Abstract:
UNLABELLED:Hepatitis C virus (HCV) requires proteins from the NS3-NS5B polyprotein to create a replicase unit for replication of its genome. The replicase proteins form membranous compartments in cells to facilitate replication, but little is known about their functional organization within these structures. We recently reported on intragenomic replicons, bicistronic viral transcripts expressing an authentic replicase from open reading frame 2 (ORF2) and a second duplicate nonstructural (NS) polyprotein from ORF1. Using these constructs and other methods, we have assessed the polyprotein requirements for rescue of different lethal point mutations across NS3-5B. Mutations readily tractable to rescue broadly fell into two groupings: those requiring expression of a minimum NS3-5A and those requiring expression of a minimum NS3-5B polyprotein. A cis-acting mutation that blocked NS3 helicase activity, T1299A, was tolerated when introduced into either ORF within the intragenomic replicon, but unlike many other mutations required the other ORF to express a functional NS3-5B. Three mutations were identified as more refractile to rescue: one that blocked cleavage of the NS4B5A boundary (S1977P), another in the NS3 helicase (K1240N), and a third in NS4A (V1665G). Introduced into ORF1, these exhibited a dominant negative phenotype, but with K1240N inhibiting replication as a minimum NS3-5A polyprotein whereas V1665G and S1977P only impaired replication as a NS3-5B polyprotein. Furthermore, an S1977P-mutated NS3-5A polyprotein complemented other defects shown to be dependent on NS3-5A for rescue. Overall, our findings suggest the existence of two interdependent sets of protein complexes supporting RNA replication, distinguishable by the minimum polyprotein requirement needed for their formation. IMPORTANCE:Positive-strand RNA viruses reshape the intracellular membranes of cells to form a compartment within which to replicate their genome, but little is known about the functional organization of viral proteins within this structure. We have complemented protein-encoded defects in HCV by constructing subgenomic HCV transcripts capable of simultaneously expressing both a mutated and functional polyprotein precursor needed for RNA genome replication (intragenomic replicons). Our results reveal that HCV relies on two interdependent sets of protein complexes to support viral replication. They also show that the intragenomic replicon offers a unique way to study replication complex assembly, as it enables improved composite polyprotein complex formation compared to traditional trans-complementation systems. Finally, the differential behavior of distinct NS3 helicase knockout mutations hints that certain conformations of this enzyme might be particularly deleterious for replication.
journal_name
J Viroljournal_title
Journal of virologyauthors
Gomes RG,Isken O,Tautz N,McLauchlan J,McCormick CJdoi
10.1128/JVI.01931-15subject
Has Abstractpub_date
2015-12-30 00:00:00pages
2868-83issue
6eissn
0022-538Xissn
1098-5514pii
JVI.01931-15journal_volume
90pub_type
杂志文章abstract::Previous recombinant Epstein-Barr virus molecular genetic experiments with specifically mutated LMP1 genes indicate that LMP1 is essential for primary B-lymphocyte growth transformation and that the amino-terminal cytoplasmic and first transmembrane domains are together an important mediator of transformation. EBV rec...
journal_title:Journal of virology
pub_type: 杂志文章
doi:10.1128/JVI.68.7.4369-4376.1994
更新日期:1994-07-01 00:00:00
abstract::Autographa californica nuclear polyhedrosis virus (AcMNPV) is a double-stranded DNA virus that expresses several immediate-early genes under the control of different promoters. The expression of one of these transcription units, IE-N, is shown here, by a transient expression assay, to be regulated by both cis- and tra...
journal_title:Journal of virology
pub_type: 杂志文章
doi:10.1128/JVI.65.2.945-951.1991
更新日期:1991-02-01 00:00:00
abstract::Interleukin-1 (IL-1) plays an important role in the inflammatory process. Some studies have demonstrated that IL-1 production was impaired in patients with chronic infections of hepatitis C virus (HCV), implying that IL-1 may play a role in viral clearance. Using an HCV subgenomic replicon cell line, we demonstrate th...
journal_title:Journal of virology
pub_type: 杂志文章
doi:10.1128/jvi.77.9.5493-5498.2003
更新日期:2003-05-01 00:00:00
abstract::To investigate the genetic stability of vaccinia virus DNA, we have tested whether alterations occurred in the polypeptide composition of this complex virus during persistent infections. We found that variants isolated at various passages in Friend erythroleukemia cells persistently infected with vaccinia virus contai...
journal_title:Journal of virology
pub_type: 杂志文章
doi:10.1128/JVI.61.8.2642-2647.1987
更新日期:1987-08-01 00:00:00
abstract::Bacteriophage phi105 is a temperate bacteriophage for Bacillus subtilis 168. Temperature-sensitive and plaque mutants of phi105 were isolated. The results of two- and three-factor crosses with these mutants suggest the vegetative map of phi105 to be circular. The location of prophage phi105 between bacterial markers p...
journal_title:Journal of virology
pub_type: 杂志文章
doi:10.1128/JVI.3.1.38-44.1969
更新日期:1969-01-01 00:00:00
abstract::Tetherin (CD317/BST-2), an interferon-induced membrane protein, restricts the release of nascent retroviral particles from infected cell surfaces. While human immunodeficiency virus type 1 (HIV-1) encodes the accessory gene vpu to overcome the action of tetherin, the lineage of primate lentiviruses that gave rise to H...
journal_title:Journal of virology
pub_type: 杂志文章
doi:10.1128/JVI.01515-09
更新日期:2009-11-01 00:00:00
abstract::Murine gammaherpesvirus 68 (gammaHV68; also known as MHV-68) can establish a latent infection in both inbred and outbred strains of mice and, as such, provides a tractable small-animal model to address mechanisms and cell types involved in the establishment and maintenance of chronic gammaherpesvirus infection. Latenc...
journal_title:Journal of virology
pub_type: 杂志文章
doi:10.1128/jvi.77.15.8310-8321.2003
更新日期:2003-08-01 00:00:00
abstract::Hepatitis C virus (HCV) core protein forms the nucleocapsid of the HCV particle. Although many functions of core protein have been reported, how the HCV particle is assembled is not well understood. Here we show that the nucleocapsid-like particle of HCV is composed of a disulfide-bonded core protein complex (dbc-comp...
journal_title:Journal of virology
pub_type: 杂志文章
doi:10.1128/JVI.00402-10
更新日期:2010-09-01 00:00:00
abstract::Respiratory syncytial virus (RSV) infection causes substantial morbidity and some deaths in the young and elderly worldwide. There is no safe and effective vaccine available, although it is possible to reduce the hospitalization rate for high-risk children by anti-RSV antibody prophylaxis. RSV has been shown to modify...
journal_title:Journal of virology
pub_type: 杂志文章
doi:10.1128/JVI.01755-09
更新日期:2010-01-01 00:00:00
abstract::The protein kinase (PK) encoded by the Epstein-Barr Virus (EBV) BGLF4 gene is the only EBV protein kinase. The expression pattern of EBV PK during the reactivation of the viral lytic cycle and the subcellular localization of the protein were analyzed with a polyclonal antiserum raised against a peptide corresponding t...
journal_title:Journal of virology
pub_type: 杂志文章
doi:10.1128/JVI.78.22.12140-12146.2004
更新日期:2004-11-01 00:00:00
abstract::A virus (M-7) isolated from baboon placental tissue demonstrates many similarities to endogenous feline virus RD-114. Immunodiffusion analysis shows a group-specific antigen (gs-1) line of identity between M-7 and RD-114. Anti-RD-114 DNA polymerase IgG inhibits M-7 polymerase by 57% compared to 97% for RD-114. M-7 vir...
journal_title:Journal of virology
pub_type: 杂志文章
doi:10.1128/JVI.14.1.133-138.1974
更新日期:1974-07-01 00:00:00
abstract::Norovirus (NV) gastroenteritis is a major contributor to global morbidity and mortality, yet little is known about immune mechanisms leading to NV control. Previous studies using the murine norovirus (MNV) model have established a key role for T cells in MNV clearance. Despite these advances, important questions remai...
journal_title:Journal of virology
pub_type: 杂志文章
doi:10.1128/JVI.03389-12
更新日期:2013-06-01 00:00:00
abstract::Varicella-zoster virus (VZV) causes varicella and establishes latency in sensory nerve ganglia, but the characteristics of VZV latency are not well defined. Immunohistochemical detection of the VZV immediate-early 63 (IE63) protein in ganglion neurons has been described, but there are significant discrepancies in esti...
journal_title:Journal of virology
pub_type: 杂志文章
doi:10.1128/JVI.02416-09
更新日期:2010-04-01 00:00:00
abstract::Immune responses and the components of protective immunity following norovirus infection in humans are poorly understood. Although antibody responses following norovirus infection have been partially characterized, T cell responses in humans remain largely undefined. In contrast, T cells have been shown to be essentia...
journal_title:Journal of virology
pub_type: 杂志文章
doi:10.1128/JVI.00727-10
更新日期:2010-09-01 00:00:00
abstract::Immunity to human immunodeficiency virus virion-like structures or a polyprotein has been examined after DNA immunization with Rev-independent expression vectors. A Gag-Pol fusion protein stimulated cytotoxic T lymphocyte and antibody responses to Gag and Pol, while a Gag-Pol pseudoparticle did not elicit substantial ...
journal_title:Journal of virology
pub_type: 杂志文章
doi:10.1128/JVI.75.10.4947-4951.2001
更新日期:2001-05-01 00:00:00
abstract::The organization of adeno-associated virus serotype 4 (AAV 4) DNA was probed by using restriction enzymes. The cleavage sites of the following enzymes were mapped: BglII, BamI, HincII, KpnI, PstI, SalI, SstI, and XhoI. The orientation of transcription on the physical cleavage map was determined by locating the fragmen...
journal_title:Journal of virology
pub_type: 杂志文章
doi:10.1128/JVI.35.3.653-661.1980
更新日期:1980-09-01 00:00:00
abstract::Functional defects in cytotoxic CD8(+) T cell responses arise in chronic human viral infections, but the mechanisms involved are not well understood. In mice, CD4 cell-mediated interleukin-21 (IL-21) production is necessary for the maintenance of CD8(+) T cell function and control of persistent viral infections. To in...
journal_title:Journal of virology
pub_type: 杂志文章
doi:10.1128/JVI.02030-10
更新日期:2011-01-01 00:00:00
abstract::Fluorosulfonylbenzoyl 5'-adenosine (FSBA) bound to one site in simian virus 40 large T antigen (T) and covalently modified greater than 95% of the molecules in a complete reaction. This analog for ATP specifically cross-links to the Mg-phosphate pocket in ATP-binding sites. Cyanogen bromide cleavage and tryptic digest...
journal_title:Journal of virology
pub_type: 杂志文章
doi:10.1128/JVI.64.10.4939-4947.1990
更新日期:1990-10-01 00:00:00
abstract::Recombinant adeno-associated virus (rAAV) vectors have been shown to be useful for efficient gene delivery to a variety of dividing and nondividing cells. Mechanisms responsible for the long-term, persistent expression of the rAAV transgene are not well understood. In this study we investigated the kinetics of rAAV-me...
journal_title:Journal of virology
pub_type: 杂志文章
doi:10.1128/jvi.74.8.3555-3565.2000
更新日期:2000-04-01 00:00:00
abstract::NS5A of the hepatitis C virus (HCV) is a highly phosphorylated protein involved in resistance against interferon and required most likely for replication of the viral genome. Phosphorylation of this protein is mediated by a cellular kinase(s) generating multiple proteins with different electrophoretic mobilities. In t...
journal_title:Journal of virology
pub_type: 杂志文章
doi:10.1128/JVI.73.9.7138-7146.1999
更新日期:1999-09-01 00:00:00
abstract::Venezuelan equine encephalitis virus (VEE) is an important equine and human pathogen of the Americas. In the adult mouse model, cDNA-derived, virulent V3000 inoculated subcutaneously (s.c.) causes high-titer peripheral replication followed by neuroinvasion and lethal encephalitis. A single change (G to A) at nucleotid...
journal_title:Journal of virology
pub_type: 杂志文章
doi:10.1128/JVI.75.8.3706-3718.2001
更新日期:2001-04-01 00:00:00
abstract::Genetic and phylogenetic analyses suggest that the pandemic H1N1/2009 virus was derived from well-established swine influenza lineages; however, there is no convincing evidence that the pandemic virus was generated from a direct precursor in pigs. Furthermore, the evolutionary dynamics of influenza virus in pigs have ...
journal_title:Journal of virology
pub_type: 杂志文章
doi:10.1128/JVI.01679-14
更新日期:2014-10-01 00:00:00
abstract::Breast cancer is the second leading cause of cancer-related deaths in women in the United States. Triple-negative breast cancer constitutes a subset of breast cancer that is associated with higher rates of relapse, decreased survival, and limited therapeutic options for patients afflicted with this type of breast canc...
journal_title:Journal of virology
pub_type: 杂志文章
doi:10.1128/JVI.01411-19
更新日期:2019-11-13 00:00:00
abstract::Mouse mammary tumor virus (MMTV)-induced mammary adenocarcinomas can develop from several different premalignant precursors common in GR mice. Insertion mutagenesis of the mammary protooncogenes int-1 and int-2 was studied in this multistep system by analyzing samples from various stages of neoplastic development for ...
journal_title:Journal of virology
pub_type: 杂志文章
doi:10.1128/JVI.64.4.1794-1802.1990
更新日期:1990-04-01 00:00:00
abstract::By using an isothermal multiply primed rolling-circle amplification protocol, the complete genomic DNA of a novel papillomavirus was amplified from a skin lesion biopsy of a Florida manatee (Trichechus manatus latirostris), one of the most endangered marine mammals in United States coastal waters. The nucleotide seque...
journal_title:Journal of virology
pub_type: 杂志文章
doi:10.1128/JVI.78.22.12698-12702.2004
更新日期:2004-11-01 00:00:00
abstract::Mumps is a highly contagious human disease, characterized by lateral or bilateral nonsuppurative swelling of the parotid glands and neurological complications that can result in aseptic meningitis or encephalitis. A mumps vaccination program implemented since the 1960s reduced mumps incidence by more than 99% and kept...
journal_title:Journal of virology
pub_type: 杂志文章
doi:10.1128/JVI.02778-13
更新日期:2014-03-01 00:00:00
abstract::The human cytomegalovirus (HCMV) immediate-early 2 (IE2) transactivator has previously been shown to form intranuclear, dot-like accumulations in association with subnuclear structures known as promyelocytic leukemia protein (PML) nuclear bodies or ND10. We recently observed that IE2 can form dot-like structures even ...
journal_title:Journal of virology
pub_type: 杂志文章
doi:10.1128/JVI.01009-07
更新日期:2007-09-01 00:00:00
abstract::The four temperature-sensitive (ts) loci identified in the PB1 and PB2 gene segments of cold-adapted A/Ann Arbor/6/60 influenza virus, the master donor virus for influenza A virus (MDV-A) FluMist vaccines, were introduced into a divergent A/Puerto Rico/8/34 influenza virus strain. Recombinant A/Puerto Rico/8/34 virus ...
journal_title:Journal of virology
pub_type: 杂志文章
doi:10.1128/jvi.78.2.995-998.2004
更新日期:2004-01-01 00:00:00
abstract::Vaccinia virus (VACV) stimulates long-term immunity against highly pathogenic orthopoxvirus infection of humans (smallpox) and mice (mousepox [ectromelia virus {ECTV}]) despite the lack of a natural host-pathogen relationship with either of these species. Previous research revealed that VACV is able to induce polyfunc...
journal_title:Journal of virology
pub_type: 杂志文章
doi:10.1128/JVI.00038-12
更新日期:2012-07-01 00:00:00
abstract::Coronavirus subgenomic mRNA possesses a 5'-end leader sequence which is derived from the 5' end of genomic RNA and is linked to the mRNA body sequence. This study examined whether coronavirus transcription involves a discontinuous transcription step; the possibility that a leader sequence from mouse hepatitis virus (M...
journal_title:Journal of virology
pub_type: 杂志文章
doi:10.1128/JVI.68.4.2615-2623.1994
更新日期:1994-04-01 00:00:00