Human papillomavirus (HPV) genome replication is dependent on the expression of E1 and E2 proteins. The organotypic (raft) culture system was used to investigate changes in viral early gene expression and vegetative genome replication during the complete life cycle of HPV type 31b (HPV31b). We have previously shown the synthesis of HPV31b viral particles as early as 10 days of growth of CIN-612 9E raft tissues (Ozbun, M. A., and Meyers, C (1997)
J. Virol. 71, 5161–5172). In the present study, we investigated the structures and temporal expression levels of HPV31b G1 and E2 transcripts, as well as the replication of the viral genome during the viral life cycle. The amplification state of the HPV31b genome was maximal at 10 days of raft tissue growth. Furthermore, the expression levels of E1 and E2 RNAs correlated with vegetative viral DNA replication. Levels of E1- and E2-specific transcripts were dissimilar throughout the viral life cycle. E2 RNA levels remained relatively constant, whereas E1 RNA levels were upregulated during the maximal amplification of viral genomes and the biosynthesis of virions. These data indicate that E1 may be the major regulator of viral genome amplification in preparation for DNA packaging and virion morphogenesis.
Human cytomegalovirus transient lytic DNA replication relies on the cis-acting element oriLyt, six viral-encoded core proteins, the proposed DNA replication initiator protein UL84, IE2, IRS1 and the gene products from the UL112/113 loci. In an effort to elucidate cellular and viral-encoded factors that may play a role in oriLyt-dependent replication we used DNA-affinity purification and mass spectrometry to isolate and identify several previously unknown cellular and viral factors that interact with HCMV oriLyt DNA. These proteins include the multifunctional hnRNP-K, BUB3, HMGB1, PTB-1, UL83, UL112/113, and IRS1. Chromatin immunoprecipitation (ChIP) assays confirmed an interaction of several of these factors with oriLyt. Co-immunoprecipitation experiments detected an interaction between UL84 and hnRNP-K in infected and transfected cells. Knockdown of hnRNP K expression by siRNA inhibited the amplification of oriLyt in the transient assay. Together, these data suggest a possible regulatory role in DNA replication for several previously unidentified viral and cellular factors.
Spurred by the recent isolation of a novel hantavirus, named Imjin virus (MJNV), from the Ussuri white-toothed shrew (Crocidura lasiura), targeted trapping was conducted for the phylogenetically related Asian lesser white-toothed shrew (Crocidura shantungensis). Pair-wise alignment and comparison of the S, M and L segments of a newfound hantavirus, designated Jeju virus (JJUV), indicated remarkably low nucleotide and amino acid sequence similarity with MJNV. Phylogenetic analyses, using maximum likelihood and Bayesian methods, showed divergent ancestral lineages for JJUV and MJNV, despite the close phylogenetic relationship of their reservoir soricid hosts. Also, no evidence of host switching was apparent in tanglegrams, generated by TreeMap 2.0β.
Hantavirus; Crocidura; Shrews; Phylogeny; Jeju Island; Korea
The S2 domain of the coronavirus spike (S) protein is known to be responsible for mediating membrane fusion. In addition to a well-recognized cleavage site at the S1–S2 boundary, a second proteolytic cleavage site has been identified in the severe acute respiratory syndrome coronavirus (SARS-CoV) S2 domain (R797). C terminal to this S2 cleavage site is a conserved region flanked by cysteine residues C822 and C833. Here, we investigated the importance of this well conserved region for SARS-CoV S-mediated fusion activation. We show that the residues between C822-C833 are well conserved across all coronaviruses. Mutagenic analysis of SARS-CoV S, combined with cell–cell fusion and pseudotyped virion infectivity assays, showed a critical role for the core-conserved residues C822, D830, L831, and C833. Based on available predictive models, we propose that the conserved domain flanked by cysteines 822 and 833 forms a loop structure that interacts with components of the SARS-CoV S trimer to control the activation of membrane fusion.
The production of virus by infected cells is an essential process for the spread and persistence of viral diseases, the effectiveness of live-viral vaccines, and the manufacture of viruses for diverse applications. Yet despite its importance, methods to precisely measure virus production from cells are lacking. Most methods test infected-cell populations, masking how individual cells behave. Here we measured the kinetics of virus production from single cells. We combined simple steps of liquid-phase infection, serial dilution, centrifugation, and harvesting, without specialized equipment, to track the production of virus particles from BHK cells infected with vesicular stomatitis virus. Remarkably, cell-to-cell differences in latent times to virus release were within a factor of two, while production rates and virus yields spanned over 300-fold, highlighting an extreme diversity in virus production for cells from the same population. These findings have fundamental and technological implications for health and disease.
single-cell sampling; virus-cell interactions; cell heterogeneity; kinetics; stochastic; intracellular resources; vesicular stomatitis virus; virus yield
Adenoviral infection induces nucleoplasmic redistribution of a nucleolar nucleophosmin 1/NPM1/B23.1. NPM1 is preferentially localized in the nucleoli of normal cells, whereas it is also present at the nuclear matrix in cancer cells. However, the biological roles of NPM1 during infection are unknown. Here, by analyzing a pV-deletion mutant, Ad5-dV/TSB, we demonstrate that pV promotes the NPM1 translocation from the nucleoli to the nucleoplasm in normal cells, and the NPM1 translocation is correlated with adenoviral replication. Lack of pV causes a dramatic reduction of adenoviral replication in normal cells, but not cancer cells, and Ad5-dV/TSB was defective in viral assembly in normal cells. NPM1 knockdown inhibits adenoviral replication, suggesting an involvement of NPM1 in adenoviral biology. Further, we show that NPM1 interacts with empty adenovirus particles which are an intermediate during virion maturation by immunoelectron microscopy. Collectively, these data implicate that pV participates in a process of viral assembly through NPM1.
adenovirus; adenoviral assembly; cancer gene therapy; nucleophosmin 1; protein V
Roles of complement factors in prion infection of the central nervous system remain unclear. In this study, we assessed the strain-dependent reactivity of complement factors in prion infections of Neuro2a (N2a) cells and mouse brains. N2a cells persistently infected with either Chandler or 22L scrapie strains were cultured in the presence of normal mouse serum (NMS), followed by staining with the phosphatidylserine binding protein and early apoptosis marker Annexin V. The proportion of Annexin V positive cells was increased both in Chandler- and 22L-infected cells. Preincubation of NMS with anti-C1q, C3 and/or C9 antibodies reduced Annexin V positive cells in Chandler-infected cells, while only anti-C3 antibodies were effective on 22L-infected cells. The immunohistochemistry showed that deposition of C1q and C3 was different between Chandler- and 22L-infected mouse brains. These results indicate that the reactivity of complement factors differs between prion strains both in vitro and in vivo.
prion; scrapie; complement factors; strain difference
Noroviruses are the major cause of food- or water-borne gastroenteritis outbreaks in humans. The norovirus protease that cleaves a large viral polyprotein to nonstructural proteins is essential for virus replication and an attractive target for antiviral drug development. Noroviruses show high genetic diversity with at least five genogroups, GI–GV, of which GI and GII are responsible for the majority of norovirus infections in humans. We cloned and expressed proteases of Norwalk virus (GI) and MD145 virus (GII) and characterized the enzymatic activities with fluorescence resonance energy transfer substrates. We demonstrated that the GI and GII proteases cleaved the substrates derived from the naturally occurring cleavage site in the open reading frame (ORF) 1 of G1 norovirus with similar efficiency, and that enzymatic activity of both proteases were inhibited by commercial protease inhibitors including chymostatin. The interaction of chymostatin to Norwalk virus protease was validated by nuclear magnetic resonance (NMR) spectroscopy.
antiviral; viral proteases; fluorescence resonance energy transfer; Norwalk virus; MD145 virus; nuclear magnetic resonance spectroscopy; chymostatin
It is generally believed that during the sexual transmission of HIV-1, the glycan-specific DC-SIGN receptor binds the virus and mediates its transfer to CD4+ cells. The lectins griffithsin (GRFT), cyanovirin-N (CV-N) and scytovirin (SVN) inhibit HIV-1 infection by binding to mannose-rich glycans on gp120. We measured the ability of these lectins to inhibit both the HIV-1 binding to DC-SIGN and the DC-SIGN-mediated HIV-1 infection of CD4+ cells. While GRFT, CV-N and SVN were moderately inhibitory to DC-SIGN binding, they potently inhibited DC-SIGN-transfer of HIV-1. The introduction of the 234 glycosylation site abolished HIV-1 sensitivity to lectin inhibition of binding to DC-SIGN and virus transfer to susceptible cells. However, the addition of the 295 glycosylation site increased the inhibition of transfer. Our data suggest that GRFT, CV-N and SVN can block two important stages of the sexual transmission of HIV-1, DC-SIGN binding and transfer, supporting their further development as microbicides.
Griffithsin; Cyanovirin-N; Scytovirin; mannose-rich glycans; HIV-1 gp120; DC-SIGN receptor
The HIV-1 capsid protein consists of two independently folded domains connected by a flexible peptide linker (residues 146–150), the function of which remains to be defined. To investigate the role of this region in virus replication, we made alanine or leucine substitutions in each linker residue and two flanking residues. Three classes of mutants were identified: (i) S146A and T148A behave like wild type (WT); (ii) Y145A, I150A, and L151A are noninfectious, assemble unstable cores with aberrant morphology, and synthesize almost no viral DNA; and (iii) P147L and S149A display a poorly infectious, attenuated phenotype. Infectivity of P147L and S149A is rescued specifically by pseudotyping with vesicular stomatitis virus envelope glycoprotein. Moreover, despite having unstable cores, these mutants assemble WT-like structures and synthesize viral DNA, although less efficiently than WT. Collectively, these findings demonstrate that the linker region is essential for proper assembly and stability of cores and efficient replication.
HIV-1 capsid protein; HIV-1 assembly; HIV-1 cores; VSV-G pseudotyping; interdomain linker; in vitro assembly; TRIM5 proteins; host restriction; virus disassembly; reverse transcription
Several herpes simplex virus 1 microRNAs are encoded within or near the latency associated transcript (LAT) locus, and are expressed abundantly during latency. Some of these microRNAs can repress the expression of important viral proteins and are hypothesized to play important roles in establishing and/or maintaining latent infections. We found that in lytically infected cells and in acutely infected mouse ganglia, expression of LAT-encoded microRNAs was weak and unaffected by a deletion that includes the LAT promoter. In mouse ganglia latently infected with wild type virus, the microRNAs accumulated to high levels, but deletions of the LAT promoter markedly reduced expression of LAT-encoded microRNAs and also miR-H6, which is encoded upstream of LAT and can repress expression of ICP4. Because these LAT deletion mutants establish and maintain latent infections, these microRNAs are not essential for latency, at least in mouse trigeminal ganglia, but may help promote it.
Herpes simplex virus; MicroRNAs; Latency; Gene regulation
H4N8 subtype avian influenza viruses were isolated from shorebirds in eastern Hokkaido. All the isolates shared >99.7% nucleotide homology, and all the viral genes except for PB1 were highly related to those of A/red-necked stint/Australia/1/04. Thus, the isolates were regarded as PB1 reassortants. The most similar PB1 gene was identified in A/mallard/New Zealand/1615-17/04 (H4N6) with nucleotide homology of 90.9%. BALB/c mice intranasally inoculated with the H4N8 isolates developed severe respiratory disease, which eventually led to death in some mice. Virus was isolated from the lungs, and viral antigen was detected in the lungs with pneumonia. Other H4 subtype viruses tested did not cause any symptoms in mice, although these viruses were also isolated from the lungs. The PB2 gene of the H4N8 isolates contains K482R, but not the E627K or D701N substitutions. The PB1-F2 gene of the isolates consists of a 101-amino acid unique sequence, but lacks the N66S mutation.
avian influenza virus; reassortant; H4N8; wild bird; mouse; pathogenicity
Viruses are infectious particles whose viability is dependent on the cells of living organisms, such as bacteria, plants, and animals. It is of great interest to discover how viruses function inside host cells in order to develop therapies to treat virally infected organisms. The fruit fly Drosophila melanogaster is an excellent model system for studying the molecular mechanisms of replication, amplification, and cellular consequences of human viruses. In this review, we describe the advantages of using Drosophila as a model system to study human viruses, and highlight how Drosophila has been used to provide unique insight into the gene function of several pathogenic viruses. We also propose possible directions for future research in this area.
Drosophila; viruses; GAL4/UAS system; transgenic expression; model organism; human pathogens
Adeno-associated virus is a promising vector for gene therapy. In the current study, the binding site on AAV serotype 3B for the heparan sulfate proteoglycan (HSPG) receptor has been characterized. X-ray diffraction identified a disaccharide binding site at the most positively charged region on the virus surface. The contributions of basic amino acids at this and other sites were characterized using site-directed mutagenesis. Both heparin and cell binding are correlated to positive charge at the disaccharide binding site, and transduction is significantly decreased in AAV-3B vectors mutated at this site to reduce heparin binding. While the receptor attachment sites of AAV-3B and AAV-2 are both in the general vicinity of the viral spikes, the exact amino acids that participate in electrostatic interactions are distinct. Diversity in the mechanisms of cell attachment by AAV serotypes will be important considerations for the rational design of improved gene therapy vectors.
Adeno-associated virus; heparin; heparan sulfate; receptor
Four serotypes of dengue virus (DENV 1-4) currently circulate between humans and domestic/peridomestic Aedes mosquitoes, resulting in 100 million infections per year. All four serotypes emerged, independently, from sylvatic progenitors transmitted among non-human primates by arboreal Aedes mosquitoes. This study investigated the genetic and phenotypic changes associated with emergence of human DENV-4 from its sylvatic ancestors. Analysis of complete genomes of 3 sylvatic and 4 human strains revealed high conservation of both the 5′- and 3′-untranslated regions but considerable divergence within the open reading frame. Additionally, the two ecotypes did not differ significantly in replication dynamics in cultured human liver (Huh-7), monkey kidney (Vero) or mosquito (C6/36) cells, although significant inter-strain variation within ecotypes was detected. These findings are in partial agreement with previous studies of DENV-2, where human strains produced a larger number of progeny than sylvatic strains in human liver cells but not in monkey or mosquito cells.
Dengue virus (DENV); sylvatic DENV; human DENV; phylogenetic and phenotypic analysis
The cellular protease subtilisin kexin isozyme-1 (SKI-1)/site-1 protease (S1P) is implicated in the proteolytic processing of the viral envelope glycoprotein precursor (GPC) of arenaviruses, a step strictly required for production of infectious progeny. The small molecule SKI-1/S1P inhibitor PF-429242 was shown to have anti-viral activity against Old World arenaviruses. Here we extended these studies and show that PF-429242 also inhibits GPC processing and productive infection of New World arenaviruses, making PF-429242 a broadly active anti-arenaviral drug. In combination therapy, PF-429242 potentiated the anti-viral activity of ribavirin, indicating a synergism between the two drugs. A hallmark of arenaviruses is their ability to establish persistent infection in vitro and in vivo. Notably, PF-429242 was able to efficiently and rapidly clear persistent infection by arenaviruses. Interruption of drug treatment did not result in re-emergence of infection, indicating that PF-429242 treatment lead to virus extinction.
Conventional assays of viral particle assembly and release are time consuming and laborious. We have developed an enzymatic virus-like particle (VLP) genesis assay that rapid and quantitative and is also versatile and applicable to diverse viruses including HIV-1 and Ebola virus. Using this assay, which has a dynamic range of several orders of magnitude, we show that the efficiency of VLP assembly and release, i.e. the fraction of the expressed protein that is assembled into extracellular particles, is dependent on the absolute level of expression of either HIV-1 Gag or Ebola virus VP40. We also demonstrate that the activity of the antiviral factor tetherin is dependent on the level of HIV-1 Gag expression and the numbers of VLPs generated, and appears to become saturated as these parameters are increased.
The retroviral genus Lentivirus comprises retroviruses characterised from five mammalian orders. Lentiviruses typically undergo rapid rates of evolution, a feature that has allowed recent evolutionary relationships to be elucidated, but has also obscured their distant evolutionary past. However, the slowdown in the rate of evolution associated with genome invasion, as has occurred in the European rabbit, enables longer-term lentiviral evolutionary history to be inferred. Here we report the identification of orthologous RELIK proviruses in the European hare, demonstrating a minimum age of 12 million years for the lagomorph lentiviruses. This finding indicates an association between lentiviruses and their hosts covering much of the evolutionary history of the lagomorphs, and taking place within species with a worldwide distribution.
RELIK; Hare; Lentivirus; Lepus europaeus; Evolution; Endogenous
In this study we examined the transport signals contributing to HPV16 L2 nucleocytoplasmic traffic using confocal microscopy analysis of enhanced green fluorescent protein – L2 (EGFP-L2) fusions expressed in HeLa cells. We confirmed that both nuclear localization signals (NLSs), the nNLS (1MRHKRSAKRTKR12) and cNLS (456RKRRKR461), previously characterized in vitro (Darshan et al., 2004), function independently in vivo. We discovered that a middle region rich in arginine residues (296SRRTGIRYSRIGNKQTLRTRS316) functions as a nuclear retention sequence (NRS), as mutagenesis of critical arginine residues within this NRS reduced the fraction of L2 in the nucleus despite the presence of both NLSs. Significantly, the infectivity of HPV16 pseudoviruses containing either RR297AA or RR297EE within the L2 NRS was strongly reduced both in HaCaT cells and in a murine challenge model. Experiments using Ratjadone A nuclear export inhibitor and mutation-localization analysis lead to the discovery of a leucine-rich nuclear export signal (462LPYFFSDVSL) mediating 16L2 nuclear export. These data indicate that HPV16 L2 nucleocytoplasmic traffic is dependent on multiple functional transport signals.
The adenovirus E4-ORF3 protein promotes viral replication by relocalizing cellular proteins into nuclear track structures, interfering with potential anti-viral activities. E4-ORF3 targets transcriptional intermediary factor 1 alpha (TIF1α), but not homologous TIF1β. Here, we introduce TIF1γ as a novel E4-ORF3-interacting partner. E4-ORF3 relocalizes endogenous TIF1γ in virus-infected cells in vivo and binds to TIF1γ in vitro. We used the homologous nature, yet differing binding capabilities, of these proteins to study how E4-ORF3 targets proteins for track localization. We mapped the ability of E4-ORF3 to interact with specific TIF1 subdomains, demonstrating that E4-ORF3 interacts with the Coiled-Coil domains of TIF1α, TIF1β, and TIF1γ, and that the C-terminal half of TIF1β interferes with this interaction. The results of E4-ORF3-directed TIF1 protein relocalization assays performed in vivo were verified using coimmunoprecipitation assays in vitro. These results suggest that E4-ORF3 targets proteins for relocalization through a loosely homologous sequence dependent on accessibility.
Adenovirus; Ad; E4-ORF3; nuclear tracks; relocalization; TIF1α; TIF1γ
Cholesterol and sphingolipid enriched lipid raft micro-domains in the plasma membrane play an important role in life-cycle of numerous enveloped viruses. Although human respiratory syncytial virus (RSV) proteins associate with the raft domains of infected cells and rafts are incorporated in RSV virion particles, the functional role of raft during RSV infection was unknown. In the current study we have identified rafts as an essential component of host cell that is required for RSV infection. Treatment of human lung epithelial cells with raft disrupting agent methyl-beta-cyclodextrin (MBCD) led to drastic loss of RSV infectivity due to diminished release of infectious progeny RSV virion particles from raft disrupted cells. RSV infection of raft deficient Niemann-Pick syndrome type C human fibroblasts and normal human embryonic lung fibroblasts revealed that during productive RSV infection, raft is required for release of infectious RSV particles.
Respiratory syncytial virus; cholesterol; lipid rafts; virus release
BKPyV and JCPyV are closely related, ubiquitious human pathogens that cause disease in immunocompromised patients. The DNA sequence of the regulatory regions distinguishes two forms of these viruses, designated archetype and rearranged. Although cell culture systems exist for rearranged BKPyV and JCPyV, currently there is no robust cell culture system to study the archetype viruses. Large T antigen (TAg) is a virally encoded protein required to initiate viral DNA synthesis. Because archetype virus produces undetectable levels of TAg, we hypothesized that TAg overexpression would stimulate archetype virus replication. Efficient propagation of the archetype forms of BKPyV and JCPyV was observed in 293TT cells, human embryonic kidney cells overexpressing SV40 TAg. Importantly, the archetypal structure of the regulatory region was maintained during viral growth. Significant replication was not observed for Merkel cell, KI, or WU polyomaviruses. 293TT cells provide a means of propagating archetype BKPyV and JCPyV for detailed study.
BKPyV; JCPyV; archetype; 293TT cells
We examined the antiviral activity of ADAR1 against HIV-1. Our results indicated that ADAR1 in a transfection system inhibited production of viral proteins and infectious HIV-1 in various cell lines including 293T, HeLa, Jurkat T and primary CD4+ T cells, and was active against a number of X4 and R5 HIV-1 of different clades. Further analysis showed that ADAR1 inhibited viral protein synthesis without any effect on viral RNA synthesis. Mutational analysis showed that ADAR1 introduced most of the A-to-G mutations in the rev RNA, in the region of RNA encoding for Rev Response Element (RRE) binding domain and in env RNA. These mutations inhibited the binding of rev to the RRE and inhibited transport of primary transcripts like gag, pol and env from nucleus to cytoplasm resulting in inhibition of viral protein synthesis without any effect on viral RNA synthesis. Furthermore, ADAR1 induced mutations in the env gene inhibited viral infectivity.
ADAR1; HIV-1; Antiviral; Cellular Protein
Alveolar macrophages are immunoregulatory effector cells that interact directly with respiratory virus pathogens in vivo. We examined the role of alveolar macrophages in acute infection with pneumonia virus of mice (PVM), a rodent pneumovirus that replicates the clinical sequelae of severe human respiratory syncytial virus disease. We show that PVM replicates in primary mouse macrophage culture, releasing infectious virions and proinflammatory cytokines. Alveolar macrophages isolated from PVM-infected mice express activation markers Clec43 and CD86, cytokines TNFα, IL-1, IL-6, and numerous CC and CXC chemokines. Alveolar macrophage depletion prior to PVM infection results in small but statistically significant increases in virus recovery but paradoxically prolonged survival. In parallel, macrophage depleted PVM-infected mice exhibit enhanced NK cell recruitment and increased production of IFNγ by NK, CD4+ and CD8+ T cells. These results suggest a protective, immunomodulatory role for IFNγ, as overproduction secondary to macrophage depletion may promote survival despite increased virus recovery.
inflammation; cytokines; respiratory virus; infection
We previously demonstrated that dengue virus (DENV) nonstructural 4B protein (NS4B) induced dengue hemorrhagic fever (DHF)-associated immunomediators in THP-1 monocytes. Moreover, cleavage of NS4AB polyprotein by the NS2B3 protease, significantly increased immunomediator production to levels found after DENV infection. In this report using primary human microvascular endothelial cells (HMVEC) transwell permeability model and HMVEC monolayer, we demonstrate that the immunomediators secreted in the supernatants of DENV-infected monocytes increase HMVEC permeability and expression of ICAM-1, VCAM-1 and E-selectin. Moreover, maturation of NS4B via cleavage of 2KNS4B is sufficient to induce immunomediators that cause HMVEC phenotypic changes, which appear to be synergistically induced by TNFα and IL-8. These data suggest that therapies targeting the maturation steps of NS4B, particularly 2KNS4B processing, may reduce overall DHF-associated immunomediator levels, thereby reducing DHF-associated morbidity and mortality. Alternatively, TNFα inhibitors may be a valid intervention strategy during the later stages of infection to prevent DHF progression.
Dengue virus; DENV; Flavivirus; chemokines and cytokines; nonstructural 2KNS4B protein; THP-1 monocytes; human microvascular endothelial cells (HMVEC); adhesion molecules; ICAM-1; VCAM-1; E-selectin; transwell permeability model; TEER; FITC-dextran