bluuebunny/crossref_metadata_2025 huggingface dataset:
Complete nucleotide sequence of simian endogenous type D retrovirus with intact genome organization: evidence for ancestry to simian retrovirus and baboon endogenous virus
Authors: van der Kuyl A C , Mang R , Dekker J T , Goudsmit J
Published: January 2020

A complete endogenous type D viral genome has been isolated from a baboon genomic library. The provirus, simian endogenous retrovirus (SERV), is 8,393 nucleotides long and contains two long terminal repeats and complete genes for gag, pro, pol, and env. The primer binding site is complementary to tRNA(Lys)3, like in lentiviruses. The env GP70 protein is highly homologous to that of baboon endogenous virus (BaEV). PCR analysis of primate DNA showed that related proviral sequences are present in Old World monkeys of the subfamily Cercopithecinae but not in apes and humans. Analysis of virus and host sequences indicated that the proviral genomes were inherited from a common ancestor. Comparison of the evolution of BaEV, exogenous simian retrovirus types 1 to 3 (SRV1 to SRV3), and SERV suggests that SERV is ancestral to both BaEV and the SRVs.
The A34R glycoprotein gene is required for induction of specialized actin-containing microvilli and efficient cell-to-cell transmission of vaccinia virus
Authors: Wolffe E J , Katz E , Weisberg A , Moss B
Published: January 2020

The mechanisms allowing vaccinia virus to spread from cell to cell are incompletely understood. The A34R gene of vaccinia virus encodes a glycoprotein that is localized in the outer membranes of extracellular virions. The small-plaque phenotype of an A34R deletion mutant was similar to that of mutants with deletions in other envelope genes that fail to produce extracellular vaccinia virions. Transmission electron microscopy, however, revealed that the A34R mutant produced numerous extracellular particles that were labeled with antibodies to other outer-envelope proteins and with protein A-colloidal gold. Fluorescence and scanning electron microscopy indicated that expression of the A34R protein was necessary for detection of vaccinia virus-induced actin tails, which provide motility to the intracellular enveloped form of vaccinia virus, and of virus-tipped specialized microvilli that project from the cell. The ability of vaccinia virus-infected cells to form syncytia after a brief exposure to a pH below 6, known as fusion from within, failed to occur in the absence of expression of the A34R protein; nevertheless, purified A34R- virions were capable of mediating low-pH-induced fusion from without. The present study provides genetic and microscopic evidence for the involvement of a specific viral protein in the formation or stability of actin-containing microvilli and for a role of these structures in cell-to-cell spread rather than in formation of extracellular virions.
Purification of the simian virus 40 (SV40) T antigen DNA-binding domain and characterization of its interactions with the SV40 origin
Authors: Joo W S , Luo X , Denis D , Kim H Y , Rainey G J , Jones C , Sreekumar K R , Bullock P A
Published: January 2020

To better define protein-DNA interactions at a eukaryotic origin, the domain of simian virus 40 (SV40) large T antigen that specifically interacts with the SV40 origin has been purified and its binding to DNA has been characterized. Evidence is presented that the affinity of the purified T antigen DNA-binding domain for the SV40 origin is comparable to that of the full-length T antigen. Furthermore, stable binding of the T antigen DNA-binding domain to the SV40 origin requires pairs of pentanucleotide recognition sites separated by approximately one turn of a DNA double helix and positioned in a head-to-head orientation. Although two pairs of pentanucleotides are present in the SV40 origin, footprinting and band shift experiments indicate that binding is limited to dimer formation on a single pair of pentanucleotides. Finally, it is demonstrated that the T antigen DNA-binding domain interacts poorly with single-stranded DNA.
Molecular mechanism underlying the action of a novel fusion inhibitor of influenza A virus
Authors: Luo G , Torri A , Harte W E , Danetz S , Cianci C , Tiley L , Day S , Mullaney D , Yu K L , Ouellet C , Dextraze P , Meanwell N , Colonno R , Krystal M
Published: January 2020

In the initial stages of influenza virus infection, the hemagglutinin (HA) protein of influenza virus mediates both adsorption and penetration of the virus into the host cell. Recently, we identified and characterized BMY-27709 as an inhibitor of the H1 and H2 subtypes of influenza A virus that specifically inhibits the HA function necessary for virus-cell membrane fusion (G.-X. Luo, R. Colonno, and M. Krystal, Virology 226:66-76, 1996). Studies presented herein show that the inhibition is mediated through specific interaction with the HA protein. This binding represses the low-pH-induced conformational change of the HA protein which is a prerequisite for membrane fusion. In an attempt to define the binding pocket within the HA molecule, a number of drug-resistant viruses have been isolated and characterized. Sequence analyses of the HA gene of these drug-resistant viruses mapped amino acid changes responsible for drug resistance to a region located near the amino terminus of HA2. In addition, we have identified inactive analogs of BMY-27709 which are able to compete out the inhibitory activity of BMY-27709. This finding suggests that inhibition of the HA-mediated membrane fusion by this class of compounds is not solely the result of binding within the HA molecule but requires specific interactions.
55- and 75-kilodalton tumor necrosis factor receptors mediate distinct actions in regard to human immunodeficiency virus type 1 replication in primary human macrophages
Authors: Herbein G , Gordon S
Published: January 2020

We report in this study that repeated tumor necrosis factor alpha (TNF-alpha) pretreatment, starting before and continued after infection by human immunodeficiency virus type 1 (HIV-1), inhibits replication of the monocytotropic Ada strain in primary tissue culture-differentiated macrophages (TCDM), as assessed by sixfold lower levels of reverse transcriptase (RT) activity than that in untreated cells and absence of syncytium formation in TCDM cultures. In order to determine the pathways involved in inhibition of HIV-1 replication in primary TCDM pretreated with TNF-alpha, we tested TNF-alpha mutants T55 and T75, which recognize either the 55-kDa (TNF-R1) or the 75-kDa (TNF-R2) TNF receptor, respectively. Pretreatment of TCDM with the T75 mutant decreased the RT activity compared with that in untreated infected control cells fivefold and almost totally inhibited syncytium formation. In contrast, when TCDM were pretreated with the T55 mutant alone, syncytia were observed and RT activity was decreased about one-half. These results suggest that the inhibition of HIV-1 replication in TCDM pretreated with TNF-alpha might be mediated mainly through the 75-kDa TNF receptor (TNF-R2) rather than through the 55-kDa receptor (TNF-R1). Inhibition of HIV-1 replication in TCDM was observed with both T75 mutant pretreatment and posttreatment, starting at 1 h or 3 days after infection, whereas posttreatment with the T55 mutant, but not pretreatment, stimulated HIV-1 growth in primary TCDM. Both pre- and posttreatment with TNF-alpha inhibited HIV-1 replication in primary TCDM. The stimulation of HIV-1 replication by TNF-alpha in a chronically infected promonocytic cell line, U1, which contains two copies of integrated provirus, was mediated through the 55-kDa TNF-R1 alone and not through the 75-kDa TNF-R2. These results demonstrate that the 55-kDa TNF-R1 is involved in postintegration stimulation of HIV-1 while the 75-kDa TNF-R2 is involved in the inhibition of an early step of the viral life cycle in primary human TCDM.
The cyclin encoded by Kaposi's sarcoma-associated herpesvirus stimulates cdk6 to phosphorylate the retinoblastoma protein and histone H1
Authors: Godden-Kent D , Talbot S J , Boshoff C , Chang Y , Moore P , Weiss R A , Mittnacht S
Published: January 2020

Kaposi's sarcoma-associated herpesvirus (KSHV or human herpesvirus 8) is a novel gammaherpesvirus implicated in the cause of Kaposi's sarcoma and certain malignancies of lymphatic origin. One of the candidate genes possibly involved in promoting tumor development is an open reading frame (ORF) with sequence similarity to human type D cyclin genes. This cyclin-like gene, when expressed in tissue culture cells, promotes phosphorylation and inactivation of the retinoblastoma tumor suppressor protein and thereby may result in deregulation of cell division control. We report here the biochemical characterization of this cyclin (KSHV-cyc) and the kinase activity that it elicits upon expression in tissue culture cells. We demonstrate that the kinase activity associated with KSHV-cyc is sensitive to the cdk inhibitor p27 (KIP) and due to activation of cdk6. However, in contrast to cdk6 activated by cellular type D cyclins, the cdk6 activated by KSHV-cyc is capable of phosphorylating not only the retinoblastoma protein but also histone H1. This finding implies that activation by KSHV-cyc alters the substrate preference of this cdk. This may have important physiological consequences in that the kinase activity triggered by this viral cyclin may abrogate cell cycle checkpoints in addition to those targeted by cellular cyclin D-cdk6 kinase.
The adeno-associated virus type 2 p40 promoter requires a proximal Sp1 interaction and a p19 CArG-like element to facilitate Rep transactivation
Authors: Pereira D J , Muzyczka N
Published: January 2020

We have identified the sequence elements that are required for adeno-associated virus type 2 p40 promoter activity. Mutation of specific promoter elements showed that two Sp1 sites at approximately -50 (Sp1-50) and -70 (GGT-70) bp upstream of the start of the p40 messages were necessary for maximal promoter activity. As expected, the TATA site at -30 was also essential. In vitro DNA binding experiments confirmed that the Sp1-50 and GGT-70 sites were bound by Sp1 or Sp1-like proteins. Two other transcription elements, the ATF-80 and AP1-40 sites, may play a role in p40 activity. Mutation of these elements resulted in a modest decrease in p40 transcription, but DNA binding experiments did not clearly demonstrate binding of transcription factors to these sites. In contrast, a major late transcription factor site at -110 was shown to bind the transcription factor, but mutation of this site had no effect on p40 activity. In a previous report, we have shown that transactivation of the p40 promoter by the viral Rep proteins required an upstream Rep binding element (in the terminal repeat or the p5 promoter), an unidentified p19 promoter element, and a p40 promoter element (D. J. Pereira and N. Muzyczka, J. Virol. 71:1747-1756, 1997). Here we demonstrate that the CArG-140 element in the p19 promoter and the Sp1-50 element in the p40 promoter are the specific p19 and p40 elements required for Rep induction of p40. As in the case of the p19 promoter, Sp1 facilitates interaction of Rep with the p40 promoter by interaction of the two proteins. Furthermore, electron microscopy experiments demonstrated that when Rep is bound to an upstream Rep binding element, it can interact with a proximal Sp1 site by protein contacts and create a loop in the intervening DNA. This finding suggests a common mechanism whereby the Rep binding element in the TR or the p5 promoter induces p19 and p40 activity by interaction with their respective Sp1 sites.
Differential susceptibility of naive and memory CD4+ T cells to the cytopathic effects of infection with human immunodeficiency virus type 1 strain LAI
Authors: Chun T W , Chadwick K , Margolick J , Siliciano R F
Published: January 2020

CD4+ T lymphocytes of individuals infected with human immunodeficiency virus type 1 (HIV-1) exhibit a qualitative defect in their ability to mount memory responses to previously encountered antigens although their responses to mitogens remain normal. T cells responsible for memory responses can be distinguished from naive T cells based on differential expression of isoforms of the tyrosine phosphatase CD45. It has been suggested that memory CD4+ T cells from infected individuals have a greater virus burden than naive CD4+ T cells and that this accounts for the loss of recall responses in infected individuals. However, it has been unclear whether naive and memory T cells are equally susceptible to infection and to the cytopathic effects of the virus. We therefore infected highly purified resting naive and memory CD4+ T cells from HIV-1-seronegative individuals with HIV-1(LAI). Infected cells were then stimulated with phytohemagglutinin to render them permissive for viral replication. Cell viability and growth rate were monitored for 8 to 10 days as indicators of cytopathic effects induced by HIV-1(LAI). Our results indicated that naive and memory CD4+ T cells display marked differences in susceptibility to the cytopathic effects induced by HIV-1(LAI), infection. The cytopathic effects induced by HIV-1(LAI) were much more severe in memory CD4+ T cells than in naive CD4+ T cells. Differential cytopathic effects in naive and memory T cells were not due to differences in virus entry into and replication in these cell populations. Rather, memory cells were more susceptible to cytopathic effects. Pronounced cytopathic effects in memory cells were clearly detectable at 7 day postinfection. Cell death occurred at the single-cell level and was not accompanied by syncytium formation. The growth rate of infected memory CD4+ T cells was also severely compromised compared to that of naive CD4+ T cells, whereas the growth rates of both uninfected naive and memory CD4+ T cells were approximately the same. At least a portion of the dying cells exhibited biochemical changes characteristic of apoptosis. These results suggest that the selective functional defects present in the memory CD4+ T-cell subset of HIV-1-infected individuals may in part be the result of the greater susceptibility of memory T cells to cytopathic effects induced by HIV-1.
Broad spectrum of in vivo fitness of human immunodeficiency virus type 1 subpopulations differing at reverse transcriptase codons 41 and 215
Authors: Goudsmit J , de Ronde A , de Rooij E , de Boer R
Published: January 2020

Viral populations in a human immunodeficiency virus type 1 (HIV-1)-infected individual behave as a quasispecies with a rated distribution of fitness variants. Fitness distributions in naturally occurring viral populations have been difficult to study due to the lack of markers for individual virus clones and complicating inter- and intrahost factors like the presence of multiple cell types with distinct tropisms, differences in route of transmission, and intervening immunity. Here, we quantitated the relative fitness in vivo of three subpopulations of HIV-1 marked by mutations at codons 41 and 215 of reverse transcriptase (RT) directly related to zidovudine resistance in an untreated individual who was infected by a zidovudine-resistant strain transmitted from a donor on therapy. The transmission event did not have a substantial impact on the distribution of mutants within the dominant virus population replicating to high levels in the recipient. The evolution of the RT gene was monitored for 20 months. All 102 clones obtained from the donor and the recipient at the different time points contained the M41L mutation, which is associated with a fourfold reduction in zidovudine sensitivity. The leucine at position 41 was stable, although it was encoded by TTG and CTG triplets that fluctuated in abundance partially due to founder effects of clones with nonsilent mutations at codon 215. Of the three subpopulations in the patient, distinguished by a tyrosine (TAC), aspartic acid (GAC), or serine (TCC) at the 215 position of RT, the relative fitness of the GAC variant was calculated to be 10 to 25% higher than the initial TAC variant, and the relative fitness of the TCC variant was 1% higher than that of the GAC variant. Similar to other RNA viruses, lentivirus populations like HIV-1 in patients with a high virus load apparently consist of a broader spectrum of fitness variants than the 1 to 2% fitness difference sufficient for significant replicative advantage.
Characterization of the antibody response specific for the human endogenous retrovirus HTDV/HERV-K
Authors: Boller K , Janssen O , Schuldes H , Tönjes R R , Kurth R
Published: January 2020

Differentiated human teratocarcinoma cell lines produce the human teratocarcinoma-derived virus (HTDV) particles encoded by the human endogenous retrovirus sequence HERV-K. We screened almost 2,000 human sera for antibodies against this endogenous human retrovirus, HTDV/HERV-K. Specificity of the immunofluorescence reactions using particle producing teratocarcinoma cells was confirmed by immunoelectron microscopy of ultrathin frozen sections. Immunoblot analyses using lysates of HTDV-producing cells revealed a 80-kDa HERV-K Gag precursor and a 90-kDa putative viral Env protein after incubation with positive sera. No processed Gag protein could be observed. Virus-specific bands were not detected in lysates of nonproducing cells. High antibody titers were found in about 60% of male patients with germ cell tumors. Antibody reactivity declined after tumor removal. In healthy blood donors, anti-HTDV reactivity was found only at low titers in a small percentage (3.9%) of individuals. A slightly elevated but statistically significant percentage of HTDV positivity was also observed for sera of pregnant women, whereas human immunodeficiency virus-positive individuals exhibited no peculiarity compared to normal blood donors. Our results provide evidence that HTDV particles are expressed in vivo and that the immune reaction against HTDV/HERV-K is specific for defined viral proteins.
Characterization of LMP-1's association with TRAF1, TRAF2, and TRAF3
Authors: Sandberg M , Hammerschmidt W , Sugden B
Published: January 2020

The latent membrane protein 1 (LMP-1) of Epstein-Barr virus (EBV) contributes to the immortalizing activity of EBV in primary, human B lymphocytes. LMP-1 is targeted to the plasma membrane, where it influences signaling pathways of infected cells. LMP-1 has been found to associate with members of the tumor necrosis factor receptor-associated factor (TRAF) family of proteins. As with LMP-1, the TRAF molecules have been shown to participate in cell signaling pathways. We have characterized and mapped in detail a region of LMP-1 that associates with TRAF1, TRAF2, and TRAF3. TRAF3 alone associates with LMP-1 in a yeast two-hybrid assay, whereas all three TRAF molecules associate with LMP-1 under various conditions when they are assayed in extracts of human cells. TRAF1, TRAF2, and TRAF3 appear to associate independently with LMP-1 but bind an overlapping target site. TRAF3 associates with LMP-1 most avidly and can compete with TRAF1 and TRAF2 for binding to LMP-1. TRAF2 associates with truncated derivatives of the carboxy terminus of LMP-1 more efficiently than with the intact terminus, indicating that LMP-1's conformation may regulate its association with TRAF2. Finally, point mutations that decrease LMP-1's association with the three TRAF molecules to 3 to 20% of wild-type levels do not detectably affect otherwise intact LMP-1's induction of NF-kappaB activity. Therefore, these associations are not necessary for the majority of intact LMP-1's induction of this signaling pathway.
Molecular cloning of Mus dunni endogenous virus: an unusual retrovirus in a new murine viral interference group with a wide host range
Authors: Bonham L , Wolgamot G , Miller A D
Published: January 2020

Mus dunni endogenous virus (MDEV) is activated from cells of the Asian wild mouse M. dunni (also known as Mus terricolor) in response to treatment with either 5-iodo-2'-deoxyuridine or hydrocortisone. MDEV represents a new murine retrovirus interference group and thus appears to use a different receptor for entry into cells than do other murine retroviruses. Here we show that MDEV is also not in the gibbon ape leukemia virus or RD114 virus interference groups. A retroviral vector with an MDEV pseudotype was capable of efficiently infecting a wide variety of cells from different species, indicating that the MDEV receptor is widely expressed. We isolated a molecular clone of this virus which exhibited no hybridization to any cloned retrovirus examined, suggesting that MDEV has an unusual genome. One copy of a possible retrovirus element that weakly hybridized with MDEV was present in the genomes of laboratory strains of mice, while no such elements were present in other species examined. A virus activated by 5-iodo-2'-deoxyuridine from cells of a BALB/c mouse, however, was not related to MDEV by either hybridization or interference analyses.
Human immunodeficiency virus-associated vasculopathy in transgenic mice
Authors: Tinkle B T , Ngo L , Luciw P A , Maciag T , Jay G
Published: January 2020

There is substantial clinical evidence for the development of vascular disorders in human immunodeficiency virus (HIV)-infected individuals, particularly in the form of vasculitis. Transgenic mice carrying a replication-defective HIV-1 provirus with selective deletion of the gag, pol, and env genes developed extensive vasculopathy. Restricted expression of HIV nonstructural genes in smooth muscle cells was accompanied by the migration and proliferation of these cells in blood vessels of all sizes and at different body sites. The frequent infiltration observed in the hypertrophic vessel walls occurred predominantly in the adventitia and was composed of primarily T cells and occasionally plasma cells. The intimal thickening generated significant luminal narrowing in some vessels, and the restricted blood flow led to ischemia in the affected tissues. Interestingly, the endothelium did not appear to support HIV gene expression or be involved in the pathological process. This transgenic model provides an opportunity to dissect the mechanism underlying HIV-associated vasculopathy.
Uracil DNA glycosylase specifically interacts with Vpr of both human immunodeficiency virus type 1 and simian immunodeficiency virus of sooty mangabeys, but binding does not correlate with cell cycle arrest
Authors: Selig L , Benichou S , Rogel M E , Wu L I , Vodicka M A , Sire J , Benarous R , Emerman M
Published: January 2020

The Vpr protein encoded by human immunodeficiency virus type 1 (HIV-1) is important for growth of virus in macrophages and prevents infected cells from passing into mitosis (G2 arrest). The cellular target for these functions is not known, but Vpr of HIV-1 and the related Vpr from simian immunodeficiency virus of sooty mangabeys (SIV(SM)) bind the DNA repair enzyme UNG, while the Vpx protein of SIV(SM) does not. Nonetheless, a mutational analysis of Vpr showed that binding to UNG is neither necessary nor sufficient for the effect of Vpr on the cell cycle.
Transformation by bovine papillomavirus type 1 E6 is independent of transcriptional activation by E6
Authors: Ned R , Allen S , Vande Pol S
Published: January 2020

We have generated mutants of bovine papillomavirus type 1 E6 (BE6) that are defective for transcriptional activation and have analyzed these mutants for transformation of contact-inhibited cells and association with the mammalian protein E6-AP. These BE6 mutants demonstrate that transformation by BE6 does not require transcriptional activation and that association of BE6 with E6-AP is a function separable from transcriptional activation by BE6. Association of BE6 with E6-AP appears to be necessary but not sufficient for transformation by BE6. In addition, the mutational analysis of BE6 demonstrates that transactivation, transformation, and association with E6-AP all require sequences throughout the BE6 gene, indicating that the papillomavirus E6 proteins do not have a simple domain structure.
Mutations in reovirus outer-capsid protein sigma3 selected during persistent infections of L cells confer resistance to protease inhibitor E64
Authors: Baer G S , Dermody T S
Published: January 2020

Mutations selected in reoviruses isolated from persistently infected cultures (PI viruses) affect viral entry into cells. Unlike wild-type (wt) viruses, PI viruses can grow in the presence of ammonium chloride, a weak base that blocks acid-dependent proteolysis of viral outer-capsid proteins in cellular endosomes during viral entry. In this study, we show that E64, an inhibitor of cysteine proteases such as those present in the endocytic compartment, blocks growth of wt reovirus by inhibiting viral disassembly. To determine whether PI viruses can grow in the presence of an inhibitor of endocytic proteases, we compared yields of wt and PI viruses in cells treated with E64. Prototype PI viruses L/C, PI 2A1, and PI 3-1 produced substantially greater yields than wt viruses type 1 Lang (T1L) and type 3 Dearing (T3D) in E64-treated cells. To identify viral genes that segregate with growth of PI viruses in the presence of E64, we tested reassortant viruses isolated from independent crosses of T1L and each of the prototype PI viruses for growth in cells treated with E64. Growth of reassortant viruses in the presence of E64 segregated exclusively with the S4 gene, which encodes viral outer-capsid protein sigma3. These results suggest that mutations in sigma3 protein selected during persistent infection alter its susceptibility to cleavage during viral disassembly. To determine the temporal relationship of acid-dependent and protease-dependent steps in reovirus disassembly, cells were infected with wt strain T1L or T3D, and medium containing either ammonium chloride or E64d, a membrane-permeable form of E64, was added at various times after adsorption. Susceptibility to inhibition by both ammonium chloride and E64 was abolished when either inhibitor was added at times greater than 60 min after adsorption. These findings indicate that acid-dependent and protease-dependent disassembly events occur with similar kinetics early in reovirus replication, which suggests that these events take place within the same compartment of the endocytic pathway.
Genomic diversity and evolution of papillomaviruses in rhesus monkeys
Authors: Chan S Y , Bernard H U , Ratterree M , Birkebak T A , Faras A J , Ostrow R S
Published: January 2020

We are studying the diversity of and relationships among papillomaviruses (PVs) to understand the modes and timescales of PV evolution and in the hope of finding animal PVs that may serve as model systems for disease caused by human PVs (HPVs). Toward this goal, we have examined 326 genital samples from rhesus monkeys and long-tailed macaques with a PCR protocol optimized for detecting genital HPV types. In 28 of the rhesus monkey samples, we found amplicons derived from 12 different and novel PV genomes, RhPV-a to RhPV-m, with the likely taxonomic status of "type." The frequency with which novel RhPVs were detected suggests that rhesus monkeys may play host to PVs with a diversity similar to that of humans. In phylogenetic trees, all 12 of the different RhPVs and the previously described type RhPV-1 were members of the genital HPV supergroup and formed three minor branches distinct from the 11 branches formed by genital HPVs. We also identified a novel PV amplicon, MfPV-a, from a long-tailed macaque, a species belonging to the same genus as rhesus monkeys. MfPV-a turned out to be a close relative of five RhPVs. It appears that the evolution of primate lineages leading to the genus Macaca and to humans created transmission barriers for PVs, resulting in viral evolution closely linked to the host. Additional support for the linked-evolution hypothesis comes from considering the phylogenetic association of two other ape and monkey PVs with the genital HPVs, the supergroup formed by at least seven ungulate PVs, and the isolated phylogenetic position of the only known bird PV.
Analysis of the gene start and gene end signals of human respiratory syncytial virus: quasi-templated initiation at position 1 of the encoded mRNA
Authors: Kuo L , Fearns R , Collins P L
Published: January 2020

The gene start (GS) and gene end (GE) transcription signals of human respiratory syncytial virus (RSV) strain A2 were analyzed in helper-dependent monocistronic and dicistronic minireplicons which were complemented by a standard RSV strain. The GS signal, which is the start site for mRNA synthesis, is highly conserved for the first nine genes: 3'-CCCCGUUUA(U/C) (negative sense). This conserved version of the signal was analyzed by "saturation" mutagenesis, in which all 10 positions, as well as one downstream and one upstream position, were changed one at a time into each of the other three nucleotides. Most of the positions appear to contribute to the signal: positions 1, 3, 6, 7, and, in particular, 9 were the most sensitive, whereas position 5 was relatively insensitive. The effect of nucleotide substitution in the first position of the signal was examined further by cDNA cloning and sequence analysis of the residual mRNA which was produced. For the two mutants examined (1C to U, and 1C to A), the site of initiation was unchanged. However, the mRNAs were dimorphic with regard to the assignment of the 5'-terminal nucleotide: two-thirds contained the predicted mutant substitution, and one-third contained the parental assignment. Intracellular minigenome contained only the mutant assignment, indicating that the heterogeneity was at the level of transcription by the RSV polymerase. This suggests that the templated mutant assignment at position 1 can sometimes be overridden by an innate preference for the parental assignment, a phenomenon which we dubbed quasi-templated initiation. The GS signal of the L gene, encoding the 10th RSV mRNA, contains three differences (3'-CCCUGUUUUA) compared to the conserved version. It was shown to be equal in efficiency to the conserved version. This was unexpected, since the saturation mutagenesis described above indicated that U in place of A at position 9 should be highly inhibitory. Instead, the A at position 10 of the L GS signal was found to be critical for activity, indicating that an essential A residue indeed was present in both versions of the GS signal but that its spacing differed. The GE signal, which directs termination and polyadenylation, has more sequence diversity in nature than does the GS signal. The naturally occurring GE signals of strain A2 were compared by their individual incorporation into a dicistronic minigenome. They were similar in the ability to produce translatable mRNA except in the cases of NS1 and NS2, which were approximately 60% as efficient.
Construction of a vaccinia virus deficient in the essential DNA repair enzyme uracil DNA glycosylase by a complementing cell line
Authors: Holzer G W , Falkner F G
Published: January 2020

The vaccinia virus D4R open reading frame, encoding the essential DNA repair enzyme uracil DNA glycosylase, was expressed in two permanent cell lines, the rabbit kidney cell line RK13 and the human fibroblast cell line 293. The temperature-sensitive vaccinia virus mutant ts4149, which maps within D4R, was able to grow under restrictive conditions in both of these transformed cell lines. Cell clones complemented D4R function to various degrees, demonstrating complementation of an essential vaccinia virus gene by a cell line constitutively expressing the essential function. Thus, the complementing host cells allowed the rescue of a virus defective in the D4R gene, demonstrating that this system may be used for the propagation of defective cytoplasmic DNA viruses. The defective virus grew to high yields only in the engineered cell lines. The data support the hypothesis that early gene products, such as uracil DNA glycosylase, supplied in trans can fully complement essential viral functions.
In vivo dynamics of equine infectious anemia viruses emerging during febrile episodes: insertions/duplications at the principal neutralizing domain
Authors: Zheng Y H , Sentsui H , Nakaya T , Kono Y , Ikuta K
Published: January 2020

Equine infectious anemia virus (EIAV) is a good model for studying mechanisms generating escaped retrovirus variants. We previously sequenced the entire gp90-encoding region of 22 cDNA clones obtained from five antigenically distinct isolates (F1V to F5V) recovered during febrile episodes in horse 493 experimentally infected with the Japanese virulent EIAV strain V70. The results showed that the mutations occurred in the principal neutralizing domain (PND) by insertions/duplications. In this study, we further characterized the PND of virus isolates sequentially recovered during 22 febrile episodes in seven horses newly infected with V70 or one of the V70-derived variants. Sequencing of 70 cDNA clones derived from the 22 episodes confirmed the generation of various new viral quasispecies with insertions/duplications in the PND. Although the insertion/duplication sequences in a total of 92 cDNA clones were extensively heterogeneous, we hypothesized that all the insertions/duplications occurred during reverse transcription from viral genomic RNA to minus strand DNA. The insertion/duplication regions were derived from a part of the PND sequence, which consisted of five small units. These small units, some with various substitutions and/or deletions, were also generated, especially in regions with insertions/duplications. Of particular note was that all these virus variants, except for two cDNA variants, were generated by essentially four different duplication pathways. Thus, these results extend the significance of insertions/duplications in the PND to the novel generation of EIAV in vivo during febrile episodes.
A mechanism for negative gene regulation in Autographa californica multinucleocapsid nuclear polyhedrosis virus
Authors: Leisy D J , Rasmussen C , Owusu E O , Rohrmann G F
Published: January 2020

The Autographa californica multinucleocapsid nuclear polyhedrosis virus (AcMNPV) ie-1 gene product (IE-1) is thought to play a central role in stimulating early viral transcription. IE-1 has been demonstrated to activate several early viral gene promoters and to negatively regulate the promoters of two other AcMNPV regulatory genes, ie-0 and ie-2. Our results indicate that IE-1 negatively regulates the expression of certain genes by binding directly, or as part of a complex, to promoter regions containing a specific IE-1-binding motif (5'-ACBYGTAA-3') near their mRNA start sites. The IE-1 binding motif was also found within the palindromic sequences of AcMNPV homologous repeat (hr) regions that have been shown to bind IE-1. The role of this IE-1 binding motif in the regulation of the ie-2 and pe-38 promoters was examined by introducing mutations in these promoters in which the central 6 bp were replaced with BglII sites. GUS reporter constructs containing ie-2 and pe-38 promoter fragments with and without these specific mutations were cotransfected into Sf9 cells with various amounts of an ie-1-containing plasmid (pIe-1). Comparisons of GUS expression produced by the mutant and wild-type constructs demonstrated that the IE-1 binding motif mediated a significant decrease in expression from the ie-2 and pe-38 promoters in response to increasing pIe-1 concentrations. Electrophoretic mobility shift assays with pIe-1-transfected cell extracts and supershift assays with IE-1-specific antiserum demonstrated that IE-1 binds to promoter fragments containing the IE-1 binding motif but does not bind to promoter fragments lacking this motif.
Specific cytotoxic T lymphocytes are involved in in vivo clearance of infectious bronchitis virus
Authors: Seo S H , Collisson E W
Published: January 2020

Cytotoxic T-lymphocyte (CTL) responses to infectious bronchitis virus (IBV) were determined at regular intervals between 3 and 30 days postinfection (p.i.). The maximum response with 82% lysis of labeled target cells was detected at 10 days p.i. The specific CTL response did not begin to decline until the amount of virus, which peaked at day 8 p.i. in both the kidneys and lungs, started to decrease. Clinical respiratory signs of illness also correlated with amount of virus. CTL activity was shown to be major histocompatibility complex (MHC) class restricted because the lysis of MHC-mismatched targets was negligible, and lysis was mediated by CD8+ CD4- T cells, as the CTL response could be abolished with removal of CD8+ CD4- but not CD4+ CD8- lymphocytes. In contrast, immunoglobulin M (IgM) antibody was not detected until day 10 p.i., and levels peaked at day 12 p.i.; IgG antibody levels were minimal until day 15 p.i. but continued to increase exponentially until day 30 p.i., the last day examined. In summary, CTL responses correlated with initial decreases in infection and illness.
Characterization of a novel third member of the human cytomegalovirus glycoprotein H-glycoprotein L complex
Authors: Huber M T , Compton T
Published: January 2020

A prerequisite for understanding the molecular function of the human cytomegalovirus (HCMV) gH (UL75)-gL (UL115) complex is a detailed knowledge of the structure of this complex in its functional form, as it is present in mature virions. The gH protein is known to be a component of a 240-kDa envelope complex designated as gCIII (D. R. Gretch, B. Kari, L. Rasmussen, R. C. Gehrz, and M. F. Stinski, J. Virol. 62:875-881, 1988). However, the exact composition of the gCIII complex remains unknown. In this report, we attempted reconstitution of the gCIII complex by coexpression of gH and gL in the baculovirus expression system. Formation of recombinant gH-gL complexes of approximately 115 kDa was demonstrated; however, no higher-molecular-mass (approximately 240-kDa) recombinant gH-gL complexes were detected, suggesting that the presence of gH and gL alone is not sufficient for reconstitution of the gCIII complex. To identify other mammalian and/or HCMV factors which may be necessary for gCIII formation, immunoprecipitates of gH and gL from HCMV-infected fibroblasts and purified HCMV virions were examined. This analysis did reveal a number of coprecipitating proteins which associate either transiently or integrally with gH and gL. One coprecipitating protein of 145 kDa was shown to be an integral component of gCIII, along with gH and gL. Characterization of the 145-kDa protein demonstrates that it is structurally and antigenically unrelated to gH and gL and that it appears to be virally encoded. Together, these data indicate that the 145-kDa protein is a third novel component of the mature HCMV gH-gL complex.
Effects of nucleotide changes on the ability of hepatitis delta virus to transcribe, process, and accumulate unit-length, circular RNA
Authors: Wu T T , Netter H J , Lazinski D W , Taylor J M
Published: January 2020

The circular RNA genome of hepatitis delta virus (HDV) can fold into an unbranched rodlike structure. We mutagenized the two ends of this structure and assayed the effects on the ability of the genomes to replicate and accumulate processed RNA transcripts in transfected cells. The top end, defined as that nearest to the 5' end of the putative mRNA for delta antigen, was much more sensitive than the other end, defined as the bottom. Most of the 22 mutants made at the bottom were able to accumulate RNA as well as the wild type. For deletions extending as close as 2 nucleotides (nt) from the predicted domains needed for the two ribozymes, the accumulation levels dropped to <0.1%. In one mutant, 13 nt of HDV was replaced with 57 nt of non-HDV sequences, and accumulation was at 20% of the wild-type level, consistent with the potential of HDV to act as a vector. However, after replacement with a second sequence, accumulation dropped to 1%. For most of the 14 mutants made at the top of the rod, we observed dramatic inhibitory effects. For example, after removal of 3 bp from the stem adjacent to the terminal loop, accumulation dropped to <0.06% of the wild-type genome level. The top region that we considered was adjacent to both the 5' end of the putative mRNA and the domain that has been proposed to contain a promoter for RNA-directed RNA synthesis. The RNA accumulation abilities of certain mutants were tested under additional different experimental conditions. It was found that after longer times, some mutants began to catch up with the wild type. Also, it was found that certain top mutants gave much greater levels of accumulation when transfected into cells containing the small delta antigen. One interpretation of these data is that certain features at the top of the rod are needed for the accumulation of essential delta antigen mRNA species.
Complete nucleotide sequence of the new simian T-lymphotropic virus, STLV-PH969 from a Hamadryas baboon, and unusual features of its long terminal repeat
Authors: Van Brüssel M , Goubau P , Rousseau R , Desmyter J , Vandamme A M
Published: January 2020

A third type of primate T-lymphotropic virus, PTLV-L, with STLV-PH969 as a prototype, has recently been isolated from an African baboon (Papio hamadryas). Classification of this virus has been based on partial sequence analysis of cDNA from a virus-producing cell line, PH969. We obtained the complete nucleotide sequence of this virus with a proviral genome of 8,916 bp. All major genes, homologous in all human T-cell lymphotropic virus (HTLV)-related viruses, and their corresponding mRNAs, including appropriate splicing, were identified. One additional nonhomologous open reading frame in the proximal pX region is accessible for translation through alternative splicing. Sequence comparison shows that STLV-PH969 is equidistantly related to HTLV type 1 (HTLV-1) and HTLV-2. In all coding regions, the similarity tends to be the lowest between STLV-PH969 and HTLV-1. However, in the long terminal repeat (LTR) region, the lowest similarity was found between STLV-PH969 and HTLV-2. The U3-R and R-U5 boundaries of the STLV-PH969 LTR were experimentally determined at nucleotides 268 and 524, respectively. This 695-bp LTR is 60 and 73 bp shorter than the LTRs of HTLV-1 and HTLV-2, respectively, but its general organization is similar to the one found in the HTLV-bovine leukemia virus genus. In the long region between the polyadenylation signal and the poly(A) site, sequence similarity with the HTLV-1 Rex-responsive element (RexRE) core and secondary structure prediction suggest the presence of a RexRE. The presence of three 21-bp repeats is conserved within the U3 region of HTLV-1, HTLV-2, and BLV. Only two direct repeats with similarity to these Tax-responsive elements were found in the STLV-PH969 LTR, which might suggest differences in the Tax-mediated transactivation of this virus. We conclude that STLV-PH969 has all the genes and genomic regions to suggest a replication cycle comparable to that of HTLV-1 and HTLV-2.
pp60c-src binding to polyomavirus middle T-antigen (MT) requires residues 185 to 210 of the MT sequence
Authors: Brewster C E , Glover H R , Dilworth S M
Published: January 2020

Interaction with the src family of tyrosine kinases is crucial to the transforming action of polyomavirus middle T-antigen (MT). Association with MT activates the tyrosine kinase activity of pp60(c-src) and, through subsequent MT phosphorylation, creates binding sites for signalling molecules whose stimulation culminates in cell transformation. Despite this importance, and many studies, little is known of the mechanisms by which pp60(c-src) binds to MT. We report here isolation of the first MT mutants that disrupt pp60(c-src) binding without affecting the interaction between MT and protein phosphatase 2A (PP2A). Through deletion analysis we established that interaction with pp60(c-src) requires the sequences between amino acids 185 and 210 of MT, but these residues have no effect on PP2A binding. Cells expressing these mutants showed few altered properties, indicating that the PP2A-MT interaction alone has little influence on cell phenotype. Subcellular location of these mutant MT molecules was indistinguishable by immunofluorescence analysis from that of wild-type MT but was altered markedly on loss of PP2A binding. This suggests a possible role for PP2A in specifying subcellular distribution.
Genotypic and phenotypic characterization of long terminal repeat sequences from long-term survivors of human immunodeficiency virus type 1 infection
Authors: Zhang L , Huang Y , Yuan H , Chen B K , Ip J , Ho D D
Published: January 2020

Human immunodeficiency virus type 1 (HIV-1)-infected individuals who remain asymptomatic despite prolonged infection present a unique opportunity to understand virologic and immunologic factors involved in the pathogenesis of AIDS. We have previously identified a group of long-term survivors (LTS) who are clinically healthy and immunologically normal despite 13 to 15 years of HIV-1 infection. In this study, we examined the 5' long terminal repeat (5' LTR) sequences in eight of these LTS. A total of 29 nucleotide sequences were obtained from their peripheral blood mononuclear cells (PBMC). Analysis of these sequences revealed no gross deletions within the 5' LTR. Seven of the eight subjects shared nearly identical consensus sequences in the binding sites for NF-kappaB, Sp1, and the viral trans-activator Tat. In multiple samples from one individual (Pt 5), however, G-to-A hypermutations were found throughout the entire region, suggesting a genetically defective 5' LTR. The effects of the observed genetic variations on LTR transcription were studied by transient transfection of an LTR-driven luciferase reporter gene and by infection with a full-length recombinant HIV-1 containing a luciferase reporter (HIVHXBLTRluc). A wide range of basal and Tat-induced transcriptional activities was found among the 5' LTR from seven of the eight LTS in both transfected 293 cells and donor PBMC, suggesting a functionally intact 5' LTR in these individuals. It is therefore unlikely that defects in the 5' LTR are the underlying explanation for the benign clinical course associated with these seven individuals. However, functional abnormalities were found in the LTR from Pt 5 in directing both heterologous and viral gene expression, providing a possible genetic explanation for the low viral load and prolonged asymptomatic state of this individual. Last, a similar overall degree of genetic diversity was found among viruses from the LTS compared to those from patients with AIDS, reinforcing the notion that a strong correlation between the degree of genetic diversity and the rate of disease progression is unlikely.
The UL20 gene product of pseudorabies virus functions in virus egress
Authors: Fuchs W , Klupp B G , Granzow H , Mettenleiter T C
Published: January 2020

The UL20 open reading frame is positionally conserved in different alphaherpesvirus genomes and is predicted to encode an integral membrane protein. A previously described UL20- mutant of herpes simplex virus type 1 (HSV-1) exhibited a defect in egress correlating with retention of virions in the perinuclear space (J. D. Baines, P. L. Ward, G. Campadelli-Fiume, and B. Roizman, J. Virol. 65:6414-6424, 1991). To analyze UL20 function in a related but different herpesvirus, we constructed a UL20- pseudorabies virus (PrV) mutant by insertional mutagenesis. Similar to HSV-1, UL20- PrV was found to be severely impaired in both cell-to-cell spread and release from cultured cells. The severity of this defect appeared to be cell type dependent, being more prominent in Vero than in human 143TK- cells. Surprisingly, electron microscopy revealed the retention of enveloped virus particles in cytoplasmic vesicles of Vero cells infected with UL20- PrV. This contrasts with the situation in the UL20- HSV-1 mutant, which accumulated virions in the perinuclear cisterna of Vero cells. Therefore, the UL20 gene products of PrV and HSV-1 appear to be involved in distinct steps of viral egress, acting in different intracellular compartments. This might be caused either by different functions of the UL20 proteins themselves or by generally different egress pathways of PrV and HSV-1 mediated by other viral gene products.