bluuebunny/crossref_metadata_2025 huggingface dataset:
Genetic analysis of the nsP3 region of Sindbis virus: evidence for roles in minus-strand and subgenomic RNA synthesis
Authors: LaStarza M W , Lemm J A , Rice C M
Published: January 2020

Sindbis virus nonstructural polyproteins and their cleavage products are believed to be essential components of viral RNA replication and transcription complexes. Although numerous studies have investigated the effect of mutations in nsP1-, nsP2-, and nsP4-coding regions on Sindbis virus-specific RNA synthesis, relatively little is known about the function of the region encoding nsP3. nsP3 is a phosphoprotein comprising two regions: an N-terminal portion which is highly conserved among alphaviruses and a C-terminal portion which is not conserved, varying both in sequence and in length. We have constructed a library of random linker insertion mutations in the nsP3-coding region and characterized selected viable mutants. Initially, 126 mutants containing insertions in the conserved region and 23 with insertions in the nonconserved region were screened for temperature-sensitive (ts) plaque formation or for significant differences in plaque morphology. All nonconserved-region mutants were similar to the parental virus, whereas 13 of those in the conserved region were either ts or exhibited altered plaque phenotypes. Ten of these 13 mutants were ts for plaque formation as well as RNA accumulation at 40 degrees C. Highly ts mutants CR3.36 and CR3.39 were defective in their ability to synthesize minus-strand RNAs at the nonpermissive temperature. The CR3.36 and CR3.39 insertion mutations localized to different regions near nsP3 residues 58 and 226, respectively. CR3.39 was able to complement ts mutants from Sindbis virus complementation groups A, B, F, and G. Another mutant isolated from the library, CR3.34, while not ts for plaque formation or RNA synthesis, formed smaller plaques and was defective in subgenomic RNA synthesis at all temperatures examined. These results suggest a role for nsP3 or nsP3-containing polyproteins in the synthesis of viral minus-strand and subgenomic RNAs.
Sequence diversity of small, round-structured viruses in the Norwalk virus group
Authors: Wang J , Jiang X , Madore H P , Gray J , Desselberger U , Ando T , Seto Y , Oishi I , Lew J F , Green K Y
Published: January 2020

We have determined the nucleotide sequences of a highly conserved region of the RNA-dependent RNA polymerase of the prototype Snow Mountain agent (SMA) and of four other small, round-structured viruses (antigenically Norwalk virus [NV]-like or SMA-like) following reverse transcription-PCR amplification of viral RNA obtained from human stools. The stool samples were either from volunteers administered SMA or from sporadic cases and outbreaks of gastroenteritis that occurred in Japan and the United Kingdom between 1984 and 1992. The GLPSG and YGDD RNA polymerase motifs were in the proper locations in the sequences of the five SRSVs, but each sequence was distinct from the 8FIIa prototype NV sequence and from each other. Analysis of the sequences and reactivities in a new NV antigen enzyme-linked immunosorbent assay showed that the five viruses could be divided into two groups (serogroups) with NV and SMA, respectively, being the prototypes. The sequences of the capsid region and a nonstructural region (2C) were determined from one strain from each group. One virus (SRSV-KY-89/89/J), isolated in Japan and antigenically similar to the prototype NV (isolated 21 years earlier in Ohio), showed a remarkable level of sequence similarity to NV. KY-89 and the 8FIIa NV showed 87.2% nucleotide similarity over 2,516 continuous nucleotides amounting to 96 to 98.9% amino acid similarity in three distinct domains in two open reading frames. Between the prototype SMA and NV, the polymerase region showed 63% nucleotide and 59% amino acid similarity, respectively. Two other antigenically SMA-like isolates (SRSV-925/92/UK and SRSV-OTH-25/89/J), from the United Kingdom and Japan, showed 80% nucleotide and 88 to 92% amino acid similarity in the polymerase region to the prototype SMA isolated 16 and 13 years earlier in the United States. The capsid region of the antigenically SMA-like OTH-25 virus showed 53% nucleotide and 65% amino acid similarity to the prototype NV capsid region. Domains of sequence diversity and conversation were identified within the capsid protein of these two distinct prototype serotypes of virus. These results indicate that NV-like and SMA-like agents are still circulating, and sequence comparisons will be useful to identify and classify distinct viruses in the NV group.
Cell-free assembly of the herpes simplex virus capsid
Authors: Newcomb W W , Homa F L , Thomsen D R , Ye Z , Brown J C
Published: January 2020

Herpes simplex virus type 1 (HSV-1) capsids were found to assemble spontaneously in a cell-free system consisting of extracts prepared from insect cells that had been infected with recombinant baculoviruses coding for HSV-1 capsid proteins. The capsids formed in this system resembled native HSV-1 capsids in morphology as judged by electron microscopy, in sedimentation rate on sucrose density gradients, in protein composition, and in their ability to react with antibodies specific for the HSV-1 major capsid protein, VP5. Optimal capsid assembly required the presence of extracts containing capsid proteins VP5, VP19, VP23, VP22a, and the maturational protease (product of the UL26 gene). Assembly was more efficient at 27 degrees C than at 4 degrees C. The availability of a cell-free assay for HSV-1 capsid formation will be of help in identifying the morphogenetic steps that occur during capsid assembly in vivo and in evaluating candidate antiherpes therapeutics directed at capsid assembly.
Isolation of Epstein-Barr virus (EBV)-negative cell clones from the EBV-positive Burkitt's lymphoma (BL) line Akata: malignant phenotypes of BL cells are dependent on EBV
Authors: Shimizu N , Tanabe-Tochikura A , Kuroiwa Y , Takada K
Published: January 2020

During cultivation of the Epstein-Barr virus (EBV)-positive Burkitt's lymphoma (BL) line Akata, it was noted that EBV DNA is lost from some of the cells. Isolation of EBV-positive and EBV-negative clones with the same origin made it possible to examine the effects of EBV in BL cells. The results indicate that malignant phenotypes of BL, such as growth in low serum, anchorage-independent growth in soft agar, and tumorigenicity in nude mice, are dependent on the presence of EBV genomes and underline the oncogenic function of EBV in human cancer.
A switch region determines the cell type-specific positive or negative action of YY1 on the activity of the human papillomavirus type 18 promoter
Authors: Bauknecht T , Jundt F , Herr I , Oehler T , Delius H , Shi Y , Angel P , Zur Hausen H
Published: January 2020

YY1 is a zinc finger transcription factor which acts as either a repressor or an activator dependent on the promoter context. YY1 is a potent activator of the genuine human papillomavirus type 18 (HPV-18) upstream regulatory region (URR) in HeLa cells, which are known for high-level expression of the HPV-18 early genes. The activating activity of YY1 is dependent on the presence of a newly identified switch region located upstream of the YY1 binding site. Deletion of this region causes YY1 to act as a repressor of HPV-18 promoter activity. In vivo footprinting of the HPV-18 URR and an in vitro electrophoretic mobility shift assay identified proteins binding to the switch region. Site-directed mutagenesis of the switch region and YY1 binding sites suggests that these two regions work in concert to yield high-level HPV-18 URR activity in HeLa cells but not in HepG2 cells, where HPV-18 is almost inactive. These data identified a novel mode of cell type-specific regulation of HPV-18 promoter activity by positive or negative action of YY1, determined by the switch region binding factor(s).
Pol gene quasispecies of human immunodeficiency virus: mutations associated with drug resistance in virus from patients undergoing no drug therapy
Authors: Nájera I , Holguín A , Quiñones-Mateu M E , Muñoz-Fernández M A , Nájera R , López-Galíndez C , Domingo E
Published: January 2020

The nucleotide sequences of two pol gene regions (codons 41 to 108 and 181 to 219 of reverse transcriptase) of 60 human immunodeficiency virus type 1 genomes obtained directly from primary lymphocytes from infected individuals are reported. In addition, the mutant spectra of several quasispecies have been sampled by repetitive sequencing of molecular clones representing the same pol genomic regions. Average mutation frequencies ranged from 1.6 x 10(-2) to 3.4 x 10(-2) substitutions per nucleotide for independent samples (relative to their consensus nucleotide sequence) and from 3.6 x 10(-3) to 1.1 x 10(-2) substitutions per nucleotide for individual quasispecies distributions. Several mutations leading to amino acid substitutions related to loss of sensitivity to reverse transcriptase inhibitors have been identified in samples from patients not subjected to antiretroviral therapy. Mutation frequencies in the codons previously identified as involved in resistance to reverse transcriptase inhibitors were very similar to the average mutation frequencies in the pol region analyzed. Thus, the finding of mutations related to drug resistance (even in the absence of positive selection by the corresponding drugs) is the expected consequence of the statistical distribution of mutations along the pol gene. The presence of such critical amino acid replacements in human immunodeficiency virus type 1 populations underscores the importance of viral quasispecies as reservoirs of phenotypic virus variants and has a number of implications for AIDS control.
NF-Y controls transcription of the minute virus of mice P4 promoter through interaction with an unusual binding site
Authors: Gu Z , Plaza S , Perros M , Cziepluch C , Rommelaere J , Cornelis J J
Published: January 2020

Electrophoretic mobility shift assays performed with nuclear extracts from human fibroblasts revealed the formation of two major protein complexes with an oligonucleotide (nucleotides 78 to 107) from the palindromic region located upstream from the minute virus of mice (MVM) P4 promoter. It was shown that this oligonucleotide bound USF at the enhancer E box CACATG. The second complex contained the transcription factor NF-Y, whose association was surprising because its target sequence lacks the canonical CCAAT motif present in all mammalian NF-Y binding sites identified so far. The MVM NF-Y recognition element instead contains the CCAAC sequence. USF and NF-Y had distinct but overlapping sequence requirements for binding, suggesting that their associations with MVM DNA were mutually exclusive. Because of the palindromic nature of MVM DNA terminal sequences, NF-Y associated with the three nucleotide configurations corresponding to the hairpin structure and to the external and internal arms of the extended duplex replication form, respectively. However, owing to the imperfection of the palindrome, the binding of USF was restricted to the internal arm. Point mutations that suppressed the in vitro binding of NF-Y to the internal palindromic arm reduced the activity of the resident P4 promoter, while those preventing complex formation with USF did not, as determined by transient expression assays using the luciferase reporter gene. The data led to the identification of a novel P4 upstream regulatory region capable of interacting with two transcription factors, from which one (NF-Y) appeared to upmodulate the activity of the promoter.
Epstein-Barr virus nuclear protein 2 transactivation of the latent membrane protein 1 promoter is mediated by J kappa and PU.1
Authors: Johannsen E , Koh E , Mosialos G , Tong X , Kieff E , Grossman S R
Published: January 2020

Expression of the Epstein-Barr virus (EBV) latent membrane protein 1 (LMP-1) oncogene is regulated by the EBV nuclear protein 2 (EBNA-2) transactivator. EBNA-2 is known to interact with the cellular DNA-binding protein J kappa and is recruited to promoters containing the GTGGGAA J kappa recognition sequence. The minimal EBNA-2-responsive LMP-1 promoter includes one J kappa-binding site, and we now show that mutation of that site, such that J kappa cannot bind, reduces EBNA-2 responsiveness by 60%. To identify other factors which interact with the LMP-1 EBNA-2 response element (E2RE), a -236/-145 minimal E2RE was used as a probe in an electrophoretic mobility shift assay. The previously characterized factors J kappa, PU.1, and AML1 bind to the LMP-1 E2RE, along with six other unidentified factors (LBF2 to LBF7). Binding sites were mapped for each factor. LBF4 is B- and T-cell specific and recognizes the PU.1 GGAA core sequence as shown by methylation interference. LBF4 has a molecular mass of 105 kDa and is probably unrelated to PU.1. LBF2 was found only in epithelial cell lines, whereas LBF3, LBF5, LBF6, and LBF7 were not cell type specific. Mutations of the AML1- or LBF4-binding sites had no effect on EBNA-2 transactivation, whereas mutation of the PU.1-binding site completely eliminated EBNA-2 responses. A gst-EBNA-2 fusion protein specifically depleted PU.1 from nuclear extracts and bound in vitro translated PU.1, providing biochemical evidence for a direct EBNA-2-PU.1 interaction. Thus, EBNA-2 transactivation of the LMP-1 promoter is dependent on interaction with at least two distinct sequence-specific DNA-binding proteins, J kappa and PU.1. LBF3, LBF5, LBF6, or LBF7 may also be involved, since their binding sites also contribute to EBNA-2 responsiveness.
Negative regulation of the bovine papillomavirus E5, E6, and E7 oncogenes by the viral E1 and E2 genes
Authors: Vande Pol S B , Howley P M
Published: January 2020

Papillomaviruses induce benign squamous epithelial lesions that infrequently are associated with uncontrolled growth or malignant conversion. The virus-encoded oncogenes are clearly under negative regulation since papillomaviruses can latently infect cells and since different levels of viral oncogene expression are seen within the layers of differentiating infected epitheliomas. We used bovine papillomavirus type 1 (BPV-1) to investigate the mechanisms involved in the negative regulation of transformation. We found that the following two distinct and interacting mechanisms negatively regulate BPV-1 transformation effected by virally encoded trans-acting factors: (i) E2 repressors suppress transformation by the E6 and E7 oncogenes, and (ii) E1 and the E2 transactivator suppress transformation by the E6, E7, and E5 oncogenes. These systems interact in that the E2 repressors function to relieve the transformation suppression effected by the E1 and E2 transactivator genes. A BPV-1 mutant that lacked E2 repressors and E1 had greatly augmented transformation capacity. Analysis of this mutant revealed that the enhanced transformation was due to expression of the E6 and E7 genes in the absence of E5, revealing a previously unappreciated potency and synergy for the BPV-1 E6 and E7 oncogenes.
Effect of different donor cells on human immunodeficiency virus type 1 replication and selection in vitro
Authors: Spira A I , Ho D D
Published: January 2020

We sought to determine the effects of different host cells on human immunodeficiency virus type 1 (HIV-1) infection in vitro. First, 17 primary viruses of various phenotypes were examined for replicative capacity in peripheral blood mononuclear cells (PBMC) from 10 healthy donors. While the range of infection was variable over a 40-fold range, it was substantially less than that previously reported (L. M. Williams and M. W. Cloyd, Virology 184:723-728, 1991). In particular, no donor cells demonstrated total resistance to HIV-1 infection. We next cocultured PBMC from an HIV-1-infected patient with stimulated PBMC from three healthy donors to determine the effect of host cells on selection for a particular HIV-1 quasispecies. By using DNA sequencing, it was found that the dominant quasispecies (AD30-15) after culture was nearly identical in the cells of different donors. Furthermore, after 6 months in vivo, the patient developed a dominant proviral population in PBMC that was most closely related to the quasispecies preferentially selected in vitro, although this quasispecies was only a minor fraction of the sequences present earlier in PBMC. In subsequent biological characterizations, it was found that AD30-15 grew much better in PBMC and macrophages than did other related quasispecies. Hence, we conclude that the primary mechanism of in vitro selection for a particular HIV-1 variant in this case is mediated by the phenotypic properties of the virus and is less dependent on host cell origin. The findings reported here have important practical implications for studies of HIV-1 replication in primary cells derived from healthy donors.
Viral RNA modulates the acid sensitivity of foot-and-mouth disease virus capsids
Authors: Curry S , Abrams C C , Fry E , Crowther J C , Belsham G J , Stuart D I , King A M
Published: January 2020

Foot-and-mouth disease virus (FMDV) manifests an extreme sensitivity to acid, which is thought to be important for entry of the RNA genome into the cell. We have compared the low-pH-induced disassembly in vitro of virions and natural empty capsids of three subtypes of serotype A FMDV by enzyme-linked immunosorbent assay and sucrose gradient sedimentation analysis. For all three subtypes (A22 Iraq 24/64, A10(61), and A24 Cruzeiro), the empty capsid was more stable by 0.5 pH unit on average than the corresponding virion. Unexpectedly, in the natural empty capsids used in this study, the precursor capsid protein VP0 was found largely to be cleaved into VP2 and VP4. For picornaviruses the processing of VP0 is closely associated with encapsidation of viral RNA, which is considered likely to play a catalytic role in the cleavage. Investigation of the cleavage of VP0 in natural empty capsids failed to implicate the viral RNA. However, it remains possible that these particles arise from abortive attempts to encapsidate RNA. Empty capsids expressed from a vaccinia virus recombinant showed essentially the same acid lability as natural empty capsids, despite differing considerably in the extent of VP0 processing, with the synthetic particles containing almost exclusively uncleaved VP0. These results indicate that it is the viral RNA that modulates acid lability in FMDV. In all cases the capsids dissociate at low pH directly into pentameric subunits. Comparison of the three viruses indicates that FMDV A22 Iraq is about 0.5 pH unit more sensitive to low pH than types A10(61) and A24 Cruzeiro. Sequence analysis of the three subtypes identified several differences at the interface between pentamers and highlighted a His-alpha-helix dipole interaction which spans the pentamer interface and appears likely to influence the acid lability of the virus.
The Epstein-Barr virus nuclear protein 2 acidic domain can interact with TFIIB, TAF40, and RPA70 but not with TATA-binding protein
Authors: Tong X , Wang F , Thut C J , Kieff E
Published: January 2020

The Epstein-Barr virus nuclear antigen 2 (EBNA-2) acidic domain is essential for B-lymphocyte growth transformation and can activate transcription when brought to a promoter by a sequence-specific DNA-binding domain. We now show that the EBNA-2 acidic domain has slightly less activity than the proteotypic acidic transactivator VP16 in depleting nuclear extracts of basal transcription activity. Like VP16, EBNA-2 associates with TFIIB, TAF40, and RPA70. However, EBNA-2 has much less avidity for TATA-binding protein. A Trp-to-Thr mutation within the acidic domain abolishes EBNA-2 transactivating activity and greatly compromises the association with TFIIB, TAF40, and RPA70, establishing a genetic linkage between transactivating activity and these associations.
Immunization with nonstructural proteins E1 and E2 of cottontail rabbit papillomavirus stimulates regression of virus-induced papillomas
Authors: Selvakumar R , Borenstein L A , Lin Y L , Ahmed R , Wettstein F O
Published: January 2020

Cottontail rabbit papillomavirus is the major animal model for cancer-associated papillomaviruses. Here we show that vaccination with the nonstructural proteins E1 and E2 induces the regression of virus-induced papillomas and that vaccination is equally effective when proteins are given with and without adjuvant. There was no correlation between antibody levels and regression, suggesting that tumor regression may be due to a cell-mediated response.
Major histocompatibility complex class I-restricted cytotoxic T-lymphocyte responses in horses infected with equine herpesvirus 1
Authors: Allen G , Yeargan M , Costa L R , Cross R
Published: January 2020

An experimental system that permits sensitive and reproducible detection of equine herpesvirus 1 (EHV-1)-specific cytotoxic T-lymphocyte (CTL) activity in the horse was developed. Peripheral blood mononuclear cells (PBMC) collected from immune horses were restimulated in vitro by culture with live EHV-1. Cytotoxic activity against virus-infected, pokeweed mitogen-stimulated lymphoblast targets was assessed in a 4-h 51Cr release assay. The optimal conditions for in vitro stimulation of equine memory CTLs and for preparation of EHV-1-infected target cells expressing viral antigens were systematically identified by individually testing the effects of variations in responder cell concentration, culture medium composition, serum type, incubation time, antigen form, and exogenous mediator content. By using this optimized system for generation and assay of equine CTLs, the development of EHV-1-specific cytotoxic responses in 12 horses was evaluated after experimental viral infection. CTLs with the capacity for killing EHV-1-infected target cells were detected in equine PBMC as early as 1 week postinfection, reached maximal levels by 2 to 3 weeks, and remained detectable for a year after infection. Equine effector cells mediating lysis of EHV-1-infected targets were predominantly CD8+ T lymphocytes, and the cytotoxicity was specific for virus and restricted by major histocompatibility complex class I molecules. The results define a reliable and convenient experimental system for generation and assay of EHV-1 CTLs which can now be used for more-detailed characterization of the equine CTL response to infection by this herpesvirus pathogen.
Integration of viral sequences into the c-myc gene in two mammary adenocarcinomas induced by polyomavirus in athymic nude mice
Authors: Berebbi M , Cajean-Feroldi C , Apiou F , Couturier J , Garcette M , Emanoil-Ravier R , Cabannes J , Perricaudet M , Blangy D
Published: January 2020

We report the analysis of polyomavirus (Py) DNA integration into chromosomal DNA of two Py-induced mammary adenocarcinomas of athymic nude mice. Prior observations had established that these tumors had high levels of episomal Py DNA, making analysis of integration sites difficult. Propagation of tumor cells in culture allows the isolation of lines which have lost episomal Py DNA but are still tumorigenic and thus can be used for in situ and Southern analysis of Py sequences. The data reported here support the conclusion that Py DNA integrated into and next to the c-myc gene, adding further importance to this tumor system which, in its modifications of c-myc expression, appears to be similar to some human mammary cancers. In situ hybridization experiments on metaphase chromosomes of tumor cells showed that (i) in both cases, there was a single integration site at the same position on the same chromosome in all cells of a given tumor, and (ii) integration sites were different in the two tumors; in one, it was located on chromosome 15, near the c-myc proto-oncogene, and in the other, it was situated in the distal part of chromosome 1. We have demonstrated a probable rearrangement between chromosome 1 and chromosome 15, in the region of Py insertion, thus suggesting that a specific site on chromosome 15 is involved in tumorigenesis. The discovery that Py DNA was integrated at specific sites in host chromosomes raised the questions of whether such integrations were correlated with the activation of specific oncogenes. The rearrangements of the c-myc proto-oncogene observed on Southern blot analysis for both tumors, along with similar integration patterns of Py sequences, the overexpression of the c-myc gene, and the synthesis of abnormal oversized hybrid transcripts between c-myc and Py genes, favor this hypothesis. Finally, the analysis of episomal Py DNA in various tumors shows viral populations presenting a specific deletion in a part of the Py late region. This deleted region in the episomal virus genome was systematically found integrated in chromosomal DNA, thus arguing for the importance of Py integration in the induction of mammary tumor.
Human immunodeficiency virus type 1 can use different tRNAs as primers for reverse transcription but selectively maintains a primer binding site complementary to tRNA(3Lys)
Authors: Wakefield J K , Wolf A G , Morrow C D
Published: January 2020

The initiation of human immunodeficiency virus type 1 (HIV-1) reverse transcription occurs at a site in the viral RNA genome which is designated the primer-binding site (PBS). The HIV-1 PBS is an 18-nucleotide sequence that is complementary to the 3'-terminal 18 nucleotides of tRNA(3Lys), which is used as the primer for reverse transcription. All HIV-1 isolates sequenced to date contain a PBS complementary to tRNA(3Lys), suggesting that other cellular tRNAs might not function as primers for reverse transcription. To investigate this possibility, we have substituted the HIV-1 PBS with sequences predicted to be complementary to the 3'-terminal nucleotides of tRNA(1,2Lys), tRNA(Ile), and tRNA(His), which previous studies have identified to be packaged into HIV-1 virions along with tRNA(3Lys). We demonstrate that infectious viruses which utilized tRNA(1,2Lys), tRNA(Ile), and tRNA(His) in reverse transcription can be recovered. However, the appearances of viruses with PBSs complementary to these alternate tRNAs were delayed compared with the wild type. After extended in vitro culture, viruses containing the PBSs complementary to these different tRNAs reverted back to the wild-type PBS complementary to tRNA3(Lys). Furthermore, only the first 9 nucleotides of the 18 nucleotide PBSs were sufficient for HIV-1 to utilize the alternate tRNA primers in reverse transcription, demonstrating that HIV-1 does not require the complete 18-nucleotide PBS to utilize these tRNA primers for reverse transcription. These results suggest that factors other than complementarity between the PBS and the primer tRNA contribute to the selectivity of tRNA3(Lys) to initiate HIV-1 reverse transcription.
Human plasma enhances the infectivity of primary human immunodeficiency virus type 1 isolates in peripheral blood mononuclear cells and monocyte-derived macrophages
Authors: Wu S C , Spouge J L , Conley S R , Tsai W P , Merges M J , Nara P L
Published: January 2020

Physiological microenvironments such as blood, seminal plasma, mucosal secretions, or lymphatic fluids may influence the biology of the virus-host cell and immune interactions for human immunodeficiency virus type 1 (HIV-1). Relative to media, physiological levels of human plasma were found to enhance the infectivity of HIV-1 primary isolates in both phytohemagglutinin-stimulated peripheral blood mononuclear cells and monocyte-derived macrophages. Enhancement was observed only when plasma was present during the virus-cell incubation and resulted in a 3- to 30-fold increase in virus titers in all of the four primary isolates tested. Both infectivity and virion binding experiments demonstrated a slow, time-dependent process generally requiring between 1 and 10 h. Human plasma collected in anticoagulants CPDA-1 and heparin, but not EDTA, exhibited this effect at concentrations from 90 to 40%. Furthermore, heat-inactivated plasma resulted in a loss of enhancement in peripheral blood mononuclear cells but not in monocyte-derived macrophages. Physiological concentrations of human plasma appear to recruit additional infectivity, thus increasing the infectious potential of the virus inoculum.
Human T-lymphotropic virus type 1 peptides in chimeric and multivalent constructs with promiscuous T-cell epitopes enhance immunogenicity and overcome genetic restriction
Authors: Lairmore M D , DiGeorge A M , Conrad S F , Trevino A V , Lal R B , Kaumaya P T
Published: January 2020

Conventional strategies of viral peptide immunizations often elicit low-affinity antibody responses and have limited ability to elicit immune responses in outbred animals of diverse major histocompatibility (MHC) haplotypes. This genetically restricted T-cell-stimulatory activity of peptides is a serious obstacle to vaccine design. However, the use of promiscuous T-cell epitopes may circumvent this problem. Promiscuous T-cell epitopes from tetanus toxin (amino acids [aa] 580 to 599) and the measles virus F protein (aa 288 to 302) that bind to several isotypic and allotypic forms of human MHC class II molecules have been identified and have been used in highly immunogenic constructs to overcome haplotype-restricted immune responses. Chimeric and beta-template peptide constructs incorporating known human T-lymphotropic virus type 1 (HTLV-1) B- and T-cell epitopes from the surface envelope protein gp46 (SP2 [aa 86 to 107] and SP4a [aa 190 to 209]) and promiscuous T-cell peptides were synthesized, and their immunogenicities were evaluated in both rabbits and mouse strains of divergent haplotypes (C3H/HeJ [H-2k], C57BL/6 [H-2b], and BALB/c [H-2d]). In addition, peptide preparations were structurally characterized by analytical high-performance liquid chromatography, mass spectrometry, and circular dichroism. In contrast to their linear forms, the chimeric constructs of both the SP2 and SP4a epitopes displayed alpha-helical secondary structures. Immunogenicity of the peptide constructs was evaluated by direct and competitive enzyme-linked immunosorbant assay (ELISA), as well as by radioimmunoprecipitation, syncytium inhibition, and antigen-induced lymphocyte proliferation assays. Immunization with the SP4a peptide without conjugation to a carrier protein produced antibodies specific for SP4a in two mouse strains (C3H/HeJ and C57BL/6). However, BALB/c mice failed to respond to the peptide, indicating that the T-cell epitope of the SP4a sequence is MHC restricted. In contrast, the chimeric constructs MVF-SP2 and SP4a-measles virus F protein were highly immunogenic, producing elevated ELISA titers after only two immunizations. Elicited antibodies recognized native forms of gp46 in ELISAs and radioimmunoprecipitation assays, as well as inhibited HTLV-1-mediated syncytium formation. In addition, chimeric constructs were effective at induction of lymphocyte proliferation to the T-cell epitope, SP4a, in each strain of immunized mice. Our data demonstrate that the antibody response to retroviral peptides is enhanced by promiscuous peptide constructs, in part because of the ability of such constructs to promote appropriate secondary structural forms of viral epitopes. In addition, these constructs promote virus-specific helper T-cell responses, thereby overcoming genetically restricted immune responses to the synthetic peptides.
Efficient excision of the upstream large intron from P4-generated pre-mRNA of the parvovirus minute virus of mice requires at least one donor and the 3' splice site of the small downstream intron
Authors: Zhao Q , Gersappe A , Pintel D J
Published: January 2020

We have previously shown that efficient excision of the upstream large intron from P4-generated pre-mRNA of the autonomous parvovirus minute virus of mice depends upon at least the initial presence of sequences within the downstream small intron (Q. Zhao, R. V. Schoborg, and D. J. Pintel, J. Virol. 68:2849-2859, 1994). In this report, we show that the requirement of downstream small intron sequences is complex and that efficient excision of the upstream intron requires at least one small intron donor and the 3' splice site. In the absence of both small intron donors, a new spliced product is produced in which the intervening exon is skipped and the large intron donor at nucleotide 514 is joined to a small intron acceptor. Exon skipping caused by the loss of the two small intron donors can be overcome, and the excision of the large intron can be regained by mutations that improve the large intron polypyrimidine tract. These results are consistent with a model in which the binding of multiple splicing factors that assemble at both a downstream donor and acceptor facilitates the binding of splicing factors to the weak polypyrimidine tract of the upstream large intron, thereby defining the intervening exon and promoting excision of the upstream intron.
Replication of avian leukosis viruses with mutations at the primer binding site: use of alternative tRNAs as primers
Authors: Whitcomb J M , Ortiz-Conde B A , Hughes S H
Published: January 2020

We have tested whether avian leukosis viruses (ALVs) can use tRNAs other than tRNATrp to initiate reverse transcription. The primer binding site (PBS) of a wild-type ALV provirus, which is complementary to the 3' end of tRNA(Trp), was replaced with sequences homologous to the 3' ends of six different chicken tRNAs (tRN(APro), tRNA(Lys), tRNA(Met), tRNA(Ile), tRNA(Phe), and tRNA(Ser)). Transfection of these proviruses into chicken embryo fibroblasts resulted in the production of infectious viruses, all of which apparently used the tRNA specified by the mutated PBS to replicate. However, growth of these viruses resulted in reversion to the wild-type (tRNA(Trp)) PBS. Some of the viruses revert quite quickly, while others are more stable. The relative stability of a given PBS correlated with the concentration of the corresponding tRNA in the virion. We determined the percentage of viral RNA that had a tRNA bound to the PBS and found that the occupancy rate is lower in the mutants than in the wild-type virus. We conclude that many different tRNAs can be used as primers to initiate reverse transcription in ALV. However, ALVs that use tRNA(Trp) have a growth advantage over ALVs that use other tRNAs.
In vivo pathogenic properties of two clonal human immunodeficiency virus type 1 isolates
Authors: Jamieson B D , Pang S , Aldrovandi G M , Zha J , Zack J A
Published: January 2020

We have investigated the in vivo pathogenic properties of two molecularly cloned strains of human immunodeficiency virus type 1 (HIV-1), HIV-1NL4-3 and HIV-1JR-CSF, in human fetal thymus/liver implants in severe combined immunodeficient mice. Studies comparing their in vivo replication kinetics and abilities to induce CD4+ thymocyte depletion were performed. HIV-1NL4-3 replicated in vivo with faster kinetics and induced greater levels of CD4+ thymocyte depletion than did HIV-1JR-CSF. These results demonstrate that different viral isolates have different pathogenic properties in this system. In the SCID-hu model, this pathogenesis most likely occurs in the absence of an immune response. Therefore, we investigated whether the absence of immune selection resulted in extensive genetic variation and the generation of viral quasispecies. To this end, DNA corresponding to the fourth variable domain region of the viral envelope gp120 protein recovered from biopsy samples at 6 weeks postinfection was sequenced. Little genetic variation was noted in either HIV-1JR-CSF- or HIV-1NL4-3-infected implants. The mutation levels demonstrated in both viral strains were more reflective of the acute rather than the chronic phase of HIV-1 infection in humans. These results suggest that the SCID-hu mouse model can be used to study the in vivo pathogenicity of different HIV-1 isolates in the absence of host immune selective pressures.
Adenosine N1-oxide inhibits vaccinia virus replication by blocking translation of viral early mRNAs
Authors: Kane E M , Shuman S
Published: January 2020

Adenosine N1-oxide (ANO) is a potent and highly selective inhibitor of vaccinia virus replication. We examined the impact of ANO on vaccinia virus macromolecular synthesis during synchronous infection of BSC40 cells. Viral DNA replication and viral late protein synthesis were blocked completely by ANO, effects that were attributable to a defect in the expression of viral early genes. Vaccinia virus early proteins were not synthesized in the presence of ANO, even though vaccinia virus early mRNAs were produced. Cellular protein synthesis was unaffected by ANO, and virus infection in the presence of the drug did not elicit the normal shutoff of host protein synthesis. Adenosine N1-oxide triphosphate (ANO-TP), the predominant metabolite of the drug in vivo, could substitute for ATP in RNA synthesis by purified vaccinia virus RNA polymerase. ANO-TP could support early transcription by purified virions if dATP was provided as an energy source. ANO-TP did not inhibit early transcription in the presence of ATP. These findings suggest a novel antiviral mechanism whereby incorporation of a modified nucleotide into viral mRNAs might selectively block viral gene expression at the level of translation. We believe that ANO merits consideration as an antipoxvirus drug for topical treatment of molluscum contagiosum in humans.
Interaction of polypyrimidine tract-binding protein with the 5' noncoding region of the hepatitis C virus RNA genome and its functional requirement in internal initiation of translation
Authors: Ali N , Siddiqui A
Published: January 2020

Initiation of translation of the human hepatitis C virus (HCV) RNA genome occurs by internal ribosome entry into the 5' noncoding region (5'NCR) in a cap-independent manner. The internal ribosome entry site of the HCV 5'NCR has been previously defined to encompass almost the entire 5'NCR. Here we report the interaction of polypyrimidine tract-binding protein (PTB) at three distinct regions within the 5'NCR by UV cross-linking assays. All three regions contain a consensus polypyrimidine tract motif. The evidence for the interaction of recombinant PTB at multiple sites within the 5'NCR is based on the use of 5'NCR mutants as competitors and by direct UV cross-linking of the mutant RNAs. Furthermore, the PTB isomers from HeLa nuclear extracts interact with the HCV 5'NCR, as shown by immunoprecipitation of a UV cross-linked complex with anti-PTB serum. Immunodepletion of PTB from translation lysates suggested the functional requirement for PTB during translation initiation of the HCV RNA. Addition of purified PTB to immunodepleted lysates did not restore translation mediated by the HCV 5'NCR, indicating the requirement of PTB-associated factors that were removed during immunodepletion.
Sequential activation of cyclin E and cyclin A gene expression by human papillomavirus type 16 E7 through sequences necessary for transformation
Authors: Zerfass K , Schulze A , Spitkovsky D , Friedman V , Henglein B , Jansen-Dürr P
Published: January 2020

To investigate E7-dependent biochemical changes which are involved in cellular transformation, we analyzed the influence of human papillomavirus type 16 (HPV-16) E7 on the expression of cell cycle regulatory proteins. Expression of E7 in established rodent fibroblasts (NIH 3T3), which was shown to be sufficient for transformation of these cells, leads to constitutive expression of the cyclin E and cyclin A genes in the absence of external growth factors. Surprisingly, expression of the cyclin D1 gene, which encodes a major regulator of G1 progression, is unaltered in E7-transformed cells. In transient transfection experiments, the cyclin A gene promoter is activated by E7 via an E2F binding site. In 14/2 cells, which were used as a model system to analyze the role of HPV-16 E7 in the transformation of primary cells, we observed rapid E7-dependent activation of cyclin E gene expression, which can be uncoupled from activation of the cyclin A gene, since the latter requires additional protein synthesis. E7-driven induction of cyclin E and cyclin A gene expression was accompanied by an increase in the associated kinase activities. Two domains of the E7 oncoprotein, which are designated cd1 and cd2, are essential for transformation of rodent fibroblasts. It is shown here that growth factor-independent expression of the cyclin E gene requires cd2 but not cd1, while activation of cyclin A gene expression requires cd1 function in addition to that of cd2. These data suggest that cyclin A gene expression is controlled by two distinct negative signals, one of which also restricts expression of the cyclin E gene. The ability of E7 to separately override each of these inhibitory signals, via cd1 and cd2, cosegregates with its ability to fully transform rodent fibroblasts. Unlike serum growth factors, E7 induces S-phase entry without activating cyclin D1 gene expression, in keeping with the finding that cyclin D1 function is not required in cells transformed by DNA tumor viruses.
In vitro analysis of a primary, major histocompatibility complex (MHC)-restricted, cytotoxic T-lymphocyte response to avian leukosis virus (ALV), using target cells expressing MHC class I cDNA inserted into a recombinant ALV vector
Authors: Thacker E L , Fulton J E , Hunt H D
Published: January 2020

The interaction between the major histocompatibility complex (MHC) and cytotoxic T lymphocytes (CTLs) is an important component of the host's resistance to viral infections and tumor formation. In this study, an avian leukosis virus (ALV) vector system, RCASBP, expressing MHC chicken class I (B-F) cDNA was used to develop target cells expressing the chicken class I glycoproteins complexed with ALV antigens on the cell surface. Peripheral blood from chickens inoculated with ALV was shown to contain antigen-specific, MHC-restricted, CD8+ effector CTLs, using a 51Cr release assay utilizing the RCASBP B-F target cells. The stimulated effector cells were also predominantly alpha beta T-cell receptor-positive (TCR2) T cells. The CTL response varied between two haplotypes of chickens which differed in their response to Rous sarcoma virus (RSV)-induced tumors. Chickens with the B21 haplotype which regress RSV-induced tumors showed maximal cytolytic activity, while chickens with the B13 haplotype which do not regress RSV-induced tumors had minimal to no cytolytic activity. In addition to assessing the CTL response to ALV, the creation of MHC-specific immortal target cell lines will be extremely useful in evaluating CTL responses to other viral disease in chickens.
Retroviral nucleocapsid domains mediate the specific recognition of genomic viral RNAs by chimeric Gag polyproteins during RNA packaging in vivo
Authors: Berkowitz R D , Ohagen A , Höglund S , Goff S P
Published: January 2020

The retroviral nucleocapsid (NC) protein is necessary for the specific encapsidation of the viral genomic RNA by the assembling virion. However, it is unclear whether NC contains the determinants for the specific recognition of the viral RNA or instead contributes nonspecific RNA contacts to strengthen a specific contact made elsewhere in the Gag polyprotein. To discriminate between these two possibilities, we have swapped the NC domains of the human immunodeficiency virus type 1 (HIV-1) and Moloney murine leukemia virus (M-MuLV), generating an HIV-1 mutant containing the M-MuLV NC domain and an M-MuLV mutant containing the HIV-1 NC domain. These mutants, as well as several others, were characterized for their abilities to encapsidate HIV-1, M-MuLV, and nonviral RNAs and to preferentially package genomic viral RNAs over spliced viral RNAs. We found that the M-MuLV NC domain mediates the specific packaging of RNAs containing the M-MuLV psi packaging element, while the HIV-1 NC domain confers an ability to package the unspliced HIV-1 RNA over spliced HIV-1 RNAs. In addition, we found that the HIV-1 mutant containing the M-MuLV NC domain exhibited a 20-fold greater ability than wild-type HIV-1 to package a nonviral RNA. These results help confirm the notion that the NC domain specifically recognizes the retroviral genomic RNA during RNA encapsidation.
Classical swine fever virus: independent induction of protective immunity by two structural glycoproteins
Authors: König M , Lengsfeld T , Pauly T , Stark R , Thiel H J
Published: January 2020

To study which proteins of classical swine fever virus (CSFV) are able to confer protective immunity in swine, N-terminal autoprotease, viral core protein, and the three structural glycoproteins were expressed via vaccinia virus recombinants (VVR). CSFV proteins synthesized in cells infected with VVR showed migration characteristics on sodium dodecyl sulfate gels identical to those of their respective CSFV counterparts. Apparently authentic dimerization of the recombinant glycoproteins was observed. The glycoproteins E0 and E2 were detected on the surfaces of VVR-infected cells. In protection experiments, swine were immunized with the different VVR, and the generation of humoral immune response was monitored. Only animals vaccinated with VVR expressing E0 and/or E2 resisted a lethal challenge infection with CSFV. Glycoprotein E0 represents a second determinant for the induction of protective immunity against classical swine fever.
Self-assembly in vitro of purified CA-NC proteins from Rous sarcoma virus and human immunodeficiency virus type 1
Authors: Campbell S , Vogt V M
Published: January 2020

The internal structural proteins of retroviruses are proteolytically processed from the Gag polyprotein, which alone is able to assemble into virus-like particles when expressed in cells. All Gag proteins contain domains corresponding to the three structural proteins MA, CA, and NC. We have expressed the CA and NC domains together as a unit in Escherichia coli, both for Rous sarcoma virus (RSV) and for human immunodeficiency virus type 1 (HIV-1). We also expressed a similar HIV-1 protein carrying the C-terminal p6 domain. RSV CA-NC, HIV-1 CA-NC, and HIV-1 CA-NC-p6 were purified in native form by classic methods. After adjustment of the pH and salt concentration, each of these proteins was found to assemble at a low level of efficiency into structures that resembled circular sheets and roughly spherical particles. The presence of RNA dramatically increased the efficiency of assembly, and in this case all three proteins formed hollow, cylindrical particles whose lengths were determined by the size of the RNA. The optimal pH at which assembly occurred was 5.5 for the RSV protein and 8.0 for the HIV-1 proteins. The treatment of the RSV CA-NC cylindrical particles with nonionic detergent, with ribonuclease, or with viral protease caused disassembly. These results suggest that RNA plays an important structural role in the virion and that it may initiate and organize the assembly process. The in vitro system described should facilitate the dissection of assembly pathways in retroviruses.
Thymus involution induced by mouse hepatitis virus A59 in BALB/c mice
Authors: Godfraind C , Holmes K V , Coutelier J P
Published: January 2020

Mouse hepatitis virus A59 (MHV-A59) infection of adult BALB/c mice induced a severe, transient atrophy of the thymus. The effect was maximal at 1 week after infection, and thymuses returned to normal size by 2 weeks after infection. There was no effect of glucocorticoids, since thymus atrophy was also found in adrenalectomized, infected mice. In infected thymus, immature CD4+ CD8+ lymphocytes were selectively depleted, and apoptosis of lymphocytes was increased. The MHV receptor glycoprotein MHVR was detected on thymus epithelial cells but not on T lymphocytes. In a small number of stromal epithelial cells, but in very few lymphocytes, the viral genome was detectable by in situ hybridization. These observations suggested that MHV-A59-induced thymic atrophy results not from a generalized lytic infection of T lymphocytes but rather from apoptosis of immature double-positive T cells that might be caused by infection of a small proportion of thymus epithelial cells or from inappropriate secretion of some factor, such as a cytokine.