bluuebunny/crossref_metadata_2025 huggingface dataset:
Identification of latency-associated transcripts that map antisense to the ICP4 homolog gene of Marek's disease virus
Authors: Cantello J L , Anderson A S , Morgan R W
Published: January 2020

Two small RNAs (0.9 and 0.75 kb), named Marek's disease virus (MDV) small RNAs (MSRs) and a 10-kb RNA, all of which map antisense to the MDV ICP4 homolog gene, have been readily detected in MDCC-MSB1 MDV-transformed T-lymphoblastoid cells. These RNAs were not detectable in reticuloendotheliosis virus-transformed T cells. When MDV was reactivated by treatment of lymphoblastoid cells with 25 micrograms of iododeoxyuridine per ml, the relative levels of the transcripts decreased. These RNAs were not detected by Northern (RNA) hybridization in productively infected chicken embryo fibroblasts 48 h postinfection; however, they were apparent 140 h postinfection. By using Northern hybridization, RNase protection assays, and primer extension analysis, the MSRs were determined to map antisense to the predicted translational start site of the ICP4 homolog gene. The conclusion most consistent with the data is that the two MSRs are overlapping, spliced RNAs. Both small RNAs contain a latency promoter binding factor consensus recognition sequence located toward their 5' ends as well as two potential ICP4 recognition consensus sequences, one in each orientation. The region contains a number of small open reading frames on each side and within the MSRs. Although the exact endpoints are unknown, the large 10-kb species spans the entire ICP4 homolog region. We believe that this group of RNAs, which map antisense to the ICP4 homolog gene, are latency-associated transcripts of MDV.
Conditional lethal expression of the vaccinia virus L1R myristylated protein reveals a role in virion assembly
Authors: Ravanello M P , Hruby D E
Published: January 2020

Within vaccinia virus-infected cells, the product of the L1R open reading frame is covalently modified by myristic acid at the penultimate NH2-terminal glycine residue. Previously we have shown that while the L1R protein is a constituent of both intracellular mature virus particles and extracellular enveloped virions which are released from the infected cell, it is associated exclusively with the primary membranes surrounding the virion core. Given this rather specific localization, it was of interest to study the potential role of this essential gene in virus replication and morphogenesis. To this end, we have constructed a recombinant vaccinia virus in which expression of the L1R gene can be transcriptionally repressed. Without the inducer isopropylthiogalactopyranoside (IPTG), synthesis of the L1R protein was blocked, resulting in a total inhibition of plaque formation. Velocity sedimentation of viral particles labeled in the presence of [3H]thymidine, grown in the absence of IPTG, revealed a substantial reduction in viral DNA incorporation into virions. Likewise, proteolysis of the major core proteins p4a, p4b, and p25K, believed to occur during the final stages of virion maturation, was severely impaired. In the absence of L1R expression, only immature virions could be detected by electron microscopy. Transient expression of a plasmid containing the full-length L1R gene driven by its own promoter was able to complement and rescue the defective phenotype. However, a plasmid bearing a mutation in the myristyl acceptor glycine residue was unable to biologically rescue the recombinant, and the protein was not detected in purified virions.trans complementation using a truncated, myristylated form of the L1R protein partially rescued the defective mutant. Collectively, these data suggest that myristic acid mediates essential interactions of the L1R protein with viral membranes and/or other virion components that lead to the productive assembly, maturation, and release of particles.
Thermal inactivation of oral polio vaccine: contribution of RNA and protein inactivation
Authors: Rombaut B , Verheyden B , Andries K , Boeyé A
Published: January 2020

Heating the Sabin strains of poliovirus at 42 to 45 degrees C caused inactivation, loss of native antigen, and release of the viral RNA (vRNA). The loss of virion infectivity exceeded the loss of vRNA infectivity (as measured by transfection) by roughly 2 log10. Pirodavir inhibited the loss of native antigen and RNA release and reduced the loss of virion infectivity to the same level as the loss of vRNA infectivity. Thermoinactivation thus involves an RNA and a protein component, and pirodavir protected only against the latter.
Association of the reovirus S1 gene with serotype 3-induced biliary atresia in mice
Authors: Wilson G A , Morrison L A , Fields B N
Published: January 2020

A panel of serotype 3 (T3) reovirus strains was screened to determine their relative capacities to cause lethal infection and hepatobiliary disease following peroral inoculation in newborn mice. A wide range of 50% lethal doses (LD50s) was apparent after peroral inoculation of the different virus strains. Two of the strains, T3 Abney and T3 clone 31, caused mice to develop the oily fur syndrome associated with biliary atresia. The capacity to cause biliary atresia was not related to the capacity to cause lethal infection, however, because the LD50s of T3 Abney and T3 clone 31 were grossly disparate. Examination of liver and bile duct tissues revealed histopathologic evidence of biliary atresia and hepatic necrosis in T3 Abney-infected mice but not in mice inoculated with a T3 strain of similar virulence or with the hepatotropic T1 Lang strain. The consistency with which T3 Abney-infected mice developed biliary atresia-associated oily fur syndrome permitted us to determine the viral genetic basis of reovirus-induced biliary atresia. Analysis of reassortant viruses isolated from an in vitro coinfection with T3 Abney and T1 Lang indicated a strong association of the hepatobiliary disease-producing phenotype with the T3 Abney S1 gene, which encodes the viral cell attachment protein, sigma 1. Amino acid residues within the sigma 1 protein that were unique to disease-producing T3 strains were identified by comparative sequence analysis. Specific changes exist within two regions of the protein, one of which is thought to be involved in binding to host cell receptors. We hypothesize that changes within this region of the protein are important in determining the tropism of this virus for bile-ductular epithelium.
Factors involved in entry of the human immunodeficiency virus type 1 into permissive cells: lack of evidence of a role for CD26
Authors: Lazaro I , Naniche D , Signoret N , Bernard A M , Marguet D , Klatzmann D , Dragic T , Alizon M , Sattentau Q
Published: January 2020

It has been proposed recently that the cell surface peptidase CD26 acts in concert with CD4, the human immunodeficiency virus (HIV) primary receptor molecule, to mediate HIV entry into permissive cells. We have failed to detect significant levels of CD26 cell surface expression and enzymatic activity in a number of commonly propagated human CD4+ cell lines, although CD26 mRNA was present at very low levels, as detected by reverse transcription PCR. No relationship existed between the expression of CD26 and the ability of these cells to be infected with HIV or to fuse to form syncytia. We have tested two inhibitors of CD26 enzymatic activity and several anti-CD26 monoclonal antibodies and found that they inhibit neither HIV infection nor HIV-induced syncytium formation. NIH 3T3 cells stably transfected with the cDNAs for human CD4 and CD26 expressed these molecules at the cell surface and had CD26 enzymatic activity. Inoculation of the double transfectants with HIV did not result in virus entry above the background level, as verified by PCR amplification of viral DNA. We were unable to recover infectious virus from the HIV-inoculated NIH 3T3 double transfectants either by transfer of supernatants or by cocultivation with human CD4+ indicator cells. Moreover, the transfectants did not fuse with HIV-infected cells to form syncytia, nor were syncytia observed in HIV-inoculated cultures. These results are inconsistent with the CD26 molecule being a cofactor for entry of HIV in CD4+ cells.
Synergistic activation of simian immunodeficiency virus and human immunodeficiency virus type 1 transcription by retinoic acid and phorbol ester through an NF-kappa B-independent mechanism
Authors: Maciaszek J W , Talmage D A , Viglianti G A
Published: January 2020

The activation of human immunodeficiency virus type 1 (HIV-1) expression in latently infected cells by exogenous agents is believed to be important in the progression of AIDS. Most factors that are known to activate HIV-1 gene expression increase the binding of NF-kappa B or NF-kappa B-like transcription factors to the HIV-1 core enhancer region. In this report, we demonstrate that retinoic acid (RA) treatment of promonocytic U937 cells stimulates expression from the simian immunodeficiency virus (SIVmac) long terminal repeat (LTR). Furthermore, RA and phorbol 12-myristate 13-acetate (PMA) synergistically stimulated both SIVmac and HIV-1 LTRs to levels of expression comparable to that achieved by the viral transactivator Tat. The cis-acting elements required for a response to RA and PMA cotreatment are located between nucleotides -50 and +1 of SIVmac and between nucleotides -83 and +80 of HIV-1. Thus, the synergistic stimulation induced by RA and PMA is NF-kappa B independent. Analysis of deletion mutants of the SIVmac LTR demonstrates that RA and PMA stimulation cooperates with NF-kappa B and Sp1. An SIVmac LTR-reporter gene construct [pLTR(-50/+466)CAT] lacking NF-kappa B and Sp1 binding sites was not activated by Tat in untreated cells but was activated in cells that were cotreated with RA and PMA. Furthermore, gel retardation assays demonstrated that RA treatment causes a change in the pattern of a cellular factor(s) which binds to the -50 through +1 region of the SIVmac LTR. These data suggest that RA induces a PMA-activatable cellular factor that cooperates with NF-kappa B, Sp1, or Tat to stimulate LTR-directed transcription.
Dual tropism for macrophages and lymphocytes is a common feature of primary human immunodeficiency virus type 1 and 2 isolates
Authors: Valentin A , Albert J , Fenyö E M , Asjö B
Published: January 2020

We have investigated the ability of human immunodeficiency virus type 1 (HIV-1) and HIV-2 isolates to infect and replicate in primary human macrophages. Monocytes from blood donors were allowed to differentiate into macrophages by culture in the presence of autologous lymphocytes and human serum for 5 days before infection. A panel of 70 HIV-1 and 12 HIV-2 isolates were recovered from seropositive individuals with different severities of HIV infection. A majority of isolates (55 HIV-1 and all HIV-2) were obtained from peripheral blood mononuclear cells, but isolates from cerebrospinal fluid, monocytes, brain tissue, plasma, and purified CD4+ lymphocytes were also included. All isolates were able to infect monocyte-derived macrophages, even though the replicative capacity of the isolates varied. Interestingly, isolates with a rapid/high, syncytium-inducing phenotype did not differ from slow/low, non-syncytium-inducing isolates in their ability to replicate in monocyte-derived macrophages. Others have reported that rapid/high, syncytium-inducing isolates have a reduced ability to infect and replicate in monocytes. However, different methods to isolate and culture the monocytes/macrophages were used in these studies and our study. Thus, the ability of HIV isolates to replicate in monocytes/macrophages appears to be strongly influenced by the isolation and culture procedures. It remains to be determined which culture procedure is more relevant for the in vivo situation.
Characterization of a cleavage mutant of the measles virus fusion protein defective in syncytium formation
Authors: Alkhatib G , Roder J , Richardson C , Briedis D , Weinberg R , Smith D , Taylor J , Paoletti E , Shen S H
Published: January 2020

Membrane fusion caused by measles virus (MV) is a function of the fusion (F) protein. This process is essential for penetration into the host cell and subsequent initiation of the virus replicative cycle. The biological activity of the MV F protein is generated by endoproteolytic cleavage of a precursor protein (F0) into a large F1 subunit and a smaller F2 subunit held together by disulfide bonds. The cleavage site consists of a cluster of five basic amino acids (amino acids 108 to 112) within the predicted primary structure of the F protein. To investigate the role of the arginine residue at the carboxy terminus of the F2 subunit (arginine 112), site-directed mutagenesis was used to construct a cleavage mutant of the MV F protein in which this arginine residue was changed to a leucine residue. The mutated F gene, encoding four out of the five basic amino acids at the cleavage site, was inserted into the genome of vaccinia virus. The resulting recombinant virus was used to study expression of the mutant F protein in infected cells. Analysis of the Leu-112 mutant protein made in infected cells demonstrated that this single-amino-acid substitution resulted in a reduced rate of transport of the mutant protein to the cell surface, despite its efficient cleavage to yield F1 and F2 subunits. However, the electrophoretic mobilities of the Leu-112 polypeptides suggested that the protein was cleaved incorrectly. This aberrant cleavage appears to have abolished the ability of the F protein to cause syncytium formation. The data indicate that the arginine 112 residue is critical for the correct proteolytic cleavage that is required for the membrane fusion activity of the MV F protein.
The transactivator gene of human T-cell leukemia virus type I is more variable within and between healthy carriers than patients with tropical spastic paraparesis
Authors: Niewiesk S , Daenke S , Parker C E , Taylor G , Weber J , Nightingale S , Bangham C R
Published: January 2020

Human T-cell leukemia virus type I (HTLV-1) causes T-cell leukemia and tropical spastic paraparesis (TSP) in a minority of infected people, whereas the majority remain healthy. No association between a particular HTLV-I sequence and disease manifestation has been found in previous studies. We studied here the sequence variability of the gene for the HTLV-I Tax protein, which is the dominant target antigen of the very strong cytotoxic T-lymphocyte response to the virus. In HTLV-I infection, the intraisolate nucleotide variability is much greater than the variability between isolates. The predicted protein sequence of Tax was significantly more variable in the healthy seropositive individuals' provirus than in those of the patients with TSP. Thus, tax sequence heterogeneity, rather than the presence of particular sequences, distinguishes healthy HTLV-I-seropositive individuals from patients with TSP.
Recombinant rabbit hemorrhagic disease virus capsid protein expressed in baculovirus self-assembles into viruslike particles and induces protection
Authors: Laurent S , Vautherot J F , Madelaine M F , Le Gall G , Rasschaert D
Published: January 2020

VP60, the unique component of rabbit hemorrhagic disease virus capsid, was expressed in the baculovirus system. The recombinant VP60, released in the supernatant of infected insect cells, assembled without the need of any other viral component to form viruslike particles (VLPs), structurally and immunologically indistinguishable from the rabbit hemorrhagic disease virion. Intramuscular vaccination of rabbits with the VLPs conferred complete protection in 15 days; this protection was found to be effective from the fifth day after VLP injection and was accompanied by a strong humoral response.
Tyrosine kinase activity may be necessary but is not sufficient for c-erbB1-mediated tissue-specific tumorigenicity
Authors: Connolly D C , Toutenhoofd S L , Maihle N J
Published: January 2020

Expression of mutant avian c-erbB1 genes results in tissue-specific transformation in chickens. Site-directed mutagenesis was used to generate kinase-defective mutants of several tissue-specific v-erbB transforming mutants by replacement of the ATP-binding lysine residue in the kinase domain with an arginine residue. These kinase-defective v-erbB mutants were analyzed for their in vitro and in vivo transforming potentials. Specifically, kinase-defective mutants of erythroleukemogenic, hemangioma-inducing, and sarcomagenic v-erbB genes were assessed for their oncogenic potential. In vitro transformation potential was assessed by soft-agar colony formation in primary cultures of chick embryo fibroblasts (CEF). In vivo transformation potential was determined by infection of 1-day-old line 0 chicks with concentrated recombinant retrovirus and then monitoring of birds for tumor formation. These transformation assays demonstrate that kinase activity is absolutely essential for transformation by tissue-specific transforming mutants of the avian c-erbB1 gene. Since all of the tissue-specific v-erbB mutants characterized to date exhibit tyrosine kinase activity in vitro but do not transform all tissues in which they are expressed, we conclude that v-erbB-associated tyrosine kinase activity may be necessary but is not sufficient to induce tumor formation.
Modulation of cellular macromolecular synthesis by coronavirus: implication for pathogenesis
Authors: Kyuwa S , Cohen M , Nelson G , Tahara S M , Stohlman S A
Published: January 2020

Infection with the murine coronavirus strain JHM decreases cell surface expression of major histocompatibility complex class I antigens. Northern blots showed that JHM virus infection rapidly reduced the level of actin mRNA, whereas the levels of major histocompatibility complex class I and tubulin mRNAs were reduced only slightly. By contrast, the mRNA levels of interleukin 1 beta, colony-stimulating factor 1 receptor, and tumor necrosis factor alpha increased following infection.
Analysis of the primary T-cell response to Sendai virus infection in C57BL/6 mice: CD4+ T-cell recognition is directed predominantly to the hemagglutinin-neuraminidase glycoprotein
Authors: Cole G A , Katz J M , Hogg T L , Ryan K W , Portner A , Woodland D L
Published: January 2020

Sendai virus infection of C57BL/6 mice elicits a strong CD4+ and CD8+ T-cell response in the respiratory tract. To investigate the specificity of the CD4+ T-cell response, a panel of hybridomas was generated from cells recovered from the respiratory tracts of infected mice. Using vaccinia virus recombinants expressing individual Sendai virus proteins, we found that the majority of these hybridomas (34 of 37) were specific for the hemagglutinin-neuraminidase (HN) glycoprotein. The hybridomas were then analyzed for reactivity to a set of overlapping peptides spanning the entire length of the hemagglutinin-neuraminidase glycoprotein. At least five H-2 I-Ab-restricted epitopes were defined in HN. The strong bias toward recognition of class II epitopes derived from a single viral protein contrasts with T-cell recognition of epitopes of several proteins in influenza A virus as found previously by others.
Membrane and protein interactions of a soluble form of the Semliki Forest virus fusion protein
Authors: Klimjack M R , Jeffrey S , Kielian M
Published: January 2020

Semliki Forest virus is an enveloped alphavirus that infects cells by a membrane fusion reaction triggered by the low pH present in endocytic vacuoles. Fusion is mediated by the E1 spike protein subunit. During fusion, several conformational changes occur in E1 and E2, the two transmembrane subunits of the spike protein. These changes include dissociation of the E1-E2 dimer, alteration of the trypsin sensitivity and monoclonal antibody binding patterns of E1, and formation of a sodium dodecyl sulfate (SDS)-resistant E1 homotrimer. A critical characteristic of Semliki Forest virus fusion is also its dependence on the presence of both cholesterol and sphingomyelin in the target membrane. We have here examined the conformational changes induced by low pH treatment of E1*, the water-soluble, proteolytically truncated ectodomain of the E1 subunit. Following low pH treatment, E1* was shown to bind efficiently to artificial liposomes. Similar to virus fusion, optimal E1*-liposome binding required low pH, cholesterol, and sphingomyelin. The E1 ectodomain, although monomeric in its neutral pH form, assembled into an SDS-resistant oligomer following treatment at low pH. This low pH-induced oligomerization required target membranes containing both cholesterol and sphingomyelin. Our results demonstrate that the E1 ectodomain responds to low pH similarly to the full-length E1 subunit. The ectodomain facilitates the characterization of conformational changes and membrane binding in the absence of virus fusion or other virus components.
Role of flanking E box motifs in human immunodeficiency virus type 1 TATA element function
Authors: Ou S H , Garcia-Martínez L F , Paulssen E J , Gaynor R B
Published: January 2020

Human immunodeficiency virus type 1 (HIV-1) gene expression is dependent on a number of cis-acting DNA elements present in the HIV-1 long terminal repeat. Previous studies have demonstrated that the TATA element is critical for basal and Tat-induced HIV-1 gene expression. The HIV-1 TATA region has an unusual structure in that the TATA sequence is flanked by two palindromic sequence motifs (CANNTG) known as E boxes which can serve as binding sites for the basic helix-loop-helix (bHLH) class of DNA-binding proteins. In this study, we performed site-directed mutagenesis of both the TATA and the flanking E box sequences of HIV-1. We also substituted the sequences flanking the adenovirus E3 promoter TATA sequence for those flanking the HIV-1 TATA sequence. Constructs were assayed for their levels of basal and Tat-induced gene expression by both in vitro transcription and transient expression assays. Both the TATA box and flanking sequences including the E box motifs were found to be important in modulating both basal gene expression and Tat-induced HIV-1 gene expression. Gel retardation analysis demonstrated that binding of both the recombinant TATA-binding protein (TBP) and the TFIID fraction which contains both TBP and TBP-associated factors was dependent primarily on the TATA element. However, competition analysis suggested that the E boxes may play a role in stabilizing the binding of TFIID but not recombinant TBP. Two proteins representing different classes of bHLH proteins, E47 and AP-4, were assayed for their ability to bind to the flanking E box motifs. We isolated a cDNA clone encoding the complete AP-4 protein and demonstrated that both AP-4 and E47 bound specifically to the 3' E box motif, which contains sequences that correspond to the consensus binding site (CAGCTG). Gel retardation analysis indicated that the binding of AP-4 to the E boxes excluded the binding of TBP to the TATA box. These studies are consistent with a model in which different classes of cellular bHLH proteins may be involved in regulating HIV-1 TATA element function by either inhibiting or promoting the assembly of different preinitiation transcriptional complexes.
Secondary herpes simplex virus latent infection in transplanted ganglia
Authors: Tenser R B , Edris W A , Gaydos A , Hay K A
Published: January 2020

Sensory ganglia latently infected with herpes simplex virus (HSV) were transplanted beneath the renal capsule of syngeneic recipients, and the latent infection remaining was investigated. HSV latency-associated transcript (LAT) expression and reactivation of HSV after explant of transplanted dorsal root ganglia were monitored as markers of latency. Two to four weeks after transplantation, both indicated evidence of HSV latency in transplants. At those times, infectious virus was not detected in direct ganglion homogenates. In addition, viral antigen and infected cell polypeptide 4 RNA were not detected. Taken together, the results suggested that HSV latent infection rather than persistent infection was present in transplants. From these results, two explanations seemed possible: latency was maintained in transplanted neurons, or alternatively, latency developed after transplantation, in neurons not previously latently infected. The latter was considered putative secondary latency and was investigated in three ways. First, evidence of reactivation which might serve as a source for secondary latency was evaluated. Reactivation of HSV in transplants was evident from HSV antigen expression (52% of transplants) and the presence of cell-free virus (38% of transplants) 3 to 5 days after transplantation. Second, putative secondary latency was investigated in recipients immunized with HSV prior to receiving latently infected ganglia. Reactivation was not detected 3 to 5 days after transplantation in immunized recipients, and LAT expression was rare in these recipients after 3 to 4 weeks. Lastly, the possibility of secondary latency was investigated by comparing results obtained with standard HSV and with reactivation-defective thymidine kinase-negative (TK-) HSV. Defective reactivation of TK- HSV was demonstrated by immunohistochemistry and by the inability to isolate infectious virus. Donor dorsal root ganglia latently infected with TK+ HSV showed many LAT-positive neurons 2 or more weeks after transplantation (average, 26 per transplant). However, LAT expression was undetectable or minimal > 2 weeks after transplantation in donor ganglia latently infected with TK- HSV (average, 0.2 per transplant). Impaired reactivation of TK- HSV-infected donor ganglia after transplantation, therefore, was correlated with subsequent limited LAT expression. From these results, the occurrence of secondary latency was concluded for ganglia latently infected with TK+ HSV and transplanted beneath the kidney capsule. In vivo reactivation in this transplant model may provide a more useful means to investigate HSV reactivation than in usual in vitro explant models and may complement other in vivo reactivation models. The occurrence of secondary latency was unique. The inhibition of secondary latency by the immune system may provide an avenue to evaluate immunological control of HSV latency.
Autophosphorylation is required for high kinase activity and efficient transformation ability of proteins encoded by host range alleles of v-src
Authors: Woods K M , Verderame M F
Published: January 2020

pp60v-src is a nonreceptor protein tyrosine kinase that can transform both chicken and rodent fibroblasts. The src homology 2 (SH2) domain of this protein serves a critical role in the regulation of protein tyrosine kinase activity. The host range proteins pp60v-src-L, which contains a deletion of a highly conserved residue (Phe-172) in the SH2 domain, and pp60v-src-PPP, which contains a change from a Leu to a Phe at amino acid 186 in the SH2 domain, transform chicken but not rat cells and have slightly reduced kinase activity measured in vitro. The data presented here show that these altered proteins require autophosphorylation on Tyr-416 for high kinase activity and transforming ability. In the absence of autophosphorylation, there is a further decrease of at least threefold in in vitro kinase activity relative to the phosphorylated host range parental protein, no morphological transformation, a reduction in anchorage independent growth, and no disruption of the actin cytoskeleton. In addition, these SH2 mutations abolish the ability of the SH2 domain to bind a phosphorylated peptide that corresponds to the autophosphorylation site of pp60src. Thus, like mutant alleles of c-src encoding transformation competent proteins, and unlike v-src, transformation by pp60v-src-F172 delta and pp60v-src-L186F is dependent on phosphorylation of Y-416 for high kinase activity and transformation ability. The dependence of transformation on phosphotyrosine is not a reflection of an intramolecular interaction between the autophosphorylation site and the SH2 domains since purified SH2 domains are incapable of binding phosphorylated autophosphorylation site peptides in vitro.
Epstein-Barr virus latency in blood mononuclear cells: analysis of viral gene transcription during primary infection and in the carrier state
Authors: Tierney R J , Steven N , Young L S , Rickinson A B
Published: January 2020

Epstein-Barr virus (EBV) can display different forms of latent infection in B-cell lines in vitro; however, the types of infection normally established by the virus in vivo remain largely unexplored. Here we have approached this question by analyzing the types of viral RNAs present in mononuclear cells freshly isolated from the blood of 14 infectious mononucleosis patients undergoing primary EBV infection and 6 long-term virus carriers. Reverse transcription-PCR amplifications were carried out with a panel of oligonucleotide primers and probes which specifically detect (i) the EBER1 RNA common to all forms of latency, (ii) transcripts either from the Cp and Wp promoters generating all six nuclear antigen (EBNA1, -2, -3A, -3B, -3C, -LP) mRNAs or from the Fp promoter generating a uniquely spliced EBNA1 mRNA, (iii) the latent membrane protein (LMP1 and 2A) mRNAs, and (iv) the BZLF1 mRNA, an immediate-early marker of lytic cycle. Viral transcription in infectious mononucleosis mononuclear cells (and in the B-cell-enriched fraction) regularly included the full spectrum of latent RNAs seen during EBV-induced B-cell growth transformation in vitro, i.e., EBER1, Cp/Wp-initiated EBNA mRNAs, and LMP1/LMP2 mRNAs, in the absence of lytic BZLF1 transcripts. In addition, transcripts with the splice pattern of Fp-initiated EBNA1 mRNA, hitherto seen only in vivo in certain EBV-positive tumors, were frequently detected. In long-term virus carriers, the mononuclear cells were again positive for latent (EBER1) and negative for lytic (BZLF1) markers; Cp/Wp-initiated RNAs were not detected in these samples, but in several individuals it was possible to amplify both Fp-initiated EBNA1 mRNA and LMP2A mRNA signals. We suggest (i) that primary infection is associated with a transient virus-driven expansion of the infected B-cell pool through a program of virus gene expression like that seen in in vitro-transformed cells and (ii) that long-term virus carriage is associated with a switch from Cp/Wp to Fp usage and thus to a more restricted form of latent protein expression that may render the infected cells less susceptible to recognition by the virus-specific cytotoxic T-cell response.
Crucial sequences within the Epstein-Barr virus TP1 promoter for EBNA2-mediated transactivation and interaction of EBNA2 with its responsive element
Authors: Meitinger C , Strobl L J , Marschall G , Bornkamm G W , Zimber-Strobl U
Published: January 2020

EBNA2 is one of the few genes of Epstein-Barr virus which are necessary for immortalization of human primary B lymphocytes. The EBNA2 protein acts as a transcriptional activator of several viral and cellular genes. For the TP1 promoter, we have shown previously that an EBNA2-responsive element (EBNA2RE) between -258 and -177 relative to the TP1 RNA start site is necessary and sufficient for EBNA2-mediated transactivation and that it binds EBNA2 through a cellular factor. To define the critical cis elements within this region, we cloned EBNA2RE mutants in front of the TP1 minimal promoter fused to the reporter gene for luciferase. Transactivation by EBNA2 was tested by transfection of these mutants in the absence and presence of an EBNA2 expression vector into the established B-cell line BL41-P3HR-1. The analysis revealed that two identical 11-bp motifs and the region 3' of the second 11-bp motif are essential for transactivation by EBNA2. Methylation interference experiments indicated that the same cellular factor in the absence of EBNA2 binds either one (complex I) or both (complex III) 11-bp motifs with different affinities, giving rise to two different specific protein-DNA complexes within the left-hand 54 bp of EBNA2RE. A third specific complex was shown previously to be present only in EBNA2-expressing cells and to contain EBNA2. Analysis of this EBNA2-containing complex revealed the same protection pattern as for complex III, indicating that EBNA2 interacts with DNA through binding of the cellular protein to the 11-bp motifs. Mobility shift assays with the different mutants demonstrated that one 11-bp motif is sufficient for binding the cellular factor, whereas for binding of EBNA2 as well as for efficient transactivation by EBNA2, both 11-bp motifs are required.
Novel features of the respiratory tract T-cell response to influenza virus infection: lung T cells increase expression of gamma interferon mRNA in vivo and maintain high levels of mRNA expression for interleukin-5 (IL-5) and IL-10
Authors: Baumgarth N , Brown L , Jackson D , Kelso A
Published: January 2020

Analysis of the respiratory tract before and after primary influenza virus infection revealed a virus-induced preferential accumulation of a CD8+ T-cell population that coexpresses mRNA for interleukin-5 (IL-5) and IL-10 with virus dose-dependent high levels of gamma interferon. However, cytokine production in lung tissues was not restricted to the T-cell population, since CD3- cells were found to express mRNA for various cytokines, including IL-4 and particularly IL-6 and granulocyte-macrophage colony-stimulating factor. These data provide in vivo evidence for a local respiratory tract immune response to influenza virus infection dominated by cytokine-producing CD8+ T cells.
Amino acid sequence analysis of the proteolytic cleavage products of the bovine immunodeficiency virus Gag precursor polypeptide
Authors: Tobin G J , Sowder R C , Fabris D , Hu M Y , Battles J K , Fenselau C , Henderson L E , Gonda M A
Published: January 2020

Bovine immunodeficiency virus Gag proteins were purified from virions, and their amino acid sequences and molecular masses were determined. The matrix, capsid, and nucleocapsid (MA, CA, and NC, respectively) and three smaller proteins (p2L, p3, and p2) were found to have molecular masses of 14.6, 24.6, and 7.3 and 2.5, 2.7, and 1.9 kDa, respectively. The order of these six proteins in the Gag precursor, Pr53gag, is NH2-MA-p2L-CA-p3-NC-p2-COOH. In contrast to other retroviral MA proteins, the bovine immunodeficiency virus MA retains its N-terminal methionine and is not modified by fatty acids. In addition, the bovine immunodeficiency virus NC migrates as a 13-kDa protein in denaturing gel electrophoresis; however, its molecular mass was determined to be 7.3 kDa.
Herpes simplex virus inhibits host cell splicing, and regulatory protein ICP27 is required for this effect
Authors: Hardy W R , Sandri-Goldin R M
Published: January 2020

While the majority of metazoan genes and those of the DNA viruses which infect them contain introns which require RNA splicing, herpes simplex virus type 1 (HSV-1) encodes relatively few spliced products. We previously showed that the HSV-1 immediate-early protein ICP27 decreased the levels of spliced target mRNAs in transfections and spliced cellular mRNAs during infection, suggesting that ICP27 may function in impairing host cell splicing. Here, we show that during infections with the wild type, but not in infections with an ICP27 viral mutant termed 27-LacZ, precursor RNA accumulated for a virus transcript which contained introns. Pre-mRNA accumulation in the nucleus was greater than that in the cytoplasm, indicating that splicing rather than transport was affected. Furthermore, splicing of a beta-globin pre-mRNA substrate was inhibited in nuclear extracts from wild-type-infected cells but not in extracts from cells infected with 27-LacZ. The inhibitory activity in extracts from wild-type-infected cells was able to reduce the splicing efficiency of competent extracts in biochemical complementation assays. ICP27 appeared to be responsible for this decrease, because the splicing activity of an extract from cells infected with an ICP27 ts mutant was significantly reduced after incubation of the extract at the permissive temperature to allow renaturation of the conformationally defective ICP27 protein. These results strongly suggest that HSV-1 infection inhibits host cell splicing through the action of ICP27.
Tula virus: a newly detected hantavirus carried by European common voles
Authors: Plyusnin A , Vapalahti O , Lankinen H , Lehväslaiho H , Apekina N , Myasnikov Y , Kallio-Kokko H , Henttonen H , Lundkvist A , Brummer-Korvenkontio M
Published: January 2020

A novel hantavirus has been discovered in European common voles, Microtus arvalis and Microtus rossiaemeridionalis. According to sequencing data for the genomic RNA S segment and nucleocapsid protein and data obtained by immunoblotting with a panel of monoclonal antibodies, the virus, designated Tula virus, is a distinct novel member of the genus Hantavirus. Phylogenetic analyses of Tula virus indicate that it is most closely related to Prospect Hill, Puumala, and Muerto Canyon viruses. The results support the view that the evolution of hantaviruses follows that of their primary carriers. Comparison of strains circulating within a local rodent population revealed a genetic drift via accumulation of base substitutions and deletions or insertions. The Tula virus population from individual animals is represented by quasispecies, indicating the potential for rapid evolution of the agent.
The Epstein-Barr virus EBNA-2 gene in oral hairy leukoplakia: strain variation, genetic recombination, and transcriptional expression
Authors: Walling D M , Perkins A G , Webster-Cyriaque J , Resnick L , Raab-Traub N
Published: January 2020

Oral hairy leukoplakia (HLP) lesions frequently contain defective Epstein-Barr virus (EBV) genomes with deletions in the EBNA-2 gene that abundantly replicate and persist within the lesion. To characterize these viral strains and recombinant variants, the EBNA-2 gene in EBV DNA from several different HLP biopsy specimens was analyzed. Amplification of EBNA-2 coding sequences by PCR demonstrated the presence in HLP of intact EBNA-2 genes as well as a variety of internally deleted variants of both EBNA-2A and EBNA-2B. Some of the deletion variants evolved within the HLP lesion from intact EBNA-2 genes, while other variants appeared to be transmissible strains that directly infected the lesion. Intrastrain recombination within the HLP lesion also generated variation within the EBNA-2 polyproline region. Cloning and sequencing of HLP cDNA demonstrated transcription from the internally deleted EBNA-2 open reading frame, indicating that these variant genes are expressed in HLP. Comparative analysis of the HLP EBNA-2 sequences confirmed previous findings of EBV coinfection with multiple types and strains. Sequence variation of these wild-type genes demonstrated that EBNA-2A sequences distinguish at least two separate strains and a variety of substrains of EBV type 1. Two of the HLP EBNA-2A sequences contained amino acid changes in a cytotoxic T-cell epitope within an otherwise highly conserved region of the gene. These data indicate that EBV coinfection, strain variation, and recombination within the EBNA-2 gene are common features of HLP and suggest that the expression of internally deleted EBNA-2 variants could contribute to EBV pathogenesis in permissive infection.
The activated Mlvi-4 locus in Moloney murine leukemia virus-induced rat T-cell lymphomas encodes an env/Mlvi-4 fusion protein
Authors: Patriotis C , Tsichlis P N
Published: January 2020

A genomic DNA probe derived from the region immediately 3' of the clusters of integrated proviruses in the Mlvi-4 locus detects a 5.5-kb mRNA transcript which is specifically expressed in normal rat thymus and spleen. The same probe detects two tumor-specific mRNA transcripts 2.5 and 10 kb long, both of which are expressed only in tumors carrying a provirus in the Mlvi-4 locus. Sequence analysis of two cDNA clones (LE3a and B1.1) of the 2.5-kb tumor-specific mRNA, obtained from two independent tumors (6889 and B1), revealed that they are both derived from hybrid env/Mlvi-4 mRNA transcripts. The splicing of env to Mlvi-4 sequences linked a cryptic splice donor site at nucleotide position 6397 of the viral genome with a splice acceptor site in the region immediately 3' of the integrated provirus. The mRNA that gives rise to cDNA clone B1.1 terminates 1,005 bases 3' of the splice acceptor site without additional splicing. The mRNA that gives rise to cDNA clone LE3a terminates in the same site but undergoes differential splicing of an 81-base-long intron. The resulting mRNAs contain 247-amino-acid (clone B1.1) or 226-amino-acid (clone LE3a) open reading frames sharing 221 N-terminal amino acids, of which 207 are derived from the viral env gene and 14 are derived from Mlvi-4. RNase protection assays using 6889 tumor cell RNA and a probe derived from the cDNA clone LE3a detected both mRNA transcripts. More abundant of the two, however, was the one encoding the putative 247-amino-acid protein. Transient transfections of a construct expressing the RNA transcript defined by clone B1.1 into D17 cells led to the expression of an Env/Mlvi-4 fusion protein with an apparent molecular mass of 33 kDa. Given that cells with provirus insertions in the Mlvi-4 locus are selected and that retroviral env gene products may have profound effects in the biology of hematopoietic cells, we suggest that the detected fusion proteins may contribute to the growth of T-cell lymphomas.
Molecular and biological characteristics of echovirus 22, a representative of a new picornavirus group
Authors: Stanway G , Kalkkinen N , Roivainen M , Ghazi F , Khan M , Smyth M , Meurman O , Hyypiä T
Published: January 2020

Recent sequence analysis revealed that the human pathogen echovirus 22 (EV22) is genetically distant from all the other picornaviruses studied to date (T. Hyypiä, C. Horsnell, M. Maaronen, M. Khan, N. Kalkkinen, P. Auvinen, L. Kinnunen, and G. Stanway, Proc. Natl. Acad. Sci. USA 89:8847-8851, 1992). We have further characterized the biological properties of the virus and show here that the virion has properties similar to those of other picornaviruses. However, the protein composition is unique, in that most copies of one of the three major capsid proteins, VP0, do not undergo the further processing to VP2 and VP4 observed during the maturation of the virus in previously studied picornaviruses. Alignment of the capsid protein sequences with those of other picornaviruses revealed, furthermore, that the VP3 polypeptide contains an apparent insertion of approximately 25 amino acids at its amino terminus. An arginine-glycine-aspartic acid (RGD) motif is found in VP1, and by using synthetic peptides, it was shown that this sequence plays a role in cell surface receptor recognition. Finally, EV23 was shown to share remarkable identity with EV22 in certain parts of the genome and also belongs to this previously unrecognized picornavirus group.
Priming of duck hepatitis B virus reverse transcription in vitro: premature termination of primer DNA induced by the 5'-triphosphate of fialuridine
Authors: Staschke K A , Colacino J M
Published: January 2020

Hepadnaviruses employ a unique mechanism for the initiation of RNA-directed DNA synthesis. Initially, four bases (5'-GTAA-3') are added to a tyrosine residue of the viral polymerase by reverse transcription of a bulge sequence in epsilon, a stem-loop structure which functions as the packaging signal for pregenomic RNA. This protein-DNA complex acts as the primer for minus-strand elongation from the 3' sequence, DR1. To understand this process in greater detail, we investigated whether the protein-mediated priming of viral DNA synthesis is affected by nucleotide analogs. By using cell-free expression of duck hepatitis B virus (DHBV) reverse transcriptase (G.-H. Wang and C. Seeger, Cell 71:663-670, 1992), the 5'-triphosphate of the thymidine analog fialuridine (FIAU) was shown to inhibit the incorporation of radiolabeled TMP into primer DNA in a dose-dependent manner. Inhibition by the 5'-triphosphate of FIAU (FIAU-TP) was nearly complete at a concentration of 10 microM. The dideoxynucleotide analogs ddGTP, ddTTP, and 3'-azidodeoxythymidine triphosphate, known inhibitors of DHBV endogenous DNA polymerase, did not affect substantially the synthesis of primer DNA. Alternate substrate analysis suggested that FIAU is incorporated efficiently into nascent primer DNA as an analog of thymidine. Using site-directed mutagenesis to construct a mutant RNA template yielding a primer with the sequence 5'-GTAC-3', we demonstrated that FIAU-TP inhibited the incorporation of TMP, had no effect on that of dAMP, and decreased markedly the incorporation of dCMP. These results show that the synthesis of full-length DHBV primer DNA is inhibited by FIAU-TP but not by the dideoxynucleotide analogs that we tested. The significance of these findings as they relate to HBV DNA replication is discussed.
A family of retroviruses that utilize related phosphate transporters for cell entry
Authors: Miller D G , Miller A D
Published: January 2020

The amphotropic murine retrovirus receptor Ram-1 shows significant sequence similarity to the gibbon ape leukemia virus (GALV) receptor Glvr-1, and both of these cell surface virus receptors normally function as sodium-dependent phosphate symporters. However, Ram-1 from humans or rats does not serve as a receptor for GALV, and Glvr-1 from humans does not serve as a receptor for amphotropic virus. Here we show that the murine retrovirus 10A1 can enter cells by using either Glvr-1 or Ram-1. Furthermore, we have constructed Ram-1/Glvr-1 hybrid receptors that allow entry of both GALV and amphotropic virus. While GALV and amphotropic virus are in separate interference groups when assayed on human cells, they do interfere with each other in cells expressing the hybrid receptor. These results indicate a close functional relationship between retroviruses that utilize members of this newly defined receptor family and provide a molecular explanation for nonreciprocal and cell type-specific interference observed for some retrovirus classes.
DNA-damaging agents greatly increase the transduction of nondividing cells by adeno-associated virus vectors
Authors: Alexander I E , Russell D W , Miller A D
Published: January 2020

None of the vector systems currently available for gene therapy applications have been shown to be capable of both efficient gene transfer into nondividing cells and long-term expression through stable integration into host cell DNA. While integrating vectors based on adeno-associated virus are capable of mediating gene transfer into nondividing cells, this process is 200-fold less efficient than transduction of dividing cells. We demonstrate that the transduction efficiency of adeno-associated virus vectors can be increased by treatment with DNA-damaging agents. Nondividing cells are especially responsive, with increases in transduction efficiency of up to 750-fold. This finding has the potential to facilitate gene therapy applications requiring gene transfer to nondividing cells.
Cytotoxic activity against maedi-visna virus-infected macrophages
Authors: Lee W C , McConnell I , Blacklaws B A
Published: January 2020

The cell type predominantly infected by maedi-visna virus (MVV) is the macrophage, and we have looked at the ability of MVV-infected macrophages to interact with cytotoxic T lymphocytes (CTL), important effector cells against virus infections. MVV-specific CTL precursors were detected, after in vitro culture with MVV antigen and recombinant human interleukin-2, in peripheral blood lymphocytes of all MVV-infected sheep. MVV-infected monocyte-derived macrophages and alveolar macrophages were able to stimulate CTL activity in vitro and were targets for these activated CTL. The major effector cell population using MVV-infected macrophage targets was CD8+ lymphocytes, although another population, lymphokine-activated killer cells, may also have been involved. There was no direct cytotoxic activity found in alveolar lymphocytes from MVV-infected sheep without lung lesions.