pinecone/core-2020-05-10-deduplication huggingface dataset:
Interactions between Tombusviruses and Satellite RNAs of Tomato Bushy Stunt Virus: A Defect in sat RNA B1 Replication Maps to ORF1 of a Helper Virus

AbstractThe biological properties of two recently described satellite RNAs of tomato bushy stunt virus (TBSV) were analyzed in natural and experimental hosts. Full-length cDNA clones were constructed for sat RNAs B1(822 nt) and B10 (612 nt) and used in inoculations with satellite-free transcripts of different tombusviruses. In all virus-host combinations tested, TBSV sat RNA B10 drastically reduced the accumulation of viral genomic RNA and attenuated symptoms. In contrast, sat RNA B1 caused a less marked reduction of viral RNA level and did not have any effect on symptoms. Experiments with Nicotiana benthamiana protoplasts showed that the differential effects of sat RNAs B1 and B10 on TBSV titer were related to differential abilities to interfere with virus replication. Three tombusviruses tested were able to maintain both sat RNAs in N. benthamiana plants, although carnation Italian ringspot virus (CIRV) was a poor helper for sat RNA B1. Using chimeric viruses, a strong determinant for low sat RNA B1 accumulation was mapped to the 5′-terminal part of the genome of CIRV. The poor helper activity of CIRV was shown to be due to low sat RNA B1 replication. A single-nucleotide mutation in the start codon of CIRV ORF1 restored the ability to replicate sat RNA B1 to high levels. This mutant encodes an ORF1 that is 22 amino acids shorter at the N-terminus than the wild-type virus
Adenovirus E4 34k and E4 11k Inhibit Double Strand Break Repair and Are Physically Associated with the Cellular DNA-Dependent Protein Kinase

AbstractThe adenovirus oncoproteins E4 34k and E4 11k, the products of E4 open reading frames 6 and 3, respectively, individually prevent the formation of concatemers of the linear viral genome in infected cells. We show here that genome concatenation in E4 mutant-infected cells requires the cellular DNA-dependent protein kinase (DNA PK) and that E4 34k inhibits V(D)J recombination, a normal cellular process that is also dependent on DNA PK. We further show that both E4 34k and E4 11k coimmunoprecipitate with DNA PK. These observations indicate that E4 products block formation of concatemers of the viral genome by inhibiting DNA PK-dependent double strand break repair and suggest that they act by forming a physical complex with DNA PK. DNA PK also participates in activation of p53 DNA-binding activity by DNA damage. By inhibiting DNA PK function, E4 products may block p53 activation in response to the products of viral DNA replication and thus provide a new mechanism to prevent apoptosis of infected cells
Molecular Characterization of Human Immunodeficiency Virus (HIV)-1 and -2 in Individuals from Guinea-Bissau with Single or Dual Infections: Predominance of a Distinct HIV-1 Subtype A/G Recombinant in West Africa

AbstractGuinea-Bissau in West Africa has the highest prevalence of human immunodeficiency virus (HIV)-2 infection in the world, but recently the HIV-1 prevalence increased rapidly with the subsequent appearance of HIV-1 and HIV-2 dual infections. Information about the genetic subtypes of HIV in the region is limited. Therefore, we characterized the env V3 region of HIV-1 and HIV-2 variants through direct DNA sequencing of peripheral blood mononuclear cell samples from 18 individuals with HIV-1 only and 9 individuals with dual infection. Phylogenetic analyses of these new sequences and database sequences from other West African countries showed that all HIV-1 and HIV-2 sequences from singly as well as dually infected individuals, except one, clustered among HIV-1 subtype A and HIV-2 subtype A, respectively. Importantly, a majority of the HIV-1 sequences from Guinea-Bissau and neighbouring countries were closely related with the isolates IbNG, DJ263, and DJ264, which share a common subtype A/G recombination pattern. Analysis of pol gene sequences from selected HIV-1 variants showed that “IbNG-like” viruses in Guinea-Bissau are also recombinant, indicating that the HIV-1 epidemic in Guinea-Bissau and neighbouring countries is dominated by an epidemic spread of a distinct subtype A/G recombinant, which is strikingly similar to the epidemic spread of a subtype A/E recombinant in Southeast Asia. Furthermore, the HIV-1 and HIV-2 variants carried by individuals with dual infection were intermixed with variants from singly infected individuals, indicating that variants involved in dual and single infections have common epidemiological histories
Establishment of the Human Papillomavirus Type 16 (HPV-16) Life Cycle in an Immortalized Human Foreskin Keratinocyte Cell Line

AbstractThe study of human papillomaviruses (HPVs) in cell culture has been hindered because of the difficulty in recreating the three-dimensional structure of the epithelium on which the virus depends to complete its life cycle. Additionally, the study of genetic mutations in the HPV genome and its effects on the viral life cycle are difficult using the current method of transfecting molecularly cloned HPV genomes into early-passage human foreskin keratinocytes (HFKs) because of the limited life span of these cells. Unless the HPV genome transfected into the early-passage HFK extends the life span of the cell, analysis of stable transfectants becomes difficult. In this study, we have used BC-1-Ep/SL cells, an immortalized human foreskin keratinocyte cell line, to recreate the HPV-16 life cycle. This cell line exhibits many characteristics of the early-passage HFKs including the ability to stratify and terminally differentiate in an organotypic raft culture system. Because of their similarity to early-passage HFKs, these cells were tested for their ability to support the HPV-16 life cycle. The BC-1-Ep/SL cells could stably maintain two HPV genotypes, HPV-16 and HPV-31b, episomally. Additionally, when the BC-1-Ep/SL cell line was stably transfected with HPV-16 and cultured using the organotypic raft culture system (rafts), it sustained the HPV-16 life cycle. Evidence for the productive stage of the HPV-16 life cycle was provided by: DNA in situ hybridization demonstrating HPV-16 DNA amplification in the suprabasal layers of the rafts, immunohistochemical staining for L1 showing the presence of capsid protein in the suprabasal layers of the rafts, and electron microscopy indicating the presence of virus like particles (VLPs) in nuclei from cells in the differentiated layers of the rafts
Type I Interferon Is a Powerful Inhibitor of in Vivo HIV-1 Infection and Preserves Human CD4+ T Cells from Virus-Induced Depletion in SCID Mice Transplanted with Human Cells

AbstractAlthough several studies are available on the in vitro inhibitory activities of type I interferon (IFN) on HIV-1 replication, the role of these cytokines in the pathogenesis of AIDS is still matter of conjecture. Both beneficial and adverse effects have been envisaged and considered as a possible rationale for the development of either IFN or anti-IFN therapies in HIV-1-infected patients. In the present study, we have evaluated the efficacy of human type I IFN on HIV-1 infection and virus-induced depletion of human CD4 T cells in two models established in SCID mice. In SCID mice transplanted with human U937 cells (U937-SCID mouse model), continuous treatment with type I consensus IFN (CIFN) resulted in a total suppression of HIV-1 infection. This inhibitory effect was superior to that obtained after AZT treatments. Results from an ensemble of experiments in SCID mice transplanted with either control or genetically modified human U937 cells transduced with a Tat-inducible IFN-α gene (LTR-IFN-A2 U937) indicated that low levels of IFN-α, produced locally as a result of virus infection, were extremely effective in inhibiting acute HIV infection and virus replication. Of interest, LTR-IFN-A2 U937 cells conferred a strong anti-HIV-1 protection to coinjected bystander U937 cells. Notably, experiments with SCID mice reconstituted with human PBL (hu-PBL-SCID mouse model) showed that treatment with CIFN inhibited HIV-1 replication more effectively than AZT treatment. Remarkably, treatment with CIFN resulted in a clear-cut protection from the virus-induced depletion of human CD4 T cells, which was also associated with the generation of an antibody response toward HIV-1 antigens in 50% of the virus-injected xenografts. These results suggest that type I IFN efficiently preserves human CD4+ cells from virus-induced damage in hu-PBL-SCID mice, not only by inducing an antiviral state in target cells but also by stimulating anti-HIV-1 human immune responses in vivo
Membrane Topology of the Vaccinia Virus A17L Envelope Protein

AbstractThe formation of a lipoprotein membrane within specialized areas of the cytoplasm is the first visible step in poxvirus morphogenesis. The A17L viral protein, an essential nonglycosylated membrane component, was predicted to have four centrally located α-helical membrane-spanning domains. The gene was expressed as a 23-kDa protein in a cell-free transcription/translation system containing canine pancreatic microsomes. The N- and C-terminal ends of the membrane-associated protein were susceptible to proteinase digestion, whereas the central region was resistant, consistent with a model in which the first and fourth hydrophobic domains are membrane spanning. This topology was supported by the sizes of the major proteinase-resistant membrane-associated products of genes containing one or more deleted hydrophobic domains and by evidence that the C-terminus was intraluminal and glycosylated on deletion of the second, third, and fourth domains, the third and fourth domains, or just the fourth domain. Moreover, glycosylation also occurred when an N-glycosylation site was introduced into the second hydrophobic domain of the full-length A17L protein. The data indicated a predominant topology in which the N- and C-termini are cytoplasmic, the first and fourth hydrophobic domains span the microsomal membrane, and the second and third hydrophobic domains are intraluminal. This arrangement has important implications for interactions of the A17L protein with other membrane components
Genetic Diversity of Pestiviruses: Identification of Novel Groups and Implications for Classification

AbstractThe complete Npro coding sequences were determined for 16 pestiviruses isolated from cattle, pig, and several wild ruminant species including reindeer, bison, deer, and bongo. Phylogenetic analysis enabled the segregation of pestiviruses into the established species bovine viral diarrhea virus-1 (BVDV-1), BVDV-2, border disease virus (BDV), and classical swine fever virus (CSFV). For BVDV-1 five distinct subgroups were identified, while BVDV-2, BDV, and CSFV were each subdivided into two subgroups. The virus isolates from bongo and deer as well as one porcine virus isolate belong to BVDV-1. Interestingly, the isolates from reindeer and bison are distinct from the established pestivirus species. The Npro sequences from these two viruses are more similar to BDV than to the other pestivirus species. Calculation of the pairwise evolutionary distances allowed a clear separation of the categories species, subgroup, and isolate only when the reindeer/bison viruses were considered as members of an additional pestivirus species. Furthermore, the entire E2 coding sequences of a representative set of virus isolates covering all recognized species and subgroups were studied. Segregation of pestiviruses based on the E2 region was identical with that obtained with the Npro sequences
Molecular and Biological Changes Associated with HeLa Cell Attenuation of Wild-Type Yellow Fever Virus

AbstractSix passages of the mosquito-borne flavivirus yellow fever (YF) wild-type strain Asibi in HeLa cells attenuated the virus for monkeys and newborn mice and resulted in loss of mosquito competence. Attenuation after the passage in HeLa cells was not unique to YF virus strain Asibi as demonstrated by the HeLa passage attenuation of wild-type YF virus strain French viscerotropic virus and YF vaccine virus 17D-204 for newborn mice. In contrast, wild-type strain Dakar 1279 and the French neurotropic vaccine virus remained virulent for newborn mice after six passages in HeLa cells. Thus not all strains of YF virus can be attenuated by passage in HeLa cells. Attenuation of YF virus strains Asibi and French viscerotropic virus was accompanied by alterations in the antigenic and biological properties of the viruses, including changes to envelope protein epitopes. Attenuation for newborn mice was coincidental with the acquisition by the HeLa-passaged viruses of the vaccine-specific envelope protein epitope recognized by monoclonal antibody H5. This suggests that this conformational change may play a role in the attenuation process. Wild-type Dakar 1279, which remained virulent for newborn mice after passage in HeLa cells, retained its wild-type antigenic character. The genome of Asibi HeLa p6 virus differed from wild-type Asibi virus by 29 nucleotides that encoded 10 amino acid substitutions: 5 in the envelope protein, 1 in NS2A, 3 in NS4B, and 1 in NS5. The substitution at NS4B-95 is seen in three different attenuation processes of wild-type YF virus, leading us to speculate that it is involved in the attenuation of virulence of wild-type strain Asibi
Novel, Nonconsensus Cellular Splicing Regulates Expression of a Gene Encoding a Chemokine-like Protein That Shows High Variation and Is Specific for Human Herpesvirus 6

AbstractThere are few genes that are specific and diagnostic for human herpesvirus-6. U83 and U22 are two of them. U22 is unique, whereas U83 encodes distant similarity with some cellular chemokines. Reverse transcription-polymerase chain reaction, cDNA cloning, and sequence analyses show polyadenylated RNA transcripts corresponding to minor full-length and abundant spliced forms of U83 in human herpesvirus 6-infected cells. The splice donor and acceptor sites do not fit consensus sequences for either major GT-AG or minor AT-AC introns. However, the spliced form can also be detected in a U83 transfected cell line; thus the novel sites are used by cellular mechanisms. This intron may represent a new minor CT-AC splicing class. The novel splicing regulates gene expression by introducing a central stop codon that abrogates production of the chemokine-like molecule, resulting in an encoded truncated peptide. The use of metabolic inhibitors and an infection time course showed expression of the two RNA transcripts with immediate early kinetics. However, the full-length product accumulated later, dependent on virus DNA replication, similar to U22. Sequence analyses of 16 strains showed high variation (13%) in U83 , with conservation of the novel splice sites. Representative strain variants had similar kinetics of expression and spliced products
Distinct Patterns of Viral Antigen Expression in Epstein–Barr Virus and Kaposi's Sarcoma-Associated Herpesvirus Coinfected Body-Cavity-Based Lymphoma Cell Lines: Potential Switches in Latent Gene Expression Due to Coinfection

AbstractEpstein–Barr virus (EBV) and Kaposi's sarcoma-associated herpesvirus (KSHV), also referred to as human herpesvirus 8 (HHV-8), are human gammaherpesviruses associated with numerous lymphomas and proliferative diseases in humans. We were interested in the protein expression patterns of specific latent and lytic proteins from the EBV genome in two body-cavity-based lymphoma cell lines, BC-1 and BC-2, which are coinfected with EBV and KSHV. BC-1 and BC-2 were analyzed using specific antibodies to latent proteins known to be essential for EBV immortalization of human primary B-lymphocytes in vitro and lytic antigens important for EBV replication and production of viral progeny. The coinfected cell lines are compared with two singly infected KSHV cell lines to determine whether antibodies against EBV-specific proteins cross-reacted against KSHV antigens. All the KSHV-infected cell lines express the KSHV-specific latency-associated nuclear antigen (LANA) with a specific pattern in the nucleus. This staining was distinct from that seen for EBNA1 in the EBV coinfected lines BC-1 and BC-2 staining the nucleus as a diffused pattern throughout the nucleus with denser staining in some regions. The coinfected cell lines all express EBNA1 and LMP1 at lower levels compared with singly infected EBV lymphoblastoid cell lines (LCLs). However, the essential latent antigens EBNA2, EBNA3A, and EBNA3C are not expressed in BC-1 and BC-2. This indicates potential regulation of EBV latent gene expression by KSHV-encoded viral or KSHV-induced cellular gene products. Additionally, lytic gene expression analysis demonstrated that BZLF1 and BMRF1 are expressed along with other early antigens (EA-D). A specific protein is detected in a singly infected KSHV cell line with cross-reactivity to antibodies that detected the EA-D complex. Moreover, in all the cell lines infected with EBV, KSHV, or EBV and KSHV, human serum with antibodies against KSHV antigens recognizes specific viral antigens ∼110 and 41–42 kDa, suggesting that human antibodies against KSHV-specific antigens can cross-react with similar EBV antigens. Therefore these data suggest that the EBV pattern of gene expression in the coinfected cell lines is a type II pattern of latency also seen in other human tumors including nasopharyngeal carcinoma and Hodgkin's lymphoma. This distinct pattern of latent and lytic gene expression in these cell lines may provide clues as to the selection for coinfection in these body cavity based lymphomas in immunocompromised hosts
Second Site Mutations in the N-Terminus of the Major Capsid Protein (VP5) Overcome a Block at the Maturation Cleavage Site of the Capsid Scaffold Proteins of Herpes Simplex Virus Type 1

AbstractVP5, the major capsid protein of herpes simplex virus type 1 (HSV-1), interacts with the C-terminal residues of the scaffold molecules encoded by the overlapping UL26 and UL26.5 open reading frames. Scaffold molecules are cleaved by a UL26 encoded protease (VP24) as part of the normal capsid assembly process. In this study, residues of VP5 have been identified that alter its interaction with the C-terminal residues of the scaffold proteins. A previously isolated virus (KUL26-610/611) was used that encoded a lethal mutation in the UL26 and UL26.5 open reading frames and required a transformed cell line that expresses these proteins for virus growth. The scaffold maturation cleavage site between amino acids 610 and 611 was blocked by changing Ala-Ser to Glu-Phe, which generated a new EcoRI restriction site. Revertant viruses, that formed small plaques on nontransformed cells, were detected at a frequency of 1:3800. Nine revertants were isolated, and all of them retained the EcoRI site and therefore were due to mutations at a second site. The second site mutations were extragenic. Using marker-transfer techniques, the mutation in one of the revertants was mapped to the 5′ region of the gene encoding VP5. DNA sequence analysis was performed for the N-terminal 571 codons encoding VP5 for all of the revertant viruses. Six of the nine revertants showed a single base pair change that caused an amino acid substitution between residues 30 and 78 of VP5. Three of these were identical and changed Ala to Val at residue 78. The data provide a partial map of residues of VP5 that alter its interaction with scaffold proteins blocked at their normal cleavage site. The yeast two-hybrid system was used as a measure of the interaction between mutant VP5 and scaffold molecules and varied from 11% to nearly 100%, relative to wild-type VP5. One revertant gave no detectable interaction by this assay. The amount of UL26 encoded protease (VP24) in B capsids for KUL26-610/611 and for revertants was 7% and 25%, respectively, relative to the amount in capsids for wild-type virus. The lack of retention of the viral protease in the mutant virus and a fourfold increase for the revertants suggest an additional essential function for VP24 in capsid maturation, and a role in DNA packaging is indicated
Mutations in the C, D, and V Open Reading Frames of Human Parainfluenza Virus Type 3 Attenuate Replication in Rodents and Primates

AbstractHuman parainfluenza virus type 3 (HPIV3) is a single-stranded negative-sense RNA virus belonging to the Respirovirus genus of the Paramyxoviridae family in the order Mononegavirales. The P gene encodes at least four proteins, including the C protein, which is expressed from an open reading frame (ORF) that overlaps the P ORF, and the D protein, which is encoded when the P ORF is fused to the D ORF by transcriptional editing. The P mRNA also contains a third ORF for the V protein, although it is unclear how or whether this ORF is accessed. We have used recombinant DNA technology to recover five mutant viruses that either interrupt or alter the C, D, and V ORFs. In one mutant virus, rC-KO, expression of the C protein was abrogated by changing the start codon from methionine to threonine and introducing two stop codons at amino acid positions 7 and 26 of the C ORF. In a second mutant virus, rF164S, a point mutation was introduced into the C ORF changing amino acid position 164 from phenylalanine (F) to serine (S), which corresponds to the F170S mutation described in the C protein of Sendai virus (Itoh et al., J. Gen. Virol. 78, 3207–3215). rC-KO was significantly attenuated in vitro and in vivo (rodents and primates), whereas rF164S was attenuated only in vivo. Interestingly, the rF164S mutant was more attenuated in the upper than in the lower respiratory tract of hamsters and monkeys. This pattern is the converse of that seen with temperature-sensitive attenuating mutations, and thus inclusion of this novel mutation in a recombinant live-attenuated vaccine candidate might prove useful in reducing residual virulence in the upper respiratory tract. Both rC-KO and rF164S conferred protection against challenge with wild-type HPIV3. In three other viruses, the D and V ORFs were interrupted singly or in combination. Although interruption of the D and V ORFs individually did not affect virus replication in vitro or in vivo, interruption of both together attenuated replication in vivo. These results indicate that the C, D, and V proteins of HPIV3 each has a role in virus replication in vitro, in vivo, or both, and define mutations that might be useful for the development of a vaccine against HPIV3
A Single Nucleotide Insertion in the 5′-Untranslated Region of Hepatitis C Virus Leads to Enhanced Cap-Independent Translation

AbstractThe 5′-untranslated region (5′-UTR) of hepatitis C virus (HCV) contains an internal ribosome entry site (IRES) that directs translation of the viral open reading frame (ORF). The 5′-UTR consists of 341 nucleotides (nt) in most strains, and multiple segments within this region are important for its IRES activity. Sequencing analysis of a full-length HCV cDNA clone derived from a Japanese HCV1b-positive patient showed the 5′-UTR was 342 nt long due to a nucleotide T insertion at position 207. The influence of this T insertion on the IRES activity in directing cap-independent translation was investigated. The IRES of the 5′-UTR342 was approximately five- and two- to sevenfold more active in directing luciferase expression in monocistronic and bicistronic expression systems, respectively, when compared with the IRES of the 5′-UTR341 of a previously reported HCV1b strain. In addition to the T insertion, another point mutation involving an A to C transition at position 119 was also present in the 5′-UTR342. Simultaneous comparison of the IRES activities in engineered constructs that contained each of the two mutations indicated that the insertion at position 207 is responsible for the enhanced IRES activity of the 5′-UTR342. Further determination of the abilities of the engineered 5′-UTRs harbouring A, G, or C insertions at the same position to initiate translation indicated that both T and non-T nucleotide insertions lead to enhanced cap-independent translation