bluuebunny/crossref_metadata_2025_split huggingface dataset:
Generation of Replication-Competent Hepatitis B Virus Nucleocapsids in Insect Cells
Authors: Seifer Maria , Hamatake Robert , Bifano Marc , Standring David N. 
Published: December 2019

ABSTRACTThe double-stranded DNA genome of human hepatitis B virus (HBV) and related hepadnaviruses is reverse transcribed from a pregenomic RNA by a viral polymerase (Pol) harboring both priming and RNA- and DNA-dependent elongation activities. Although hepadnavirus replication occurs inside viral nucleocapsids, or cores, biochemical systems for analyzing this reaction are currently limited to unencapsidated Pols expressed in heterologous systems. Here, we describecisandtransclasses of replicative HBV cores, produced in the recombinant baculovirus system via coexpression of HBV core and Pol proteins from either a single RNA (i.e., incis) or two distinct RNAs (intrans). Upon isolation from insect cells,cisandtranscores contained Pol-linked HBV minus-strand DNA with 5′ ends mapping to the authentic elongation origin DR1 and also plus-strand DNA species. Onlytranscores, however, were highly active for the de novo priming and reverse transcription of authentic HBV minus strands in in vitro endogenous polymerase assays. This reaction strictly required HBV Pol but not the ɛ stem-loop element, although the presence of one ɛ, or better, two ɛs, enhanced minus-strand synthesis up to 10-fold. Compared to unencapsidated Pol enzymes, encapsidated Pol appeared to be (i) highly processive, able to extend minus-strand DNAs of 400 nucleotides from DR1 in vitro, and (ii) more active for HBV plus-strand synthesis. These observations suggest possible contributions to the replication process from the HBV core protein. These novel core reagents should facilitate the analysis of HBV replication in its natural environment, the interior of the capsid, and also fuel the development of new anti-HBV drug screens.
Suppressors of Cleavage-Site Mutations in the p62 Envelope Protein of Semliki Forest Virus Reveal Dynamics in Spike Structure and Function
Authors: Tubulekas Ioannis , Liljeström Peter 
Published: December 2019

ABSTRACT
The E2 spike glycoprotein of Semliki Forest virus is produced as a p62 precursor protein, which is cleaved by host proteases to its mature form, E2. Cleavage is not necessary for particle formation or release but is necessary for infectivity. Previous results had shown that phenotypic revertants of cleavage-deficient p62 mutants are generated, and here we show that these may contain second-site suppressor mutations in the vicinity of the cleavage site. These hot-spot sites were mutated to abolish the generation of such suppressor mutations; however, secondary mutations in another distant domain of the E2 protein appeared instead, all of which still caused cleavage-deficient mutations. Such mutants grew very poorly and were inefficient in virus entry and release. The mutated sites define domains of the spike protein which probably interact to regulate its structure and function. Because of their highly attenuated phenotype and the lower probability of reversion, the new mutations close to the cleavage site were used to make new helper vectors for packaging of recombinant RNA into infectious particles, thus increasing further the biosafety of the vector system based on the Semliki Forest virus replicon.
Sequential Steps in Human Immunodeficiency Virus Particle Maturation Revealed by Alterations of Individual Gag Polyprotein Cleavage Sites
Authors: Wiegers Klaus , Rutter Gabriel , Kottler Hubert , Tessmer Uwe , Hohenberg Heinz , Kräusslich Hans-Georg 
Published: December 2019

ABSTRACT
Retroviruses are produced as immature particles containing structural polyproteins, which are subsequently cleaved by the viral proteinase (PR). Extracellular maturation leads to condensation of the spherical core to a capsid shell formed by the capsid (CA) protein, which encases the genomic RNA complexed with nucleocapsid (NC) proteins. CA and NC are separated by a short spacer peptide (spacer peptide 1 [SP1]) on the human immunodeficiency virus type 1 (HIV-1) Gag polyprotein and released by sequential PR-mediated cleavages. To assess the role of individual cleavages in maturation, we constructed point mutations abolishing cleavage at these sites, either alone or in combination. When all three sites between CA and NC were mutated, immature particles containing stable CA-NC were observed, with no apparent effect on other cleavages. Delayed maturation with irregular morphology of the ribonucleoprotein core was observed when cleavage of SP1 from NC was prevented. Blocking the release of SP1 from CA, on the other hand, yielded normal condensation of the ribonucleoprotein core but prevented capsid condensation. A thin, electron-dense layer near the viral membrane was observed in this case, and mutant capsids were significantly less stable against detergent treatment than wild-type HIV-1. We suggest that HIV maturation is a sequential process controlled by the rate of cleavage at individual sites. Initial rapid cleavage at the C terminus of SP1 releases the RNA-binding NC protein and leads to condensation of the ribonucleoprotein core. Subsequently, CA is separated from the membrane by cleavage between the matrix protein and CA, and late release of SP1 from CA is required for capsid condensation.
A Novel Membrane Protein Is a Mouse Mammary Tumor Virus Receptor
Authors: Golovkina Tatyana V. , Dzuris John , van den Hoogen Bernadette , Jaffe Aron B. , Wright Paul C. , Cofer Shelagh M. , Ross Susan R. 
Published: December 2019

ABSTRACT
Mouse mammary tumor virus (MMTV) infects a number of different cell types, including mammary gland and lymphoid cells, in vivo. To identify the cellular receptor for this virus, a mouse cDNA expression library was transfected into Cos-7 monkey kidney cells, and those transfected cells able to bind virus were selected by using antibody against the virus’s cell surface envelope protein, gp52. One clone isolated from a library prepared from newborn thymus RNA, called MTVR, was able to confer virus binding to both monkey and human cells; this binding was blocked by anti-MTVR antibody. Moreover, transfection of MTVR into CV1 cells rendered them susceptible to infection by a murine leukemia virus-based retrovirus vector pseudotyped with the MMTV envelope protein. An epitope-tagged MTVR cofractionated with cellular membranes. Coimmunoprecipitation of the MMTV envelope protein and a MTVR-rabbit Fc fusion protein showed that these two proteins bound to each other. The MTVR sequence clone is unique, shows no homology to known membrane proteins, and is transcribed in many tissues.
Neutralizing Antibodies Protect against Lethal Flavivirus Challenge but Allow for the Development of Active Humoral Immunity to a Nonstructural Virus Protein
Authors: Kreil Thomas R. , Maier Elisabeth , Fraiss Sabine , Eibl Martha M. 
Published: December 2019

ABSTRACTAntibody-mediated neutralization of viruses has been extensively studied in vitro, but the precise mechanisms that account for antibody-mediated protection against viral infection in vivo still remain largely uncharacterized. The two points under discussion are antibodies conferring sterilizing immunity by neutralizing the virus inoculum or protection against the development of disease without complete inhibition of virus replication. For tick-borne encephalitis virus (TBEV), a flavivirus, transfer of neutralizing antibodies specific for envelope glycoprotein E protected mice from subsequent TBEV challenge. Nevertheless, short-term, low-level virus replication was detected in these mice. Furthermore, mice that were exposed to replicating but not to inactivated virus while passively protected developed active immunity to TBEV rechallenge. Despite the priming of TBEV-specific cytotoxic T cells, adoptive transfer of serum but not of T cells conferred immunity upon naive recipient mice. These transferred sera were not neutralizing and were predominantly specific for NS1, a nonstructural TBEV protein which is expressed in and on infected cells and which is also secreted from these cells. Results of these experiments showed that despite passive protection by neutralizing antibodies, limited virus replication occurs, indicating protection from disease rather than sterilizing immunity. The protective immunity induced by replicating virus is surprisingly not T-cell mediated but is due to antibodies against a nonstructural virus protein absent from the virion.
In Vitro Packaging of Adeno-Associated Virus DNA
Authors: Zhou Xiaohuai , Muzyczka Nicholas 
Published: December 2019

ABSTRACT
We have developed an in vitro procedure for packaging of recombinant adeno-associated virus (AAV). By using AAV replicative-form DNA as the substrate, it is possible to synthesize an infectious AAV particle in vitro that can be used to transfer a marker gene to mammalian cells. The packaging procedure requires the presence of both the AAV Rep and capsid proteins. Two kinds of in vitro products can be formed which facilitate DNA transfer. Both are resistant to heat and have a density in cesium chloride gradients that is indistinguishable from that of the in vivo-synthesized wild-type virus. This indicates that the particles formed have the appropriate protein-to-DNA ratio and a structure that shares the heat resistance of mature AAV particles. The two types of particles can be distinguished by their sensitivity to chloroform and DNase I treatment. The chloroform-resistant product is, by several criteria, an authentic AAV particle. In addition to having the correct density and being resistant to treatment with chloroform, DNase I, and heat, this particle is efficiently synthesized only if the AAV genome contains intact terminal repeats, which are known to be required for AAV packaging. It is also precipitated by a monoclonal antibody that recognizes mature virus particles but not bound by an antibody that recognizes monomeric or denatured capsid proteins. The chloroform-resistant species is not made when aphidicolin is present in the reaction mixture, suggesting that active DNA replication is required for in vitro packaging. In contrast, the chloroform-sensitive product has several features that suggest it is an incompletely assembled virus particle. It is sensitive to DNase I, does not require the presence of AAV terminal repeats, and is capable of transferring DNA that is theoretically too large to package. Sucrose gradient centrifugation of the in vitro-synthesized products reveals that the particles have sedimentation values between 60S and 110S, which is consistent with partially assembled and mature AAV particles. The in vitro packaging procedure should be useful for studying the mechanism by which a human icosahedral DNA virus particle is assembled, and it may be useful for producing recombinant AAV for gene therapy. The chloroform-sensitive particle may also be useful for transferring DNA that is too large to be packaged in mature recombinant AAV.
Mutations of the Human Immunodeficiency Virus Type 1 p6
Gag
Domain Result in Reduced Retention of Pol Proteins during Virus Assembly
Authors: Yu Xiao-Fang , Dawson Liza , Tian Chun-Juan , Flexner Charles , Dettenhofer Markus 
Published: December 2019

ABSTRACT
One of the crucial steps in the assembly of the human immunodeficiency virus type 1 (HIV-1) and other retroviruses is the incorporation and retention of all the key viral enzymes in released virions. The viral enzymes protease, reverse transcriptase, and integrase of HIV-1 are initially synthesized as Gag-Pol fusion polyproteins. It has been shown that the incorporation of Gag-Pol polyproteins during virus assembly requires the Gag domains that are shared by the Gag and Gag-Pol precursors. We now report that truncation of the C-terminal p6 domain of HIV-1 Gag, which is present in the Gag precursor but not in the Gag-Pol precursor, drastically reduced the amount of Pol proteins in the mutant virions. Mutations in the lentivirus conserved motif P(T/S)APP in p6 also drastically reduced the amount of Pol proteins in mutant virions. The steady-state levels of Gag-Pol precursors and cleaved Pol proteins in the transfected cells were not affected by mutations in p6. The incorporation of unprocessed Gag-Pol precursors into p6 mutant virions was detected when the viral protease was mutated, suggesting that the interactions among mutant Gag molecules and Gag-Pol precursors were not significantly affected. These results suggest that the p6 domain of HIV-1 Gag may play an important role in recruiting or retaining cleaved Pol proteins during virus assembly.
The Carboxyl-Terminal Region of the Human Papillomavirus Type 16 E1 Protein Determines E2 Protein Specificity during DNA Replication
Authors: Zou Nianxiang , Liu Jen-Sing , Kuo Shu-Ru , Broker Thomas R. , Chow Louise T. 
Published: December 2019

ABSTRACT
The mechanism of DNA replication is conserved among papillomaviruses. The virus-encoded E1 and E2 proteins collaborate to target the origin and recruit host DNA replication proteins. Expression vectors of E1 and E2 proteins support homologous and heterologous papillomaviral origin replication in transiently transfected cells. Viral proteins from different genotypes can also collaborate, albeit with different efficiencies, indicating a certain degree of specificity in E1-E2 interactions. We report that, in the assays of our study, the human papillomavirus type 11 (HPV-11) E1 protein functioned with the HPV-16 E2 protein, whereas the HPV-16 E1 protein exhibited no detectable activity with the HPV-11 E2 protein. Taking advantage of this distinction, we used chimeric E1 proteins to delineate the E1 protein domains responsible for this specificity. Hybrids containing HPV-16 E1 amino-terminal residues up to residue 365 efficiently replicated either viral origin in the presence of either E2 protein. The reciprocal hybrids containing amino-terminal HPV-11 sequences exhibited a high activity with HPV-16 E2 but no activity with HPV-11 E2. Reciprocal hybrid proteins with the carboxyl-terminal 44 residues from either E1 had an intermediate property, but both collaborated more efficiently with HPV-16 E2 than with HPV-11 E2. In contrast, chimeras with a junction in the putative ATPase domain showed little or no activity with either E2 protein. We conclude that the E1 protein consists of distinct structural and functional domains, with the carboxyl-terminal 284 residues of the HPV-16 E1 protein being the primary determinant for E2 specificity during replication, and that chimeric exchanges in or bordering the ATPase domain inactivate the protein.
Physiological Knockout of the Envelope Gene of the Single-Copy ERV-3 Human Endogenous Retrovirus in a Fraction of the Caucasian Population
Authors: de Parseval Nathalie , Heidmann Thierry 
Published: December 2019

ABSTRACT
ERV-3 is an evolutionarily conserved single-copy human endogenous retrovirus with a coding envelope gene potentially involved in important placental functions. We have investigated the sequence variability of this gene among 150 unrelated Caucasian individuals and found eight polymorphic sites. One of them corresponds to the introduction of a stop codon resulting in the production of a severely truncated ERV-3 envelope protein lacking both the fusion peptide and the immunosuppressive domain of the protein. The stop codon is observed in a homozygous state in approximately 1% of Caucasian individuals without evidence for counterselection, thus precluding the involvement of any essential function of the gene in placental implantation and development. This natural knockout provides a mean to investigate other potential roles for this otherwise highly conserved gene.
Alanine Substitutions of Polar and Nonpolar Residues in the Amino-Terminal Domain of CCR5 Differently Impair Entry of Macrophage- and Dualtropic Isolates of Human Immunodeficiency Virus Type 1
Authors: Rabut Gwénaël E. E. , Konner Jason A. , Kajumo Francis , Moore John P. , Dragic Tatjana 
Published: December 2019

ABSTRACT
Multiple extracellular domains of the CC-chemokine receptor CCR5 are important for its function as a human immunodeficiency virus type 1 (HIV-1) coreceptor. We have recently demonstrated by alanine scanning mutagenesis that the negatively charged residues in the CCR5 amino-terminal domain are essential for gp120 binding and coreceptor function. We have now extended our analysis of this domain to include most polar and nonpolar amino acids. Replacement of alanine with all four tyrosine residues and with serine-17 and cysteine-20 decrease or abolish gp120 binding and CCR5 coreceptor activity. Tyrosine-15 is essential for viral entry irrespective of the test isolate. Substitutions at some of the other positions impair the entry of dualtropic HIV-1 isolates more than that of macrophagetropic ones.
Two Closely Related but Distinct Retroviruses Are Associated with Walleye Discrete Epidermal Hyperplasia
Authors: LaPierre Lorie A. , Holzschu Donald L. , Wooster Greg A. , Bowser Paul R. , Casey James W. 
Published: December 2019

ABSTRACT
Walleye discrete epidermal hyperplasia (WEH) is a hyperproliferative skin disease that is prevalent on adult walleye fish throughout North America. We have identified two retroviruses associated with WEH, designated here as walleye epidermal hyperplasia virus type 1 and type 2 (WEHV1 and WEHV2), that are closely related to one another (77% identity) and to walleye dermal sarcoma virus (64% identity) within the polymerase region. WEHV1 and/or WEHV2 viral DNA was readily detected by PCR in hyperplastic tissue samples, but only low levels of viral DNA were detected in uninvolved skin. Southern blot analysis showed one to three copies of integrated WEHV2 viral DNA in lesions but did not detect WEHV2 viral DNA in uninvolved skin from the same fish. Northern blots detected abundant levels of WEHV1 and/or WEHV2 virion RNA transcripts of approximately 13 kb in hyperplastic tissue, but virion RNA was not observed in uninvolved skin and muscle. These results suggest that WEHV1 and WEHV2 are the causative agents of discrete epidermal hyperplasia.
Switch to Unusual Amino Acids at Codon 215 of the Human Immunodeficiency Virus Type 1 Reverse Transcriptase Gene in Seroconvertors Infected with Zidovudine-Resistant Variants
Authors: Yerly Sabine , Rakik Abdelrahim , Kinloch De Loes Sabine , Hirschel Bernard , Descamps Diane , Brun-Vézinet Françoise , Perrin Luc 
Published: December 2019

ABSTRACT
Sequences of the human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) domain were determined by direct sequencing of HIV-1 RNA in successive plasma samples from eight seroconverting patients infected with virus bearing the T215Y/F amino acid substitution associated with zidovudine (ZDV) resistance. At baseline, additional mutations associated with ZDV resistance were detected. Three patients had the M41L amino acid change, which persisted. Two patients had both the D67N and the K70R amino acid substitutions; reversion to the wild type was seen at both positions in one of these patients and at codon 70 in the other one. Reversion to the wild type at codon 215 was observed in only one of eight patients. Unusual amino acids, such as aspartic acid (D) and cysteine (C), appeared at position 215 in four patients during follow-up. These variants isolated by coculturing were sensitive to ZDV. Overgrowth of these variants suggests that they have better fitness than the original T215Y variant. Intraindividual nucleoside substitutions over time were 10 times more frequent in codons associated with ZDV resistance (41, 67, 70, 215, and 219) than in other codons of the RT domain. The predominance of nonsynonymous substitutions observed over time suggests that most changes reflect adaptation of the RT function. The variance in sequence evolution observed among patients, in particular at codon 215, supports a role for chance in the evolution of the RT domain.
Activation of Caspases in Pig Kidney Cells Infected with Wild-Type and CrmA/SPI-2 Mutants of Cowpox and Rabbitpox Viruses
Authors: Macen J. , Takahashi A. , Moon K. B. , Nathaniel R. , Turner P. C. , Moyer R. W. 
Published: December 2019

ABSTRACT
The cowpox virus (CPV) CrmA and the equivalent rabbitpox virus (RPV) SPI-2 proteins have anti-inflammatory and antiapoptosis activity by virtue of their ability to inhibit caspases, including the interleukin-1β-converting enzyme (ICE; caspase-1). Infection of LLC-PK1 pig kidney cells with a CPV CrmA mutant, but not with wild-type (wt) CPV, results in the induction of many of the morphological features of apoptosis (C. A. Ray and D. J. Pickup, Virology 217:384–391, 1996). In our study, LLC-PK1 cells infected with CPVΔcrmA, but not those infected with wt CPV, showed induction of poly(ADP-ribose) polymerase (PARP)- and lamin A-cleaving activities and processing of the CPP32 (caspase-3) precursor to a mature 18-kDa form. Surprisingly, infection of LLC-PK1 cells with either wt RPV (despite the presence of the SPI-2 protein) or RPVΔSPI-2 resulted in cleavage activity against PARP and lamin A and the appearance of the mature subunit of CPP32/caspase-3. The biotinylated specific peptide inhibitor Ac-Tyr-Val-Lys(biotinyl)-Asp-2,6-dimethylbenzoyloxymethylketone [AcYV(bio)KD-aomk] labeled active caspase subunits of 18, 19, and 21 kDa in extracts from LLC-PK1 cells infected with CPVΔcrmA, wt RPV, or RPVΔSPI-2 but not wt CPV. Mixed infection of LLC-PK1 cells with wt RPV and wt CPV gave no PARP-cleaving activity, and all PARP cleavage mediated by SPI-2 and CrmA mutants of RPV and CPV, respectively, could be eliminated by coinfection with wt CPV. These results suggest that the RPV SPI-2 and CPV CrmA proteins are not functionally equivalent and that CrmA, but not SPI-2 protein, can completely prevent apoptosis in LLC-PK1 cells under these conditions.
Nuclear Matrix Attachment Regions of Human Papillomavirus Type 16 Point toward Conservation of These Genomic Elements in All Genital Papillomaviruses
Authors: Tan Shyh-Han , Bartsch Dusan , Schwarz Elisabeth , Bernard Hans-Ulrich 
Published: December 2019

ABSTRACT

The gene functions, transcriptional regulation, and genome replication of human papillomaviruses (HPVs) have been extensively studied. Thus far, however, there has been little research on the organization of HPV genomes in the nuclei of infected cells. As a first step to understand how chromatin and suprachromatin structures may modulate the life cycles of these viruses, we have identified and mapped interactions of HPV DNAs with the nuclear matrix. The endogenous genomes of HPV type 16 (HPV-16) which are present in SiHa, HPKI, and HPKII cells, adhere in vivo to the nuclear matrixes of these cell lines. A tight association with the nuclear matrix in vivo may be common to all genital HPV types, as the genomes of HPV-11, HPV-16, HPV-18, and HPV-33 showed high affinity in vitro to preparations of the nuclear matrix of C33A cells, as did the well-known nuclear matrix attachment region (MAR) of the cellular beta interferon gene. Affinity to the nuclear matrix is not evenly spread over the HPV-16 genome. Five genomic segments have strong MAR properties, while the other parts of the genome have low or no affinity. Some of the five MARs correlate with known
cis
-responsive elements: a strong MAR lies in the 5′ segment of the long control region (LCR), and another one lies in the E6 gene, flanking the HPV enhancer, the replication origin, and the E6 promoter. The strongest MAR coincides with the E5 gene and the early-late intergenic region. Weak MAR activity is present in the E1 and E2 genes and in the 3′ part of L2. The in vitro map of MAR activity appears to reflect MAR properties in vivo, as we found for two selected fragments with and without MAR activity. As is typical for many MARs, the two segments with highest affinity, namely, the 5′ LCR and the early-late intergenic region, have an extraordinarily high A-T content (up to 85%). It is likely that these MARs have specific functions in the viral life cycle, as MARs predicted by nucleotide sequence analysis, patterns of A-T content, transcription factor YY1 binding sites, and likely topoisomerase II cleavage sites are conserved in similar positions throughout all genital HPVs.