bluuebunny/crossref_metadata_2025 huggingface dataset:
Mutations in the 5' end of the herpes simplex virus type 2 latency-associated transcript (LAT) promoter affect LAT expression in vivo but not the rate of spontaneous reactivation of genital herpes
Authors: Wang K , Pesnicak L , Straus S E
Published: January 2020

The primary herpes simplex virus type 2 (HSV-2) latency-associated transcript (LAT) promoter influences LAT expression and rates of virus reactivation. We explored the biological importance of particular neuronally responsive regions within the promoter by creating new recombinant viruses bearing a targeted deletion (246 bp [strain 524]) or a point mutation (2 bp [strain 167]) in this region. These recombinant viruses grew efficiently in vitro and in vivo, caused acute genital disease in guinea pigs, and, as measured by quantitative-competitive (QC) DNA PCR, established latency, all as well as did the wild-type parental HSV-2 strain 333, the rescuant strain 524R, and the previously described 624-bp LAT- promoter deletion mutant. By QC-reverse transcriptase PCR of RNA from latently infected ganglia, mutant 167 expressed wild-type levels of LAT and the deletion mutant 524 expressed 9- to 15-fold less LAT than normal, while the LAT expression of the LAT- mutant was undetectable or at least 5 log units less than that of the wild type. The rates of recurrence of genital lesions were normal for recombinant viruses 524 and 167 but reduced (as expected) for the LAT- mutant. Alteration of a subset of LAT promoter elements reduced LAT expression by 1 log unit but did not influence the rate of spontaneous disease reactivation in vivo. Far greater reductions in LAT expression are necessary before reactivation rates are noticeably changed.
The sequence and structure of the 3' arm of the first stem-loop of the human immunodeficiency virus type 2 trans-activation responsive region mediate Tat-2 transactivation
Authors: Browning C , Hilfinger J M , Rainier S , Lin V , Hedderwick S , Smith M , Markovitz D M
Published: January 2020

Human immunodeficiency virus type 2 (HIV-2) causes AIDS, but generally after a much longer asymptomatic period than that which follows infection with HIV-1. At the molecular level, HIV-2 is much more closely related to the simian immunodeficiency viruses than to HIV-1 and our previous studies have demonstrated that HIV-2 and HIV-1 enhancer stimulation is mediated by different sets of cellular proteins following T-cell activation. Similar to HIV-1, HIV-2 encodes a transactivating protein, Tat, which appears to be necessary for viral replication and stimulates viral transcriptional initiation and/or elongation. While Tat-1 binds to the RNA of the trans-activation responsive (TAR) region of HIV-1 and HIV-2, cellular factors that bind to the RNA transcript are also necessary for Tat to function in vivo. Since almost all previous investigations of cellular cofactors for Tat had focused on HIV-1, we undertook studies aimed at understanding the interaction between the TAR RNA region of the HIV-2 promoter (TAR-2) and cellular proteins. By using extension inhibition analysis (toeprinting) and RNA electrophoretic mobility shift assays, we demonstrated binding of a nuclear factor(s) in T cells to the base of the promoter-proximal stem-loop structure. Mutational analysis of this region revealed that both the sequence of the 3' arm and the stem structure itself are important for activation of the promoter by Tat-2. In contrast, the structure is necessary for activation of TAR-2 by Tat-1 but the sequence is less important. These results suggest that a cellular factor interacts with the 3' arm of the proximal stem-loop structure of TAR-2 and mediates Tat-2-induced increases in the level of HIV-2 transcripts.
A simian virus 40 large T-antigen segment containing amino acids 1 to 127 and expressed under the control of the rat elastase-1 promoter produces pancreatic acinar carcinomas in transgenic mice
Authors: Tevethia M J , Bonneau R H , Griffith J W , Mylin L
Published: January 2020

The simian virus 40 large T antigen induces tumors in a wide variety of tissues in transgenic mice, the precise tissues depending on the tissue specificity of the upstream region controlling T-antigen expression. Expression of mutant T antigens that contain a subset of the protein's activities restricts the spectrum of tumors induced. Others showed previously that expression of a mutant large T antigen containing the N-terminal 121 amino acids (T1-121) under control of the lymphotropic papovavirus promoter resulted in slow-growing choroid plexus tumors, whereas full-length T antigen under the same promoter induced rapidly growing CPR tumors, T-cell lymphomas, and B-cell lymphomas. In those instances, the alteration in tumor induction or progression correlated with inability of the mutant large T antigen to bind the tumor suppressor p53. In the study reported here, we investigated the capacity of an N-terminal T antigen segment (T1-127) expressed in conjunction with small t antigen under control of the rat elastase-1 (E1) promoter to induce pancreatic tumors. The results show that pancreases of transgenic mice expressing T1-127 and small t antigen display acinar cell dysplasia at birth that progresses to neoplasia. The average age to death in these mice is within the range reported for transgenic mice expressing full-length T antigen under control of the E1 promoter. These results indicate that sequestering p53 by binding is not required for the development of rapidly growing acinar cell carcinomas. In addition, we provide evidence that small t antigen is unlikely to be required. Finally, we show that the p53 protein in acinar cell carcinomas is wild type in conformation.
Characteristics of the Pro225His mutation in human immunodeficiency virus type 1 (HIV-1) reverse transcriptase that appears under selective pressure of dose-escalating quinoxaline treatment of HIV-1
Authors: Pelemans H , Esnouf R , Dunkler A , Parniak M A , Vandamme A M , Karlsson A , De Clercq E , Kleim J P , Balzarini J
Published: January 2020

Treatment of human immunodeficiency virus type 1 (HIV-1)-infected CEM cell cultures with escalating concentrations of the quinoxaline S-2720 resulted in an ordered appearance of single and multiple mutant virus strains that gradually became resistant to the quinoxaline and other nonnucleoside reverse transcriptase (RT) inhibitors (NNRTIs). A novel mutation, Pro225His, consistently appeared in a Val106Ala RT-mutated genetic background. The contribution of this mutation to the resistance of the mutant HIV-1 RT to NNRTIs was additive to the resistance caused by the Val106Ala mutation. Interestingly, site-directed mutagenesis studies revealed that the Pro225His-mutated RT had acquired markedly greater sensitivity to bis(heteroaryl)piperazine (BHAP U-90152) (delavirdine) but not to any of the other NNRTIs. The kinetics of inhibition of the Pro225His mutant RT by the NNRTIs (including BHAP U-90152) was not substantially different from that observed for the wild-type RT. The hypersensitivity of the mutant enzyme and virus to BHAP U-90152 could be rationally explained by the molecular-structural determinants of the RT-BHAP complex, which has recently been resolved by X-ray crystallography.
Evolutionary dynamics of genetic variation in Epstein-Barr virus isolates of diverse geographical origins: evidence for immune pressure-independent genetic drift
Authors: Khanna R , Slade R W , Poulsen L , Moss D J , Burrows S R , Nicholls J , Burrows J M
Published: January 2020

The question whether immune pressure exerted by cytotoxic T lymphocytes (CTLs) can influence the long-term evolution of genetically stable viruses such as Epstein-Barr virus (EBV) has generated considerable scientific interest, primarily due to its important implications for the overall biology of the virus. While arguing for a role of CTLs in the evolution of viruses, it is important to differentiate between genetic variation in virus and immune recognition of these variant virus by CTLs. To assess the role of genetic selection in the long-term evolution of EBV, we have analyzed a large panel of type 1 EBV isolates from African, Southeast Asian, Papua-New Guinean (PNG), and Australian Caucasian individuals. Seven different regions of the EBV genome, which include nine CTL epitopes restricted through a range of HLA class I alleles, were sequenced and compared. Although numerous nucleotide changes were identified within these isolates, comparison of synonymous and nonsynonymous substitutions in the CTL epitope indicated that the genetic variation was generated mostly independently of immune selection pressure. Surprisingly, an inverse correlation between genetic variation within certain CTL epitopes and the frequency distribution of HLA alleles that present the CTL epitopes was seen, suggesting that the evolutionary pressures on the CTL epitopes of the virus may be toward their conservation rather than their inactivation. Furthermore, molecular evolutionary genetic analysis of nucleotide sequences revealed that viral isolates from PNG are evolving as a lineage distinct from isolates from African, Southeast Asian, and Australian Caucasian individuals.
Investigation of the attenuation exhibited by a molecularly cloned chicken anemia virus isolate by utilizing a chimeric virus approach
Authors: Meehan B M , Todd D , Creelan J L , Connor T J , McNulty M S
Published: January 2020

Molecular cloning of the Cux-1 isolate of chicken anemia virus (CAV), which had been passaged 173 times in cell culture, resulted in the isolation of an attenuated strain, designated cloned isolate 10, which reverted to virulence following 10 passages in young chicks (D. Todd, T. J. Connor, V. M. Calvert, J. L. Creelan, B. M. Meehan, and M. S. McNulty, Avian Pathol. 24:171-187, 1995). The attenuated cloned isolate 10 differs from the molecularly cloned pathogenic Cux-1 isolate in that it possesses a 21-nucleotide insertion within the nontranscribed region of the CAV genome and 17 individual nucleotide substitutions dispersed throughout the genome. Comparative analyses with other published CAV sequences indicated that cloned isolate 10 was unique at nine nucleotide positions and at five amino acid positions. The molecular basis of the attenuation exhibited by cloned isolate 10 was investigated by evaluating the pathogenicities of two sets of complementary chimeric viruses. These sets were produced by transfection with chimeric double-stranded replicative-form (RF) DNA equivalents that contained DNA sequences derived from cloned isolate 10 and the pathogenic cloned Cux-1 isolate. The construction of the chimeric RFs exploited the occurrence of unique EcoRI, PstI, and BamHI restriction sites, which allowed their respective circular CAV RFs to be manipulated as three restriction fragments of 0.58, 0.93, and 0.71 kbp. Examination of the levels of anemia and gross pathology in the thymuses and bone marrows of 14 day-old specific-pathogen-free chicks following infection of 1-day-old chicks with the chimeric and cloned parental isolates indicated that nucleotide changes in each of the three genomic regions contributed towards attenuation. The significance of this result to the development and use of live attenuated CAV vaccines is discussed.
Regulation of receptor binding affinity of influenza virus hemagglutinin by its carbohydrate moiety
Authors: Ohuchi M , Ohuchi R , Feldmann A , Klenk H D
Published: January 2020

The hemagglutinin (HA) of the fowl plague virus (FPV) strain of influenza A virus has two N-linked oligosaccharides attached to Asn123 and Asn149 in the vicinity of the receptor binding site. The effect of these carbohydrate side chains on the binding of HA to neuraminic acid-containing receptors has been analyzed. When the oligosaccharides were deleted by site-specific mutagenesis, HA expressed from a simian virus 40 vector showed enhanced hemadsorbing activity. Binding was so strong under these conditions that erythrocytes were no longer released by viral neuraminidase and that release was significantly reduced when neuraminidase from Vibrio cholerae was used. Similarly, when these oligosaccharides were removed selectively from purified viruses by N-glycosidase F, such virions were unable to elute from receptors, although they retained neuraminidase activity. Thus, release of FPV from cell receptors depends on the presence of the HA glycans at Asn123 and Asn149. On the other hand, receptor binding was abolished when these oligosaccharides were sialylated after expression in the absence of neuraminidase (M. Ohuchi, A. Feldmann, R. Ohuchi, and H.-D. Klenk, Virology 212:77-83, 1995). These observations indicate that the receptor affinity of FPV HA is controlled by oligosaccharides adjacent to the receptor binding site.
Mutations in coat protein binding sites of alfalfa mosaic virus RNA 3 affect subgenomic RNA 4 accumulation and encapsidation of viral RNAs
Authors: Reusken C B , Neeleman L , Brederode F T , Bol J F
Published: January 2020

The 3'-untranslated regions (3'-UTRs) of the three RNAs of alfalfa mosaic virus (AMV) contain a specific binding site for coat protein (CP) and act as a promoter for minus-strand RNA synthesis by the purified AMV RNA-dependent RNA polymerase (RdRp) in an in vitro assay. Binding of CP to the viral RNAs is required to initiate infection. The sequence of the 3'-terminal 39 nucleotides of AMV RNA 3 can be folded into two stem-loop structures flanked by three single-stranded AUGC sequences and represents a CP binding site. Mutations in this sequence that are known to interfere with CP binding in vitro were introduced into an infectious clone of RNA 3, and mutant RNA transcripts were used as templates in the in vitro RdRp assay and to infect protoplasts and plants. Mutation of AUGC motif 2 or disruption of the stem of the 3'-proximal hairpin 1 interfered with CP binding in vitro but not with minus-strand promoter activity in vitro or replication of RNA 3 in vivo. However, hairpin 1 appeared to be essential for encapsidation of RNA 3. Reversion of three G-C base pairs in hairpin 1 had no effect on CP binding but interfered with minus-strand promoter activity in vitro and with RNA 3 replication in vivo. It is concluded that the viral RdRp and CP recognize different elements in the 3'-UTRs of AMV RNAs. Moreover, several mutations that interfered with CP binding in vitro interfered with the accumulation in vivo of RNA 4, the subgenomic messenger for CP, but not with the accumulation of RNA 3.
Promiscuous use of CC and CXC chemokine receptors in cell-to-cell fusion mediated by a human immunodeficiency virus type 2 envelope protein
Authors: Bron R , Klasse P J , Wilkinson D , Clapham P R , Pelchen-Matthews A , Power C , Wells T N , Kim J , Peiper S C , Hoxie J A , Marsh M
Published: January 2020

The CC chemokine receptors CCR5, CCR2, and CCR3 and the CXC chemokine receptor CXCR4 have been implicated as CD4-associated cofactors in the entry of primary and cell line-adapted human immunodeficiency virus type 1 (HIV-1) strains. CXCR4 is also a receptor for T-cell-line-adapted, CD4-independent strains of HIV-2. With the exception of this latter example, little has been reported on the entry cofactors used by HIV-2 strains. Here we show that a CD4-dependent, T-cell-line-adapted HIV-2 strain uses CXCR4 and, to a lesser extent, CCR3 for fusion with and infectious entry into cells. In a cell-to-cell fusion assay, the envelope protein of this virus can utilize a wider repertoire of chemokine receptors to induce fusion. These include CCR1, CCR2, CCR3, CCR4, CCR5, CXCR2, and CXCR4. Kinetic analysis indicated that cell lines expressing the receptors that support infection, CXCR4 and CCR3, form syncytia more rapidly than do cell lines expressing the other receptors. Nevertheless, although less efficient, fusion with CXCR2 expressing cells was specific, since it was inhibited by antibodies against CXCR2. The extensive use of chemokine receptors in cell-to-cell fusion has implications for understanding the molecular basis of CD4-chemokine receptor-induced lentivirus fusion and may have relevance for syncytium formation and the direct cell-to-cell transfer of virus in vivo.
Evidence that resolution of rotavirus infection in mice is due to both CD4 and CD8 cell-dependent activities
Authors: McNeal M M , Rae M N , Ward R L
Published: January 2020

The effector functions responsible for resolution of shedding in mice orally inoculated with the murine rotavirus strain EDIM were identified in B-cell-deficient and normal BALB/c mice after monoclonal antibody (MAb) depletion of CD4 and CD8 cells. When depleted of CD8 cells, B-cell-deficient muMt mice resolved their infections more slowly than nondepleted animals, but CD4 cell depletion caused chronic, high-level shedding. This finding indicated that CD4 cell-dependent immunological effectors other than, or in addition to, CD8 cells played roles in rotavirus resolution in muMt mice in the absence of antibody. The roles of CD4 and CD8 cells in resolution of rotavirus shedding were further characterized in immunologically normal BALB/c mice. Depletion of CD4 cells before EDIM inoculation resulted in rapid resolution of most shedding, but chronic, low-level shedding continued for weeks. When the CD4 cell-depleted BALB/c mice were subsequently depleted of CD8 cells, shedding levels increased significantly (P < 0.001), indicating that CD8 cells were responsible for the rapid but incomplete suppression of rotavirus shedding. Further experimentation revealed that little rotavirus antibody was made in CD4 cell-depleted BALB/c mice, and only after CD4 cells were repopulated did antibody production increase and virus shedding fully resolve. Thus, resolution of rotavirus shedding in both muMt and BALB/c mice was associated with CD4 and CD8 cell effector activities.
Poliovirus 2C region functions during encapsidation of viral RNA
Authors: Vance L M , Moscufo N , Chow M , Heinz B A
Published: January 2020

We have been exploring the mechanism of action of 5-(3,4-dichlorophenyl) methylhydantoin (hydantoin), an antiviral drug that inhibits the replication of poliovirus in culture. By varying the time of drug addition to infected cells, we found that the drug acts at a stage which is late in the replication cycle and subsequent to the step inhibited by guanidine. Furthermore, we detected normal levels of full-length plus-strand virion RNA in hydantoin-treated cultures. A new assembly intermediate in addition to the expected assembly intermediates was detected in drug-treated cultures. This intermediate has properties consistent with that of a packaging intermediate. Drug-resistant mutants were readily isolated. Sequence analysis of three independent drug-resistant mutants identified amino acid substitutions in the 2C coding region. Reconstruction by site-directed mutagenesis confirmed that these single mutations were sufficient to confer drug resistance. Taken together, these data suggest that the poliovirus 2C region is involved in virus encapsidation and that hydantoin inhibits this stage of replication.
RNA aptamers specifically interact with the prion protein PrP
Authors: Weiss S , Proske D , Neumann M , Groschup M H , Kretzschmar H A , Famulok M , Winnacker E L
Published: January 2020

We have isolated RNA aptamers which are directed against the recombinant Syrian golden hamster prion protein rPrP23-231 (rPrPc) fused to glutathione S-transferase (GST). The aptamers did not recognize the fusion partner GST or the fusion protein GST::rPrP90-231 (rPrP27-30), which lacks 67 amino acids from the PrP N terminus. The aptamer-interacting region of PrPc was mapped to the N-terminal amino acids 23 to 52. Sequence analyses suggest that the RNA aptamers may fold into G-quartet-containing structural elements. Replacement of the G residues in the G quartet scaffold with uridine residues destroyed binding to PrP completely, strongly suggesting that the G quartet motif is essential for PrP recognition. Individual RNA aptamers interact specifically with prion protein in brain homogenates from wild-type mice (C57BL/6), hamsters (Syrian golden), and cattle as shown by supershifts obtained in the presence of anti-PrP antibodies. No interaction was observed with brain homogenates from PrP knockout mice (prn-p(0/0)). Specificity of the aptamer-PrP interaction was further confirmed by binding assays with antisense aptamer RNA or a mutant aptamer in which the guanosine residues in the G tetrad scaffold were replaced by uridine residues. The aptamers did not recognize PrP27-30 in brain homogenates from scrapie-infected mice. RNA aptamers may provide a first milestone in the development of a diagnostic assay for the detection of transmissible spongiform encephalopathies.
Details of the arrangement of the outer capsid of rice dwarf phytoreovirus, as visualized by two-dimensional crystallography
Authors: Zhu Y , Hemmings A M , Iwasaki K , Fujiyoshi Y , Zhong B , Yan J , Isogai M , Omura T
Published: January 2020

Two-dimensional crystals were obtained from purified P8, an outer capsid protein of rice dwarf phytoreovirus. A filtered image of the two-dimensional crystal, in combination with the results of biochemical analysis, revealed the unit formation of the capsid protein, a capsomere structure, which appeared to be an approximately equilateral triangle with sides of approximately 6 nm and which was composed of a trimer of P8 protein. Details of the arrangements of the outer capsid of the virus are described.
Persistence of recombinant adenovirus in vivo is not dependent on vector DNA replication
Authors: Nelson J E , Kay M A
Published: January 2020

Recombinant adenovirus vectors represent an efficient means of transferring genes into many different organs. The first-generation E1-deleted vector genome remains episomal and, in the absence of host immunity, persists long-term in quiescent tissues such as the liver. The mechanism(s) which allows for persistence has not been established; however, vector DNA replication may be important because replication has been shown to occur in tissue culture systems. We have utilized a site-specific methylation strategy to monitor the replicative fate of E1-deleted adenovirus vectors in vitro and in vivo. Methylation-marked adenovirus vectors were produced by the addition of a methyl group onto the N6 position of the adenine base of XhoI sites, CTCGAG, by propagation of vectors in 293 cells expressing the XhoI isoschizomer PaeR7 methyltransferase. The methylation did not affect vector production or transgene expression but did prevent cleavage by XhoI. Loss of methylation through viral replication restores XhoI cleavage and was observed by Southern analysis in a wide variety of, but not all, cell culture systems studied, including hepatoma and mouse and macaque primary hepatocyte cultures. In contrast, following liver-directed gene transfer of methylated vector in C57BL/6 mice, adenovirus vector DNA was not cleaved by XhoI and therefore did not replicate, even after a period of 3 weeks. Although replication may occur in some tissues, these results show that stabilization of the vector within the target tissue prior to clearance by host immunity is not dependent upon replication of the vector, demonstrating that the input transduced DNA genomes were the persistent molecules. This information will be useful for the design of optimal adenovirus vectors and perhaps nonviral episomal vectors for clinical gene therapy.
Cellular recombination pathways and viral terminal repeat hairpin structures are sufficient for adeno-associated virus integration in vivo and in vitro
Authors: Yang C C , Xiao X , Zhu X , Ansardi D C , Epstein N D , Frey M R , Matera A G , Samulski R J
Published: January 2020

The human parvovirus adeno-associated virus (AAV) is unique in its ability to target viral integration to a specific site on chromosome 19 (ch-19). Recombinant AAV (rAAV) vectors retain the ability to integrate but have apparently lost this ability to target. In this report, we characterize the terminal-repeat-mediated integration for wild-type (wt), rAAV, and in vitro systems to gain a better understanding of these differences. Cell lines latent for either wt or rAAV were characterized by a variety of techniques, including PCR, Southern hybridization, and fluorescence in situ hybridization analysis. More than 40 AAV-rAAV integration junctions were cloned, sequenced, and then subjected to comparison and analysis. In both immortalized and normal diploid human cells, wt AAV targeted integration to ch-19. Integrated provirus structures consisted of head-to-tail tandem arrays with the majority of the junction sequences involving the AAV inverted terminal repeats (ITRs). No complete viral ITRs were directly observed. In some examples, the AAV p5 promoter sequence was found to be fused at the virus-cell junction. Data from dot blot analysis of PCR products were consistent with the occurrence of inversions of genomic and/or viral DNA sequences at the wt integration site. Unlike wt provirus junctions, rAAV provirus junctions mapped to a subset of non-ch-19 sequences. Southern analysis supported the integration of proviruses from two independent cell lines at the same locus on ch-2. In addition, provirus terminal repeat sequences existed in both the flip and flop orientations, with microhomology evident at the junctions. In all cases with the exception of the ITRs, the vector integrated intact. rAAV junction sequence data were consistent with the occurrence of genomic rearrangement by deletion and/or rearrangement-translocation at the integration locus. Finally, junctions formed in an in vitro system between several AAV substrates and the ch-19 target site were isolated and characterized. Linear AAV substrates typically utilized the end of the virus DNA substrate as the point of integration, whereas products derived from AAV terminal repeat hairpin structures in the presence or absence of Rep protein resembled AAV-ch-19 junctions generated in vivo. These results describing wt AAV, rAAV, and in vitro integration junctions suggest that the viral integration event itself is mediated by terminal repeat hairpin structures via nonviral cellular recombination pathways, with specificity for ch-19 in vivo requiring additional viral components. These studies should have an important impact on the use of rAAV vectors in human gene therapy.
Protein interactions during coronavirus assembly
Authors: Nguyen V P , Hogue B G
Published: January 2020

Coronaviruses assemble and obtain their envelope at membranes of the intermediate compartment between the endoplasmic reticulum and Golgi complex. Like other enveloped viruses, coronavirus assembly is presumably dependent on protein localization and protein-protein as well as protein-RNA interactions. We have used the bovine coronavirus (BCV) as a model to study interactions between the viral proteins in virus-infected cells that are important for coronavirus assembly. BCV is a prototype for the coronaviruses that express an additional major structural protein, the hemagglutinin esterase (HE), in addition to the spike (S) glycoprotein, membrane (M) glycoprotein, and nucleocapsid (N) protein. Complexes consisting of the M, S, and HE proteins were detected in virus-infected cells by coimmunoprecipitations. Kinetic analyses demonstrated that S protein and HE each quickly formed a complex with M protein after synthesis, whereas heterocomplexes consisting of all three proteins formed more slowly. The kinetics of HE biosynthesis revealed that the half-life of oligomerization was approximately 30 min, which correlated with the appearance of complexes consisting of M, HE, and S proteins, suggesting that oligomerization and/or conformational changes may be important for the S-M-HE protein complexes to form. Only HE dimers were found associated with the heterocomplexes consisting of all three proteins. S-M-HE protein complexes were detected prior to processing of the oligosaccharide chains on HE, indicating that these protein complexes formed in a premedial Golgi compartment before trimming of sugar chains. Transient coexpressions and double-labeling immunofluorescence demonstrated that HE and S proteins colocalized with M protein. This was further supported by coimmunoprecipitation of specific HE-M and S-M protein complexes from transfected cells, indicating that these proteins can form complexes in the absence of other viral proteins.
Isolation of highly fusogenic variants of simian virus 5 from persistently infected cells that produce and respond to interferon
Authors: Young D F , Didcock L , Randall R E
Published: January 2020

A series of experiments were undertaken to examine how interferon and neutralizing antibodies influence the ability of simian virus 5 (SV5) (strain W3) to establish and maintain persistent infections in murine cells. In contrast to the rapid decline in SV5 protein synthesis observed in murine BALB/c fibroblasts (BF cells), which produce and respond to interferon, between 24 and 48 h postinfection there was no inhibition of virus protein synthesis in MSFI- cells, skin fibroblasts derived from alpha/beta-interferon receptor knockout BALB/c mice. Furthermore, the addition of anti-interferon antibodies to the culture medium of infected BF cells significantly reduced the observed decline in virus protein synthesis. Following infection of untreated BF cells, the majority replicated virus but survived the infection and eventually cleared the virus after 8 to 15 days. However, not all the cells were cured, and the cultures became persistently infected. Upon passage of persistently infected cultures, the virus fluxed between active and repressed states as a consequence of interferon production. This resulted in a balance being reached in which only 5 to 20% of the cells were infected at any one time. After 30 passages of the persistently infected cells, highly fusogenic virus variants arose (one of which was isolated and termed W3-f). W3-f remained as sensitive to interferon as the parental W3 isolate but, in the absence of interferon, spread much more rapidly than the parental W3 strain through BF cell monolayers. Sequence analysis revealed no deduced amino acid differences between the F proteins of W3 and W3-f. BF cell cultures persistently infected with W3-f were rapidly cleared of virus by the addition of virus-neutralizing antibodies to the culture medium. In contrast, neutralizing antibodies had little effect on the numbers of cells persistently infected with W3 over several passages. These results suggest that the ability of paramyxoviruses to cause cell-cell fusion may be selected for in vivo as a consequence of their adaptation to the interferon response rather than their need to escape from neutralizing antibodies. The significance of these observations with regard to persistent parainfluenza virus infections in vivo is further discussed.
Human immunodeficiency virus type 1 Vif protein binds to the Pr55Gag precursor
Authors: Bouyac M , Courcoul M , Bertoia G , Baudat Y , Gabuzda D , Blanc D , Chazal N , Boulanger P , Sire J , Vigne R , Spire B
Published: January 2020

The Vif protein of human immunodeficiency virus type 1 is required for productive replication in peripheral blood lymphocytes. Previous reports suggest that vif-deleted viruses are limited in replication because of a defect in the late steps of the virus life cycle. One of the remaining questions is to determine whether the functional role of Vif involves a specific interaction with virus core proteins. In this study, we demonstrate a direct interaction between Vif and the Pr55Gag precursor in vitro as well as in infected cells. No interaction is observed between Vif and the mature capsid protein. The Pr55Gag-Vif interaction is detected (i) in the glutathione S-transferase system, with in vitro-translated proteins demonstrating a critical role of the NC p7 domain of the Gag precursor; (ii) with proteins expressed in infected cells; and (iii) by coimmunoprecipitation experiments. Deletion of the C-terminal 22 amino acids of Vif abolishes its interaction with the Pr55Gag precursor. Furthermore, point mutations in the C-terminal domain of Vif which have been previously shown to abolish virus infectivity and binding to cell membranes dramatically decrease the Gag-Vif interaction. These results suggest that the interaction between Vif and the pr55Gag precursor is a critical determinant of Vif function.
The function of the spike protein of mouse hepatitis virus strain A59 can be studied on virus-like particles: cleavage is not required for infectivity
Authors: Bos E C , Luytjes W , Spaan W J
Published: January 2020

The spike protein (S) of the murine coronavirus mouse hepatitis virus strain A59 (MHV-A59) induces both virus-to-cell fusion during infection and syncytium formation. Thus far, only syncytium formation could be studied after transient expression of S. We have recently described a system in which viral infectivity is mimicked by using virus-like particles (VLPs) and reporter defective-interfering (DI) RNAs (E. C. W. Bos, W. Luytjes, H. Van der Meulen, H. K. Koerten, and W. J. M. Spaan, Virology 218:52-60, 1996). Production of VLPs of MHV-A59 was shown to be dependent on the expression of M and E. We now show in several ways that the infectivity of VLPs is dependent on S. Infectivity was lost when spikeless VLPs were produced. Infectivity was blocked upon treatment of the VLPs with MHV-A59-neutralizing anti-S monoclonal antibody (MAb) A2.3 but not with nonneutralizing anti-S MAb A1.4. When the target cells were incubated with antireceptor MAb CC1, which blocks MHV-A59 infection, VLPs did not infect the target cells. Thus, S-mediated VLP infectivity resembles MHV-A59 infectivity. The system can be used to identify domains in S that are essential for infectivity. As a first application, we investigated the requirements of cleavage of S for the infectivity of MHV-A59. We inserted three mutant S proteins that were previously shown to be uncleaved (E. C. W. Bos, L. Heijnen, W. Luytjes, and W. J. M. Spaan, Virology 214:453-463, 1995) into the VLPs. Here we show that cleavage of the spike protein of MHV-A59 is not required for infectivity.
Cell culture adaptation of Puumala hantavirus changes the infectivity for its natural reservoir, Clethrionomys glareolus, and leads to accumulation of mutants with altered genomic RNA S segment
Authors: Lundkvist A , Cheng Y , Sjölander K B , Niklasson B , Vaheri A , Plyusnin A
Published: January 2020

This paper reports the establishment of a model for hantavirus host adaptation. Wild-type (wt) (bank vole-passaged) and Vero E6 cell-cultured variants of Puumala virus strain Kazan were analyzed for their virologic and genetic properties. The wt variant was well adapted for reproduction in bank voles but not in cell culture, while the Vero E6 strains replicated to much higher efficiency in cell culture but did not reproducibly infect bank voles. Comparison of the consensus sequences of the respective viral genomes revealed no differences in the coding region of the S gene. However, the noncoding regions of the S gene were found to be different at positions 26 and 1577. In one additional and independent adaptation experiment, all analyzed cDNA clones from the Vero E6-adapted variant were found to carry substitutions at position 1580 of the S segment, just 3 nucleotides downstream of the mutation observed in the first adaptation. No differences were found in the consensus sequences of the entire M segments from the wt and the Vero E6-adapted variants. The results indicated different impacts of the S and the M genomic segments for the adaptation process and selective advantages for the variants that carried altered noncoding sequences of the S segment. We conclude that the isolation in cell culture resulted in a phenotypically and genotypically altered hantavirus.
Poliovirus-encoded 2C polypeptide specifically binds to the 3'-terminal sequences of viral negative-strand RNA
Authors: Banerjee R , Echeverri A , Dasgupta A
Published: January 2020

The poliovirus-encoded, membrane-associated polypeptide 2C is believed to be required for initiation and elongation of RNA synthesis. We have expressed and purified recombinant, histidine-tagged 2C and examined its ability to bind to the first 100 nucleotides of the poliovirus 5' untranslated region of the positive strand and its complementary 3'-terminal negative-strand RNA sequences. Results presented here demonstrate that the 2C polypeptide specifically binds to the 3'-terminal sequences of poliovirus negative-strand RNA. Since this region is believed to form a stable cloverleaf structure, a number of mutations were constructed to examine which nucleotides and/or structures within the cloverleaf are essential for 2C binding. Binding of 2C to the 3'-terminal cloverleaf of the negative-strand RNA is greatly affected when the conserved sequence, UGUUUU, in stem a of the cloverleaf is altered. Mutational studies suggest that interaction of 2C with the 3'-terminal cloverleaf of negative-strand RNA is facilitated when the sequence UGUUUU is present in the context of a double-stranded structure. The implication of 2C binding to negative-strand RNA in viral replication is discussed.
Promonocytic U937 subclones expressing CD4 and CXCR4 are resistant to infection with and cell-to-cell fusion by T-cell-tropic human immunodeficiency virus type 1
Authors: Moriuchi H , Moriuchi M , Arthos J , Hoxie J , Fauci A S
Published: January 2020

Different strains of human immunodeficiency virus type 1 (HIV-1) vary markedly in the ability to infect cells of the monocyte/macrophage (M/M) lineage. M/M are generally resistant to infection with T-cell-tropic (T-tropic) strains of HIV-1. Recently, the chemokine receptors CCR5 and CXCR4 were identified as cofactors for fusion/entry of macrophage- and T-tropic strains of HIV-1, respectively. To investigate the mechanisms of resistance of M/M to T-tropic HIV-1 infection, we examined a number of subclones of the U937 promonocytic cell line. We found that certain subclones of U937 (plus clones) could, while others (minus clones) could not, support replication of T-tropic strains of HIV-1. We demonstrate that (i) both minus and plus clones support HIV-1 replication when transfected with an infectious molecular cDNA clone of a T-tropic HIV-1; (ii) minus clones do not, but plus clones do, efficiently support fusion with cells expressing HIV-1 IIIB Env; (iii) both plus and minus clones (with the exception of one clone) express physiologically functional CXCR4 protein as well as CD4 on the cell surface; (iv) introduction of CXCR4 into the CXCR4-negative clone does not restore fusogenicity with or susceptibility to T-tropic HIV-1; and (v) a ligand (stromal cell-derived factor 1) for or a monoclonal antibody (12G5) to CXCR4 does not effectively inhibit HIV-mediated cell-to-cell fusion of U937 cells. These data indicate that resistance to T-tropic HIV-1 infection of U937 minus clones occurs at fusion/ entry events and that expression of functional CXCR4 and CD4 is not a sole determinant for susceptibility to T-tropic HIV-1 infection; furthermore, they suggest that other factors are positively or negatively involved in HIV-mediated cell-to-cell fusion in U937 promonocytic cells.
Role of sialyloligosaccharide binding in Theiler's virus persistence
Authors: Zhou L , Lin X , Green T J , Lipton H L , Luo M
Published: January 2020

Theiler's murine encephalomyelitis viruses (TMEVs) belong to the Picornaviridae family and are divided into two groups, typified by strain GDVII virus and members of the TO (Theiler's original) group. The highly virulent GDVII group causes acute encephalitis in mice, while the TO group is less virulent and causes a chronic demyelinating disease which is associated with viral persistence in mice. This persistent central nervous system infection with demyelination resembles multiple sclerosis (MS) in humans and has thus become an important model for studying MS. It has been shown that some of the determinants associated with viral persistence are located on the capsid proteins of the TO group. Structural comparisons of two persistent strains (BeAn and DA) and a highly virulent strain (GDVII) showed that the most significant structural variations between these two groups of viruses are located on the sites that may influence virus binding to cellular receptors. Most animal viruses attach to specific cellular receptors that, in part, determine host range and tissue tropism. In this study, atomic models of TMEV chimeras were built with the known structures of GDVII, BeAn, and DA viruses. Comparisons among the known GDVII, BeAn, and DA structures as well as the predicted models for the TMEV chimeras suggested that a gap on the capsid surface next to the putative receptor binding site, composed of residues from VP1 and VP2, may be important in determining viral persistence by influencing virus attachment to cellular receptors, such as sialyloligosaccharides. Our results showed that sialyllactose, the first three sugar molecules of common oligosaccharides on the surface of mammalian cells, inhibits virus binding to the host cell and infection with the persistent BeAn virus but not the nonpersistent GDVII and chimera 39 viruses.
Structure-function analysis of the triphosphatase component of vaccinia virus mRNA capping enzyme
Authors: Yu L , Martins A , Deng L , Shuman S
Published: January 2020

The N-terminal 60 kDa (amino acids 1 to 545) of the D1 subunit of vaccinia virus mRNA capping enzyme is an autonomous bifunctional domain with triphosphatase and guanylyltransferase activities. We previously described two alanine cluster mutations, R77 to A (R77A)-K79A and E192A-E194A, which selectively inactivated the triphosphatase component. Here, we characterize the activities of 11 single alanine mutants-E37A, E39A, Q60A, E61A, T67A, T69A, K75A, R77A, K79A, E192A, and E194A-and a quadruple mutant in which four residues (R77, K79, E192, and E194) were replaced by alanine. We report that Glu-37, Glu-39, Arg-77, Glu-192, and Glu-194 are essential for gamma-phosphate cleavage. The five essential residues are conserved in the capping enzymes of Shope fibroma virus, molluscum contagiosum virus, and African swine fever virus. Probing the structure of D1(1-545) by limited V8 proteolysis suggested a bipartite subdomain structure. The essential residue Glu-192 is the principal site of V8 cleavage. Secondary cleavage by V8 occurs at the essential residue Glu-39. The triphosphatase-defective quadruple mutant transferred GMP to the triphosphate end of poly(A) to form a tetraphosphate cap structure, GppppA. We report that GppppA-capped RNA is a poor substrate for cap methylation by the vaccinia virus and Saccharomyces cerevisiae RNA (guanine-7) methyltransferases. The transcription termination factor activity of the D1-D12 capping enzyme heterodimer was not affected by mutations that abrogated ATPase activity. Thus, the capping enzyme is not responsible for the requirement for ATP hydrolysis during transcription termination.
Nef association with human immunodeficiency virus type 1 virions and cleavage by the viral protease
Authors: Bukovsky A A , Dorfman T , Weimann A , Göttlinger H G
Published: January 2020

Nef is a regulatory gene product of human immunodeficiency virus type 1 (HIV-1) and other primate lentiviruses which enhances virion infectivity by an unknown mechanism. We report here that Nef is detectable at moderate levels in preparations of HIV-1 virions which lack active viral protease (PR). Significantly smaller amounts of intact Nef were present in wild-type virion preparations. Instead, a smaller Nef-related product with an apparent molecular mass of 18 kDa was associated with wild-type virions, indicating that packaging of Nef resulted in cleavage by the viral PR. The presence of the HIV-1 PR inhibitor A77003 during virus production prevented the appearance of the 18-kDa Nef product and caused an accumulation of full-length Nef in virion preparations. Nef associated with comparable efficiency with viral particles produced by the Gag polyproteins of HIV-1 and Moloney murine leukemia virus, indicating that no specific interaction with a virion component is required for the incorporation of Nef. The N-terminal 86 amino acids of Nef were sufficient for packaging into virions. A nonmyristylated form of Nef associated with viral particles with considerably lower efficiency, suggesting that Nef gains access into nascent virions primarily as a consequence of its affinity for membranes. Our results raise the possibility that Nef enhances infectivity directly as a component of the virion.
Human T-cell leukemia virus type 1 Tax transactivates the human proliferating cell nuclear antigen promoter
Authors: Ressler S , Morris G F , Marriott S J
Published: January 2020

The human T-cell leukemia virus type 1 (HTLV-1) transforming protein, Tax, is a potent transactivator of both viral and cellular gene expression. The ability of Tax to transform cells is believed to depend on its transactivation of cellular-growth-regulatory genes. Expression of proliferating cell nuclear antigen (PCNA) is intimately linked to cell growth and DNA replication and repair. By testing a series of PCNA promoter deletion constructs, we have demonstrated that the PCNA promoter can be transactivated by Tax. The smallest construct that was activated did not include the ATF/CRE binding site at nucleotide -50, and mutations in the ATF/CRE element in the context of a larger promoter were still activated by Tax. In addition, a Tax mutant that is defective for activation of the CRE pathway retained the ability to activate the -397 promoter construct. When a series of linker scanner mutations that span the region from nucleotide -45 to -7 were assayed, mutations in and around a repeat sequence were found to abolish Tax transactivation. Multimerized copies of either half of the repeat were Tax responsive. A single protein complex was shown to bind specifically to the Tax-responsive region, and the binding of this complex was enhanced in the presence of Tax. These results demonstrate that the PCNA promoter contains a Tax-responsive element located between nucleotides -45 and -7 whose sequence is different from those of other, previously identified Tax-responsive elements. The ability of Tax to activate the PCNA promoter may play an important role in cellular transformation by HTLV-1.
Characterization of ribonucleoprotein complexes containing an abundant polyadenylated nuclear RNA encoded by Kaposi's sarcoma-associated herpesvirus (human herpesvirus 8)
Authors: Zhong W , Ganem D
Published: January 2020

Infection with Kaposi's sarcoma-associated herpesvirus (KSHV) (also called human herpesvirus 8) is strongly linked to all forms of Kaposi's sarcoma. We have previously identified two polyadenylated KSHV transcripts that are actively transcribed in Kaposi's sarcoma (KS) tumors and in KSHV-infected B-lymphoma cells. One of these RNAs (termed T1.1 or nut-1 RNA) is a 1.1-kb transcript present in a subpopulation of KS tumor cells. This RNA is localized to the nucleus of infected cells and has no open reading frames longer than 62 codons, suggesting that it may not function as an mRNA in vivo. Here we demonstrate that nut-1 RNA is a lytic-cycle gene product that is found in high-molecular-weight ribonucleoprotein complexes in infected cell nuclei. The transcript lacks the trimethylguanosine (TMG) cap found in many U-like small nuclear RNAs, but a subpopulation of nut-1 RNAs can associate with Sm protein-containing small nuclear ribonucleoproteins, as judged by immunoprecipitation analyses using monoclonal anti-Sm and anti-TMG antibodies. This interaction does not require other viral gene products, and deletion of the sole candidate Sm binding site on nut-1 RNA does not ablate this association. This finding suggests an indirect interaction with Sm-containing structures, and models for such associations are presented.