of cloned chimeric viruses, showed high viral loads and progressed to fatal immunodeficiency. Five macaques received a single molecular clone, named SHIV-40K6. The SHIV-40K6nefallele was active in CD4 and class I major histocompatibility complex downregulation and enhanced viral infectivity and replication. Notably, all of the macaques inoculated with SHIV-40K6 showed high levels of viral replication early in infection. During later stages, however, the course of infection was variable. Three animals maintained high viral loads and developed immunodeficiency. Of the remaining two macaques, which showed decreasing viral loads after the acute phase of infection, only one efficiently controlled viral replication and remained asymptomatic during 1.5 years of follow-up. The other animal showed an increasing viral load and developed signs of progressive infection during later stages. Our data demonstrate that HIV-1nefcan, to a large extent, functionally replace SIVmacnefin vivo.
Patchwork Pattern of Transcriptional Reactivation in the Lungs Indicates Sequential Checkpoints in the Transition from Murine Cytomegalovirus Latency to Recurrence
Authors: Kurz Sabine K. , Reddehase Matthias J. 
Published: December 2019

ABSTRACT

The lungs are a significant organ site of murine cytomegalovirus (mCMV) latency. We have shown that activity of the major immediate-early promoter (MIEP), which drives the transcription from the
ie1-ie3
transcription unit, does not inevitably initiate the productive cycle (S. K. Kurz, M. Rapp, H.-P. Steffens, N. K. A. Grzimek, S. Schmalz, and M. J. Reddehase, J. Virol. 73:482–494, 1999). Thus, even though MIEP activity governed by the MIEP-enhancer is unquestionably the first condition for recurrence, regulation of the enhancer by transcription factors is not the only mechanism controlling latency. Specifically, during latency, focal and stochastic MIEP activity in lung tissue was found to selectively generate IE1 transcripts, while transactivator-specifying IE3 transcripts were missing. This suggested a control of mCMV latency that is effectual at IE1-IE3 precursor mRNA cotranscriptional processing. Here we have used this model for studying the kinetics of reactivation and recurrence in individual lung tissue pieces after hematoablative, genotoxic treatment. Notably, reactivation was triggered, but the number of transcriptionally active foci in the lungs did not increase over time. This result is not compatible with a model of spontaneous reactivations accumulating after withdrawal of immune control. Instead, the data support the idea that reactivation is an induced event. In some pieces, focal reactivation generated IE3 transcripts but not gB transcripts, while other pieces contained foci that had proceeded to gB transcription, and only a few foci actually reached the state of virus recurrence. This finding indicates the existence of several sequentially ordered control points in the transition from mCMV latency to recurrence.
Targeting Human Immunodeficiency Virus (HIV) Type 2 Integrase Protein into HIV Type 1
Authors: Liu Hongmei , Wu Xiaoyun , Xiao Hongling , Kappes John C. 
Published: December 2019

ABSTRACT

Integrase (IN) is the only retroviral enzyme necessary for the integration of retroviral cDNA into the host cell’s chromosomes. The structure and function of IN is highly conserved. The human immunodeficiency virus type 2 (HIV-2) IN has been shown to efficiently support 3′ processing and strand transfer of HIV-1 DNA substrate in vitro. To determine whether HIV-2 IN protein (IN
2
) could substitute for HIV-1 IN function in vivo, we used HIV-1 Vpr to deliver the IN
2
into IN mutant HIV-1 virions by expression in
trans
as a Vpr-IN fusion protein.
Trans
-complementation with IN
2
markedly increased the infectivity of IN-minus HIV-1. Compared with the homologous
trans
-IN protein, infectivity was increased to a level of 16%. Since IN has been found to play a role in reverse transcription (Wu et al., J. Virol. 73:2126–2135, 1999), cells infected with IN
2
-complemented HIV-1 were analyzed for DNA products of reverse transcription. DNA levels of approximately 18% of that of wild type were detected. The homologous
trans
-IN protein restored the synthesis of viral cDNA to approximately 86% of that of wild-type virus. By complementing integration-defective HIV-1 IN mutant viruses, which were not impaired in cDNA synthesis, the
trans
-IN
2
protein was shown to support integration up to a level of 55% compared with that of the homologous
trans
-IN protein. The delivery of heterologous IN protein into HIV-1 particles in
trans
offers a novel approach to understand IN protein function in vivo.
Development of Murine Leukemia Virus-Based Self-Activating Vectors That Efficiently Delete the Selectable Drug Resistance Gene during Reverse Transcription
Authors: Delviks Krista A. , Pathak Vinay K. 
Published: December 2019

ABSTRACT
Expression of the selectable drug resistance gene in retroviral vectors used for gene therapy can lead to a decreased expression of the gene of interest and may induce a host immune response, resulting in a decreased efficiency of gene therapy. In this study, we demonstrate that high-frequency deletion of direct repeats, an inherent property of reverse transcriptases, can be used to efficiently excise the drug resistance gene during reverse transcription. One retroviral vector containing a direct repeat deleted the neomycin resistance expression cassette during a single replication cycle at >99% efficiency.
Walleye Dermal Sarcoma Virus: OrfA N-Terminal End Inhibits the Activity of a Reporter Gene Directed by Eukaryotic Promoters and Has a Negative Effect on the Growth of Fish and Mammalian Cells
Authors: Zhang Z. , Martineau D. 
Published: December 2019

ABSTRACT

Walleye dermal sarcoma virus (WDSV) is a fish retrovirus causing a skin tumor termed walleye dermal sarcoma, which develops and regresses on a seasonal basis. The WDSV genome contains three short open reading frames designated
orfA
,
orfB
, and
orfC
in addition to the viral structural genes,
gag
,
pol
, and
env
.
orfA
and
orfB
transcripts are detected in tumors by reverse transcription-PCR. Recently, OrfA, whose amino acid sequence is similar to that of cyclins A and D, has been shown to complement a cyclin-deficient yeast strain. We report that expression of the accessory gene
orfA
inhibited nonspecifically the activity of a reporter gene directed by various eukaryotic promoters. In addition, stable transfection with the wild-type
orfA
generated substantially fewer G418-resistant colonies in both fish and mammalian cells than the parent vector. An
orfA
mutant expressing only the first N-terminal 49 residues of the full-length protein had the same negative effect on the activity of the reporter gene and on the number of stably transfected colonies as the full-length OrfA. Thus, OrfA inhibits cell growth and/or causes cell death, and the first 49 N-terminal residues of this protein are sufficient to cause these negative effects.
Divergent Requirements for the MAPKERKSignal Transduction Pathway during Initial Virus Infection of Quiescent Primary B Cells and Disruption of Epstein-Barr Virus Latency by Phorbol Esters
Authors: Fenton Mandy , Sinclair Alison J. 
Published: December 2019

ABSTRACTQuiescent primary B lymphocytes and Epstein-Barr virus (EBV)-immortalized lymphoblastoid cell lines express components of the extracellular response kinase arm of the mitogen-activated protein kinase (MAPKERK) signal transduction pathway and transmit signals through the pathway when exposed to appropriate stimuli. Although the MAPKERKpathway is activated following infection with EBV, MAPK/ERK kinase (MEK1) activity is not required to drive the proliferation of infected cells. However, MEK1 contributes to EBV latency control.
Characterization of Intracellular Reverse Transcription Complexes of Moloney Murine Leukemia Virus
Authors: Fassati Ariberto , Goff Stephen P. 
Published: December 2019

ABSTRACT
To examine the early events in the life cycle of Moloney murine leukemia virus (MoMLV), we analyzed the intracellular complexes mediating reverse transcription. Partial purification of the reverse transcription complexes (RTCs) by equilibrium density fractionation and velocity sedimentation indicated that three distinct species of intracellular complexes are formed shortly after cell infection. Only one of these species is able to start and complete reverse transcription in the cell cytoplasm. This RTC is composed of at least the viral genome, capsid, integrase, and reverse transcriptase proteins. The RTC becomes permeable to micrococcal nuclease but not to antibodies. Shortly after initiation of reverse transcription, the viral strong stop DNA within the RTC is protected from nuclease digestion. The sedimentation velocity of the RTC decreases during reverse transcription. After entry into the nucleus, most capsid proteins are lost from the RTC and its sedimentation velocity decreases further.
A Cell Line-Based Neutralization Assay for Primary Human Immunodeficiency Virus Type 1 Isolates That Use either the CCR5 or the CXCR4 Coreceptor
Authors: Trkola Alexandra , Matthews Jamie , Gordon Cynthia , Ketas Tom , Moore John P. 
Published: December 2019

ABSTRACTWe describe here a cell line-based assay for the evaluation of human immunodeficiency virus type 1 (HIV-1) neutralization. The assay is based on CEM.NKR cells, transfected to express the HIV-1 coreceptor CCR5 to supplement the endogenous expression of CD4 and the CXCR4 coreceptor. The resulting CEM.NKR-CCR5 cells efficiently replicate primary HIV-1 isolates of both R5 and X4 phenotypes. A comparison of the CEM.NKR-CCR5 cells with mitogen-activated peripheral blood mononuclear cells (PBMC) in neutralization assays with sera from HIV-1-infected individuals or specific anti-HIV-1 monoclonal antibodies shows that the sensitivity of HIV-1 neutralization is similar in the two cell types. The CEM.NKR-CCR5 cell assay, however, is more convenient to perform and eliminates the donor-to-donor variation in HIV-1 replication efficiency, which is one of the principal drawbacks of the PBMC-based neutralization assay. We suggest that this new assay is suitable for the general measurement of HIV-1 neutralization by antibodies.