pinecone/core-2020-05-10-deduplication huggingface dataset:
The HDV Large-Delta Antigen Fused with GFP Remains Functional and Provides for Studying Its Dynamic Distribution

AbstractHepatitis D virus (HDV) requires the isoprenylated large delta antigen (LDAg) for interaction with hepatitis B surface antigen (HBsAg) to allow packaging and secretion out of the host cell. Phosphorylated LDAg has been found but, as yet, neither localization of LDAg within the nucleus nor any other function has been correlated with modification. In this study, we transfected HuH-7 or HeLa cells with plasmids encoding various lengths of LDAg [designated GFP–LD and GFP–LD(31-214) for full length and a deletion, respectively] or non-isoprenylated mutants of these [designated GFP–LDM and GFP–LD(31-214)M] fused to the green fluorescent protein (GFP). These fusion proteins were then characterized and it was found that: (i) the addition of the GFP did not interfere with the functioning of the full-length or N-terminally deleted LDAgs when interacting with HBsAg for secretion; (ii) the HDV small antigen (SDAg) together with the GFP–LD, but not the GFP–LD(31-214), could be cosecreted by HBsAg; and (iii) the GFP–LD, but not the GFP–LD(31-214), exerted a dominant-negative role on HDV genome replication. Analyses of transiently transfected cells and postmitotic permanent cells revealed the sequential appearance of GFP–LD in the nucleoplasm, then in the nucleolus, and finally in nuclear speckles (NS). Isoprenylation of LDAg seems to be important for targeting to and accumulating in the NS, which was evident from the dynamic and static localization of the non-isoprenylation mutant (GFP–LDM) and the distribution of wild-type (GFP–LD) when treated with an isoprenylation inhibitor, lovastatin, for more than 48 h. Permanently expressing GFP–LD cells allowed us to show the dynamic redistribution of dephosphorylated GFP–LD from the nucleolus to the SC-35 containing NS in the presence of dichlororibofuranosyl benzimidazole (DRB) and then the translocation back of the GFP–LD to the nucleolus within 2 h after removal of DRB. Our studies thus suggest that the various versions of the GFP–LD fusion protein, having the same function as their nonfusion counterparts, can be a powerful tool for the study of the dynamic localization of LDAg when correlated with the functional modification of this protein
Infectious cDNA Clone of Attenuated Langat Tick-Borne Flavivirus (Strain E5) and a 3′ Deletion Mutant Constructed from It Exhibit Decreased Neuroinvasiveness in Immunodeficient Mice

AbstractForty-five years ago a naturally attenuated tick-borne flavivirus, Langat (LGT) strain TP21, was recovered from ticks in Malaysia. Subsequently, it was tested as a live attenuated vaccine for virulent tick-borne encephalitis viruses. In a large clinical trial its attenuation was confirmed but there was evidence of a low level of residual virulence. Thirty-five years ago further attenuation of LGT TP21 was achieved by multiple passages in eggs to yield mutant E5. To study the genetic determinants of the further attenuation exhibited by E5 and to allow us to manipulate the genome of this virus for the purpose of developing a satisfactory live attenuated tick-borne flavivirus vaccine, we recovered infectious E5 virus from a full-length cDNA clone. The recombinant E5 virus (clone 651) recovered from a full-length infectious cDNA clone was more attenuated in immunodeficient mice than that of its biologically derived E5 parent. Increase in attenuation was associated with three amino acid substitutions, two located in the structural protein E and one in nonstructural protein NS4B. Subsequently an even greater degree of attenuation was achieved by creating a viable 320 nucleotide deletion in the 3′-noncoding region of infectious full-length E5 cDNA. This deletion mutant was not cytopathic in simian Vero cells and it replicated to lower titer than its E5-651 parent. In addition, the E5 3′ deletion mutant was less neuroinvasive in SCID mice than its E5-651 parent. Significantly, the deletion mutant proved to be 119,750 times less neuroinvasive in SCID mice than its progenitor, LGT strain TP21. Despite its high level of attenuation, the E5 3′ deletion mutant remained highly immunogenic and intraperitoneal (ip) inoculation of 10 PFU induced complete protection in Swiss mice against subsequent challenge with 2000 ip LD50 of the wild-type LGT TP21
Virion-Associated HIV-1 Vpr: Variable Amount in Virus Particles Derived from Cells upon Virus Infection or Proviral DNA Transfection

AbstractHuman immunodeficiency virus type-1 (HIV-1) Vpr is a virion-associated protein implicated to have a role in AIDS pathogenesis. In regard to the amount of Vpr incorporated into virus particles, the published data vary widely. To address this, we quantitated Vpr in virus particles derived from diverse sources that are used to evaluate the biological effect of Vpr. Virus particles from infected cells showed only a small amount of Vpr. Interestingly, virus particles from cells cotransfected with HIV-1 proviral DNA lacking Vpr coding sequences (NLΔVpr) and a Vpr expression plasmid showed a drastic increase (29.4-fold) in the incorporation of Vpr. Furthermore, cotransfection involving NLΔVpr and different concentrations of Vpr expression plasmid resulted in virus particles containing Vpr in proportion to the Vpr expression plasmid used. The differences in virus particles with respect to Vpr as revealed by these studies should be taken into account in assessing the effect of Vpr
Comparison of Murine and Human Nectin1 Binding to Herpes Simplex Virus Glycoprotein D (gD) Reveals a Weak Interaction of Murine Nectin1 to gD and a gD-Dependent Pathway of Entry

AbstractThe murine nectin1α (mNectin1α), a homolog of human nectin1α (hNectin1α, or PRR1, HveC), mediates the entry of herpes simplex virus (HSV) into cells. Previously, we reported that the binding of hNectin1 to HSV glycoprotein D (gD) was readily detectable, whereas the binding of mNectin1 to gD was not detectable, thus raising the question whether mNectin1 mediates a gD-dependent or a gD-independent pathway of entry. Here we report comparative binding studies of murine- and human-nectin1α to virions and to gD. The assays consistently showed either a very weak binding or no detectable binding of murine nectin1α to gD. They included (i) binding of soluble mNectin1-Fc or hNectin1-Fc to virions and competition of the binding by soluble gD(Δ290–299t) and by monoclonal antibodies to gD; (ii) pull-down experiments of wt gD from lysates of infected cells; and (iii) ELISA binding of soluble gD(Δ290–299t) to cells expressing mNectin1 or hNectin1. In contrast to the binding studies, the entry studies readily showed that entry mediated by mNectin1 was dependent on gD. Thus, a gDnull (gD−/−) mutant virus was unable to enter mNectin1-expressing cells, and entry of wild-type virus was inhibited by antibodies to gD or soluble gD at similar concentrations. We infer that gD represents a weak ligand in the interaction between mNectin1 and virions, whereas it represents a strong and the major ligand for hNectin1. Yet gD is required in HSV-1 entry mediated by mNectin1α. We conclude that a high-affinity binding of the receptor to gD is not a requirement in the gD-dependent pathway of HSV entry to cells
Basic Amino Acid Residues in the V3 Loop of Simian Immunodeficiency Virus Envelope Alter Viral Coreceptor Tropism and Infectivity but Do Not Allow Efficient Utilization of CXCR4 as Entry Cofactor

AbstractIn contrast to human immunodeficiency viruses type 1 and type 2 (HIV-1 and HIV-2, respectively), simian immunodeficiency virus (SIVmac) rarely uses CXCR4 (X4) for efficient entry into target cells. Basic amino acid residues in the V3 loop of HIV Env allow efficient coreceptor utilization of X4. Therefore, we investigated if similar changes in the SIVmac Env protein also mediate a coreceptor switch from CCR5 (R5) to X4. Functional analysis revealed that none of eight SIVmac variants, containing V3 regions with an overall charge between +4 and +10, efficiently utilized X4 as entry cofactor. Nonetheless, these alterations had differential effects on SIV coreceptor tropism and on Env expression levels. A single amino acid substitution of L328R, located near the tip of the V3 loop, resulted in grossly reduced Env expression levels and impaired viral infectivity. Notably, additional basic residues restored efficient Env expression and virion incorporation but not infectivity. In comparison to the L328R mutation, changes of P334K and D337K had little disruptive effects on SIVmac entry and replication. Interestingly, mutation of L320K and P321R disrupted coreceptor usage of GPR15 but not R5. These changes also impaired SIVmac replication in peripheral blood mononuclear cells (PBMC) derived from a Δ32/Δ32 donor but not in R5-expressing human or simian PBMC. Our results show that positively charged amino acid residues in the V3 loop affect SIVmac coreceptor tropism and infectivity but do not allow efficient utilization of X4
Capsid Size Determination in the P2–P4 Bacteriophage System: Suppression of sir Mutations in P2's Capsid Gene N by Supersid Mutations in P4's External Scaffold Gene sid

AbstractThe sid gene of the P2-dependent phage P4 provides an external scaffold so P2 N gene encoded protomers assemble as T = 4 capsids rather than as P2's T = 7 capsids. Mutations (sir) in the middle of N interfere with Sid's function. We describe a new P4 mutant class, nms (“supersid”) mutations, which direct also P2 sir to provide small capsids. Three different nms mutations were located near the sid end, commingled with sid− mutations. Suppression of sir by nms is not allele-specific. Our results favor this interpretation of capsid size control: (i) sir mutations reduce pN protomer flexibility and thereby interfere with the generation of T = 4 compatible hexons; (ii) the C-termini of Sid molecules link up when forming the scaffold; nms mutations strengthen these Sid–Sid contacts and thus allow the scaffold to force even sir-type protomers to form T = 4 compatible hexons. Some related findings concern suppression of N ts mutations by P4
Autographa californica Baculoviruses with Large Genomic Deletions Are Rapidly Generated in Infected Insect Cells

AbstractDefective interfering baculoviruses (DIs) lack considerable portions of the genome, interfere with the replication of helper virus, and cause the so-called “passage-effect” during serial passaging in insect cells and in bioreactor configurations. We investigated their origin by (nested) PCR and demonstrated that DIs lacking approximately 43% (d43) of their DNA are present in low-passage Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV)-E2 virus stocks and in polyhedra, but not in the authentic AcMNPV isolate obtained prior to passage in cell culture. To investigate whether DIs are rapidly generated de novo in Sf21 insect cells, a genetically homogeneous AcMNPV bacmid was serially passaged, resulting in the generation of d43 DIs within two passages. AT-rich sequences of up to 66 nucleotides of partly unknown origin were found at the deletion junctions in the d43 DI genomes. These data suggest that the rapid generation of DIs is an intrinsic property of baculovirus infection in insect cell culture and involves several recombination steps
Rack1 Binds HIV-1 Nef and Can Act as a Nef–Protein Kinase C Adaptor

AbstractNef proteins of primate immunodeficiency viruses exert pleiotropic effects, such as enhanced endocytosis of CD4 and MHC-I cell surface molecules, perturbation of signal transduction cascades, and virion infectivity enhancement. Nef function intersects that of a number of cell kinases, including C kinases (PKCs) and Src-family kinases. Here the interaction of HIV-1 Nef with Rack1 (receptor for activated C kinase 1) is reported. Nef binds the Rack1 C-terminal moiety in a yeast two-hybrid system and in cell-free pull-down assays and copurifies with in vitro translated Rack1. Nef and Rack1 partially colocalize on the trans-Golgi network and plasma membranes. The presence of Rack1 doubles Nef phosphorylation by PKCs in vitro. Our data agree with the idea that Rack1 acts as a Nef intracellular docking site, bringing Nef and PKCs together. Other signal transduction or endocytosis proteins, in particular Src-like kinases, might meet Nef by intermediation of the Rack1 adaptor
The V Protein of Human Parainfluenza Virus 2 Antagonizes Type I Interferon Responses by Destabilizing Signal Transducer and Activator of Transcription 2

AbstractType I interferon (IFN) induces antiviral responses through the activation of the ISGF3 transcription factor complex that contains the subunit proteins STAT1, STAT2, and p48/ISGF3γ/IRF9. The ability of some human paramyxoviruses to overcome IFN actions by specific proteolysis of STAT proteins has been examined. Infection of cells with type 2, but not type 1 or type 3 human parainfluenza virus (HPIV) leads to a loss of cellular STAT2 protein. Expression of a single HPIV2 protein derived from the V open reading frame blocks IFN-dependent transcriptional responses in the absence of other viral proteins. The loss of IFN response is due to V-protein-induced proteolytic degradation of STAT2. Expression of HPIV2 V causes the normally stable STAT2 protein to be rapidly degraded, and this proteolytic activity can be partially alleviated by proteasome inhibition. No V-protein-specific effects on STAT2 mRNA levels were observed. The results indicate that the V protein of HPIV2 is sufficient to recognize and target a specific cellular transcription factor for destruction by cellular machinery
Comparative Genomics of Lactococcal Phages: Insight from the Complete Genome Sequence of Lactococcus lactis Phage BK5-T

AbstractLactococcus lactis phage BK5-T and Streptococcus thermophilus phage Sfi21, two cos-site temperate Siphoviridae with 40-kb genomes, share an identical genome organization, sequence similarity at the amino acid level over about half of their genomes, and nucleotide sequence identity of 60% over the DNA packaging and head morphogenesis modules. Siphoviridae with similarly organized genomes and substantial protein sequence similarity were identified in several genera of low-GC-content Gram-positive bacteria. These phages demonstrated a gradient of relatedness ranging from nucleotide sequence similarity to protein sequence similarity to gene map similarity over the DNA packaging and head morphogenesis modules. Interestingly, the degree of relatedness was correlated with the evolutionary distance separating their bacterial hosts. These observations suggest elements of vertical evolution in phages. The structural genes from BK5-T shared no sequence relationships with corresponding genes/proteins from lactococcal phages belonging to distinct lactococcal phage species, including phage sk1 (phage species 936) that showed a closely related gene map. Despite a clearly distinct genome organization, lactococcal phages sk1 and c2 showed nine sequence-related proteins. Over the early gene cluster phage BK5-T shared nine regions of high nucleotide sequence similarity, covering at most two adjacent genes, with lactococcal phage r1t (phage species P335). Over the structural genes, the closest relatives of phage r1t were not lactococcal phages belonging to other phage species, but Siphoviridae from Mycobacteria (high-GC-content Gram-positive bacteria). Evidence for recent horizontal gene transfer between distinct phage species was obtained for dairy phages, but these transfers were limited to phages infecting the same bacterial host species
ICAM-1 Is Required for Resistance to Herpes Simplex Virus Type 1 but Not Interferon-α1 Transgene Efficacy

AbstractThe purpose of this study was to determine the role of ICAM-1 in ocular herpes simplex virus type 1 (HSV-1) infection. Wild-type and ICAM-1 knockout mice were assessed for resistance to ocular HSV-1 infection in the presence of naked DNA plasmid vector or plasmid DNA encoding interferon-α1 topically applied to the cornea of the mice. Wild-type mice showed greater resistance to HSV-1 infection compared to ICAM-1 knockout mice as measured by cumulative survival. The absence of ICAM-1 did not affect the efficacy of the interferon-α1 transgene against ocular HSV-1. Both ICAM-1 and wild-type mice treated with the transgene showed a reduction in viral load and antigen expression in the trigeminal ganglion compared to the plasmid vector-treated counterparts. In contrast, the presence of the transgene reduced the number of infiltrating cells into the cornea in comparison to plasmid vector DNA controls in the wild-type mice but not in the ICAM-1 knockout mice. Collectively, these results suggest that the IFN-α1 transgene can restore resistance against HSV-1 infection in ICAM-1-deficient mice
Molecular Characterization of the Genome of Maize Rayado Fino Virus, the Type Member of the Genus Marafivirus

AbstractThe complete nucleotide sequence of the single-stranded RNA genome of Maize rayado fino virus (MRFV), the type member of the genus Marafivirus, is 6305 nucleotides (nts) in length and contains two putative open reading frames (ORFs). The largest ORF (nt 97–6180) encodes a polyprotein of 224 kDa with sequence similarities at its N-terminus to the replication-associated proteins of other viruses with positive-strand RNA genomes and to the papainlike protease domain found in tymoviruses. The C-terminus of the 224-kDa ORF also encodes the MRFV capsid protein. A smaller, overlapping ORF (nt 302-1561) encodes a putative protein of 43 kDa with unknown function but with limited sequence similarities to putative movement proteins of tymoviruses. The nucleotide sequence and proposed genome expression strategy of MRFV is most closely related to that of oat blue dwarf virus (OBDV). Unlike OBDV, MRFV RNA does not appear to contain a poly(A) tail, and it encodes a putative second overlapping open reading frame
Vesicular Release of Ebola Virus Matrix Protein VP40

AbstractWe have analysed the expression and cellular localisation of the matrix protein VP40 from Ebola virus. Full-length VP40 and an N-terminal truncated construct missing the first 31 residues [VP40(31–326)] both locate to the plasma membrane of 293T cells when expressed transiently, while a C-terminal truncation of residues 213 to 326 [VP40(31–212)] shows only expression in the cytoplasm, when analysed by indirect immunofluorescence and plasma membrane preparations. In addition, we find that full-length VP40 [VP40(1–326)] and VP40(31–326) are both released into the cell culture supernatant and float up in sucrose gradients. The efficiency of their release, however, is dependent on the presence of the N-terminal 31 residues. VP40 that is released into the supernatant is resistant to trypsin digestion, a finding that is consistent with the formation of viruslike particles detected by electron microscopy. Together, these results provide strong evidence that Ebola virus VP40 is sufficient for virus assembly and budding from the plasma membrane
Rubella Virus RNA Replication Is cis-Preferential and Synthesis of Negative- and Positive-Strand RNAs Is Regulated by the Processing of Nonstructural Protein

AbstractRubella virus (RV) genome encodes nonstructural protein (NSP) in a large open reading frame at its 5′ end. It is translated into p200 and further processed into p150 and p90. The NSPs are responsible for viral RNA replication, during which a full-length negative-strand RNA serves as the intermediate for the replication of positive-strand genomic RNA and the transcription of subgenomic RNA. Using complementation experiments, we demonstrated that RV negative-strand RNA is synthesized preferentially in cis while positive-strand RNAs can be synthesized both in cis and in trans but with higher efficiency in cis. During virus infection, negative-strand RNA accumulates until 10 hours postinfection (hpi) and remains nearly constant thereafter. In contrast, positive-strand RNAs (both genomic and subgenomic RNA) do not increase much before 10 hpi and accumulate rapidly thereafter. Previously we demonstrated that p200 synthesizes negative- but not positive-strand RNA, whereas cleavage products p150/p90 are required for efficient production of positive-strand RNAs. In this study, we present evidence demonstrating that a higher concentration of p150/p90 is associated with lower production of negative-strand RNA. Our data support the hypothesis that p200 is the principal replicase for negative-strand RNA, as is p150/p90 for positive-strand RNA. The switch from the synthesis of negative- to positive-strand RNA is thus regulated by NSP processing, which not only activates the efficient production of positive-strand RNA, but also disables negative-strand RNA synthesis. A mechanism for NSP translation, processing, and regulation of RV RNA synthesis is proposed
Recovery of Infectious Human Parainfluenza Type 2 Virus from cDNA Clones and Properties of the Defective Virus without V-Specific Cysteine-Rich Domain

AbstractA full-length cDNA clone was constructed from the genome of the human parainfluenza type 2 virus (hPIV2). First, Vero cells were infected with recombinant vaccinia virus expressing T7 RNA polymerase, and then the plasmid encoding the antigenome sequence was transfected into Vero cells together with polymerase unit plasmids, NP, P, and L, which were under control of the T7 polymerase promoter. Subsequently, the transfected cells were cocultured with fresh Vero cells. Rescue of recombinant hPIV2 (rPIV2) from cDNA clone was demonstrated by finding the introduced genetic tag. As an application of reverse genetics, we introduced one nucleotide change (UCU to ACU) to immediate downstream of the RNA-editing site of the V gene in the full-length hPIV2 cDNA and were able to obtain infectious viruses [rPIV2V(−)] from the cDNA. The rPIV2V(−) possessed a defective V protein that did not have the unique cysteine-rich domain in its carboxyl terminus (the V-protein-specific domain). The rPIV2V(−) showed no growth in CV-1 and FL cells. Replication of the rPIV2V(−) in these cells, however, was partially recovered by adding anti-interferon (IFN)-β antibody into the culture medium, showing that the rPIV2V(−) is highly sensitive against IFN and that no growth of rPIV2V(−) in CV-1 and FL cells is mainly due to its hypersensitivity to endogenously produced IFN. These findings indicate that the V-protein-specific domain of hPIV2 is related to IFN resistance. On the other hand, the rPIV2V(−) efficiently replicated in Vero cells, which are known as a IFN-non-producers. However, the virus yields of rPIV2V(−) in Vero cells were 10- to100-fold lower than those of control rPIV2, although syntheses of the viral-specific proteins and their mRNAs in rPIV2V(−)-infected Vero cells were augmented up to 48 p.i. in comparison with those of rPIV2. Furthermore, the rPIV2V(−) virions showed anomalous in size as compared with rPIV2 virions. These results suggest that the V protein plays an important role in the hPIV2 assembly, maturation, and morphogenesis
Relationships between 80 Human Papillomavirus Genotypes and Different Grades of Cervical Intraepithelial Neoplasia: Association and Causality

AbstractTo clarify the causal relationship between human papillomavirus (HPV) and cervical intraepithelial neoplasia (CIN), we analyzed 386 unfixed biopsy specimens by blot hybridization at Tm −40°C, targeting 38 skin and 42 genital HPVs. By the recognition of PstI, BanI, and MspI cleavage patterns, single genital, but no skin's HPVs were identified with more than 10 copies per cell in 354 CIN (88 CIN I, 94 CIN II, and 172 CIN III). HPVs 40, 42, 43, 54, 62, or 71 was found in 10 CIN I, while HPVs 18, 30, 39, 51, 56, 59, 66, 68, 69, or 82 was found in 35 CIN I, 20 CIN II, or 8 CIN III. On the other hand, HPVs 16, 31, 33, 35, 52, 58, or 67 was identified in 43 CIN I, 74 CIN II, or 164 CIN III. The results are strongly indicative that most genital HPVs have potency to induce CIN I; however, HPV 16 and its closely related types are able to efficiently induce CIN III. We discuss the definition of causal HPV for CIN with regard to viral prevalence and viral load