bluuebunny/crossref_metadata_2025 huggingface dataset:
CD4-independent infection by human immunodeficiency virus type 2 strain ROD/B: the role of the N-terminal domain of CXCR-4 in fusion and entry
Authors: Potempa S , Picard L , Reeves J D , Wilkinson D , Weiss R A , Talbot S J
Published: January 2020

The human immunodeficiency virus type 2 (HIV-2) strain ROD/B can efficiently use the 7tm chemokine receptor CXCR-4 as a primary receptor to enter CD4-negative cells. We have stably expressed CXCR-4 on mink lung Mv-1-lu and feline kidney CCC cells (normally restrictive to HIV entry) and have shown efficient fusion, entry, and replication of ROD/B. Mutation of the two N-linked glycosylation sites on CXCR-4 (N11-->I, and N176-->Q) or pretreatment of CCC or Mv-1-lu cells expressing wild-type CXCR-4 with the glycosylation inhibitor tunicamycin increased fusion and entry by ROD/B. Deletion of portions of the N terminus of CXCR-4 resulted in a 3- to 10-fold decrease in cell-free infection by ROD/B and complete inhibition of cell-cell fusion by both ROD/B and another HIV-2 strain, CBL23. These data suggest that the N-terminal domain of CXCR-4 is involved in but is not essential for the efficient fusion of ROD/B with CD4-negative cells. Deletion of the C-terminal (intracellular) domain of CXCR-4 did not significantly affect entry by ROD/B, indicating that intracellular signalling through this domain does not play a significant role in entry by HIV-2.
Murine retroviruses use at least six different receptors for entry into Mus dunni cells
Authors: Miller A D , Wolgamot G
Published: January 2020

Murine retroviruses have been divided into six interference groups that use different receptors for cell entry: the ecotropic, xenotropic, polytropic, amphotropic, 10A1, and Mus dunni endogenous virus groups. Some interference is observed between xenotropic and polytropic viruses and between amphotropic and 10A1 viruses, indicating some overlap in receptor specificity between these groups, but otherwise these interference groups appear completely independent. In contrast, one study found interference among many of these groups when Mus dunni wild mouse cells were examined with an immunofluorescence assay to detect infection by the challenge virus. Here we have used a more direct assay for cell entry by using pseudotyped retroviral vectors to measure interference in M. dunni cells, and we find no evidence for extensive interference between members of different murine retrovirus groups. Indeed, our results in M. dunni cells are consistent with interference results observed in other cell types and indicate that the anomalous interference results previously observed in M. dunni cells with the immunofluorescence assay were most likely due to factors other than those that affect receptor-mediated virus entry. In summary, our results show that murine retroviruses use at least six different receptors for entry into M. dunni cells.
Role of the extracellular domain of human herpesvirus 7 glycoprotein B in virus binding to cell surface heparan sulfate proteoglycans
Authors: Secchiero P , Sun D , De Vico A L , Crowley R W , Reitz M S , Zauli G , Lusso P , Gallo R C
Published: January 2020

In an attempt to identify the human herpesvirus 7 (HHV-7) envelope protein(s) involved in cell surface binding, the extracellular domain of the HHV-7 glycoprotein B (gB) homolog protein was cloned and expressed as a fusion product with the Fc domain of human immunoglobulin G heavy chain gamma1 (gB-Fc) in an eukaryotic cell system. Indirect immunofluorescence followed by flow cytometric analysis revealed specific binding of gB-Fc to the membrane of SupT1 cells but not to other CD4+ T-lymphoblastoid cell lines, such as Jurkat or PM1, clearly indicating that gB-Fc did not bind to the CD4 molecule. This was also suggested by the ability of gB-Fc to bind to CD4-negative fibroblastoid Chinese hamster ovary (CHO) cells. The binding was abrogated by enzymatic removal of cell surface heparan sulfate proteoglycans by heparinase and heparitinase but not by treatment with condroitinase ABC. In addition, binding of the gB-Fc fusion protein to CHO cells was severely impaired in the presence of soluble heparin, as well as when heparan sulfate-deficient mutant CHO cells were used. Consistent with these findings, soluble heparin was found to block HHV-7 infection and syncytium formation in the SupT1 cell line. Although the CD4 antigen is a critical component of the receptor for the T-lymphotropic HHV-7, these findings suggest that heparin-like molecules also play an important role in HHV-7-cell surface interactions required for infection and that gB represents one of the HHV-7 envelope proteins involved in the adsorption of virus-to-cell surface proteoglycans.
The major immediate-early proteins IE1 and IE2 of human cytomegalovirus colocalize with and disrupt PML-associated nuclear bodies at very early times in infected permissive cells
Authors: Ahn J H , Hayward G S
Published: January 2020

The major immediate-early (MIE) gene products of human cytomegalovirus (HCMV) are nuclear phosphoproteins that are thought to play key roles in initiating lytic cycle gene regulation pathways. We have examined the intranuclear localization pattern of both the IE1 and IE2 proteins in virus-infected and DNA-transfected cells. When HCMV-infected human diploid fibroblast (HF) cells were stained with specific monoclonal antibodies, IE1 localized as a mixture of nuclear diffuse and punctate patterns at very early times (2 h) but changed to an exclusively nuclear diffuse pattern at later times. In contrast, IE2 was distributed predominantly in nuclear punctate structures continuously from 2 to at least 12 h after infection. These punctate structures resembled the preexisting PML-associated nuclear bodies (ND10 or PML oncogenic domains [PODs]) that are disrupted and dispersed by the IE110 protein as a very early event in herpes simplex virus (HSV) infection. However, HCMV differed from HSV by leading instead to a change in both the PML and SP100 protein distribution from punctate bodies to uniform diffuse patterns, a process that was complete in 50% of the cells at 2 h and in 90% of the cells by 4 h after infection. Confocal double-label indirect immunofluorescence assay analysis confirmed that both IE1 and IE2 colocalized transiently with PML in punctate bodies at very early times after infection. In transient expression assays, introduction of IE1-encoding plasmid DNA alone into Vero or HF cells produced the typical total redistribution of PML into a uniform nuclear diffuse pattern together with the IE1 protein, whereas introduction of IE2-encoding plasmid DNA alone resulted in stable colocalization of the IE2 protein with PML in the PODs. A truncated mutant form of IE1 gave large nuclear aggregates and failed to redistribute PML, and similarly a deleted mutant form of IE2 failed to colocalize with the punctate PML bodies, confirming the specificity of these effects. Furthermore, both Vero and U373 cell lines constitutively expressing IE1 also showed total PML relocalization together with the IE1 protein into a nuclear diffuse pattern, although a very small percentage of the cells which failed to express IE1 reverted to a punctate PML pattern. Finally, the PML redistribution activity of IE1 and the direct association of IE2 with PML punctate bodies were both confirmed by infection with E1A-negative recombinant adenovirus vectors expressing either IE1 or IE2 alone. These results confirm that transient colocalization with and disruption of PML-associated nuclear bodies by IE1 and continuous targeting to PML-associated nuclear bodies by IE2 are intrinsic properties of these two MIE regulatory proteins, which we suggest may represent critical initial events for efficient lytic cycle infection by HCMV.
The herpes simplex virus immediate-early protein ICP0 affects transcription from the viral genome and infected-cell survival in the absence of ICP4 and ICP27
Authors: Samaniego L A , Wu N , DeLuca N A
Published: January 2020

ICP4, ICP0, and ICP27 are the immediate-early (IE) regulatory proteins of herpes simplex virus that have the greatest effect on viral gene expression and growth. Comparative analysis of viral mutants defective in various subsets of these IE genes should help elucidate how these proteins affect cellular and viral processes. This study focuses on the mutant d97, which is defective for the genes encoding ICP4, ICP0, and ICP27 and expresses the bacterial beta-galactosidase (beta-gal) gene from the ICP0 promoter. Together with the d92 virus (ICP4- ICP27-) and the ICP0-complementing cell line L7, d97 provided a unique opportunity to evaluate ICP0 function in the absence of the regulatory activities specified by ICP4 and ICP27. The pattern of protein synthesis in d97-infected cells was unique relative to other IE gene mutants in that it was similar to that seen in the absence of prior viral protein synthesis, possibly approximating the effect of cellular factors and virion components alone. Inactivation of ICP0 in the absence of ICP4 produced a significant decrease in the levels of the early mRNAs ICP6 and thymidine kinase (tk). There was also a marginal reduction in the levels of the IE ICP22 mRNA, and this was most notable at low multiplicity of infection (MOI). In d97-infected L7 cells, the levels of the viral mRNAs were mostly restored to those observed in infections with d92. Nuclear runoff transcription analysis demonstrated that the presence of ICP0 resulted in an increase in the transcription rates of the analyzed genes. The transcription rates of the early genes were dramatically reduced in the absence of ICP0. At low MOI, the transcription rates of ICP6 and tk were comparable to the rate of transcription of a cellular gene. Relevant to the potential use of d97 as a transfer vector, it was also determined that the absence of ICP0 reduced the cellular toxicity of the virus compared to that of d92. The beta-gal transgene expressed from an IE promoter was detected for up to 14 days postinfection; however, the level of beta-gal expression declined dramatically after 1 day postinfection. In the presence of ICP0, the level of expression of beta-gal was increased; however the infected monolayer was destroyed by 3 days postinfection. Therefore, deletion of ICP0 in the absence of ICP4 and ICP27 reduces toxicity and lowers the level of expression of genes from the viral genome.
The cytotoxicity of the parvovirus minute virus of mice nonstructural protein NS1 is related to changes in the synthesis and phosphorylation of cell proteins
Authors: Anouja F , Wattiez R , Mousset S , Caillet-Fauquet P
Published: January 2020

Autonomous parvoviruses exert lytic and cytostatic effects believed to contribute to their antineoplastic activity. Studies with inducible clones have demonstrated a direct involvement of parvovirus nonstructural proteins (NS) in oncolysis. Human and rat fibroblasts have been stably transfected with MVM(p) (minute virus of mice prototype strain) NS genes cloned under the control of a hormone-inducible promoter. Dexamethasone-induced synthesis of the NS proteins in sensitive transformed cells results in cell killing within a few days. From these sensitive cell lines have been isolated some NS-resistant clones that also prove resistant to MVM(p) infection, suggesting that cell factors modulate NS cytotoxicity. We have previously reported that factors involved in cell cycle regulation may contribute to this modulation, since NS toxicity requires cell proliferation and correlates with a cell cycle perturbation leading to an arrest in phase S/G2. In addition to its role in cytotoxicity, NS1 can regulate transcription driven by parvovirus and nonparvovirus promoters. Since phosphorylation is a critical event in controlling the activity of many proteins, notably transcription factors and cell cycle-regulated proteins, we have examined the effect of NS1 on the synthesis and phosphorylation of cell proteins. Our results indicate that NS1 interferes, within 7 h of induction, with phosphorylation of a protein of about 14 kDa (p14). Cell synchronization has enabled us to show that phosphorylation of this protein occurs in early S phase and is prevented when NS1 is induced. This early effect of NS1 on p14 phosphorylation may be directly linked to cytotoxicity and is probably related to the previously reported inhibition of cell DNA synthesis. Late in the induction period (24 h), NS1 also alters the synthesis of a 50-kDa protein and a 35-kDa protein (p50 and p35, respectively). Microsequencing of p35 reveals sequence homology with beta-tubulin. These effects of NS1, observed only in NS1-sensitive cell lines, may be related to the protein's cytotoxicity.
RevM10-expressing T cells derived in vivo from transduced human hematopoietic stem-progenitor cells inhibit human immunodeficiency virus replication
Authors: Bonyhadi M L , Moss K , Voytovich A , Auten J , Kalfoglou C , Plavec I , Forestell S , Su L , Böhnlein E , Kaneshima H
Published: January 2020

A key feature of the pathogenesis of human immunodeficiency virus type 1 (HIV-1) infection is the gradual loss of CD4-positive T cells. A number of gene therapy strategies have been designed with the intent of inhibiting HIV replication in mature T cells. As T cells are products of hematolymphoid differentiation, insertion of antiviral genes into hematopoietic stem cells could serve as a vehicle to confer long-term protection in progeny T cells derived from transduced stem cells. One such "cellular immunization" strategy utilizes the gene coding for the HIV-1 rev trans-dominant mutant protein RevM10 which has been demonstrated to inhibit HIV-1 replication in T-cell lines and in primary T cells. In this study, we used a Moloney murine leukemia virus-based retrovirus encoding a bicistronic message coexpressing RevM10 and the murine CD8-alpha' chain (Lyt2). This vector allows rapid selection of transgene-expressing cells as well as quantitation of transgene expression. We demonstrate that RevM10-transduced CD34-enriched hematopoietic progenitor-stem cells (HPSC) isolated from human umbilical cord blood or from granulocyte colony-stimulating factor-mobilized peripheral blood can give rise to mature thymocytes in the SCID-hu thymus/liver mouse model. The phenotypic distribution of HPSC-derived thymocytes is normal, and expression of the transgene can be detected by flow cytometric analysis. Moreover, we demonstrate that RevM10 can inhibit HIV replication in T cells derived from transduced HPSC after expansion in vitro. This is the first demonstration of anti-HIV efficacy in T cells derived from transduced human HPSC.
Cold-adapted poliovirus mutants bypass a postentry replication block
Authors: Dove A W , Racaniello V R
Published: January 2020

In the current model of poliovirus entry, the initial interaction of the native virion with its cellular receptor is followed by a transition to an altered form, which then acts as an intermediate in viral entry. While the native virion sediments at 160S in a sucrose gradient, the altered particle sediments at 135S, has lost the coat protein VP4, and has become more hydrophobic. Altered particles can be found both associated with cells and in the culture medium. It has been hypothesized that the cell-associated 135S particle releases the viral genome into the cell cytoplasm and that nonproductive transitions to the 135S form are responsible for the high particle-to-PFU ratio observed for polioviruses. At 25 degrees C, a temperature at which the transition to 135S particles does not occur, the P1/Mahoney strain of poliovirus was unable to replicate, and cold-adapted (ca) mutants were selected from the population. These mutants have not gained the ability to convert to 135S particles at 25 degrees C, and the block to wild-type (wt) infection at low temperatures is not at the level of cellular entry. The particle-to-PFU ratio of poliovirus does not change at 25 degrees C in the absence of alteration. Three independent amino acid changes in the 2C coding region were identified in ca mutants, at positions 218 (Val to Ile), 241 (Arg to Ala), and 309 (Met to Val). Introduction of any of these mutations individually into wt poliovirus by site-directed mutagenesis confers the ca phenotype. All three serotypes of the Sabin vaccine strains and the P3/Leon strain of poliovirus also exhibit the ca phenotype. These results do not support a model of poliovirus entry into cells that includes an obligatory transition to the 135S particle.
Open reading frame 26 of human herpesvirus 8 encodes a tetradecanoyl phorbol acetate- and butyrate-inducible 32-kilodalton protein expressed in a body cavity-based lymphoma cell line
Authors: O'Neill E , Douglas J L , Chien M L , Garcia J V
Published: January 2020

DNA sequences corresponding to a novel herpesvirus (human herpesvirus 8 [HHV8]) are associated with Kaposi's sarcoma (KS), Castleman's disease, and body cavity-based lymphomas (BCBL). Studies of a BCBL-derived cell line suggest a direct correlation between seropositivity against antigens specifically present in such lines and the development of KS. We have generated recombinant proteins corresponding to open reading frame (ORF) 26 of HHV8 and have produced affinity-purified antibodies. Using these antibodies, we studied the expression of HHV8 ORF26 in a BCBL-derived cell line and found that it encodes a cytoplasmic protein whose expression is induced 16-fold by treatment with phorbol ester or sodium butyrate. This protein induction correlates with a significant induction of viral RNA transcripts. Interestingly, under our experimental conditions minimal increases in viral DNA were observed. No antibodies to the ORF26 protein of HHV8 were found in the sera from two human immunodeficiency virus-positive patients with KS as determined by immunoprecipitation analysis. However, antibodies in the sera from the two KS patients immunoprecipitated a 34-kDa protein found in extracts from induced BCBL1 cells that was not recognized by the control sera.
Hepatitis B virus replication is cell cycle independent during liver regeneration in transgenic mice
Authors: Guidotti L G , Matzke B , Chisari F V
Published: January 2020

The content of hepatitis B virus (HBV) replicative forms and HBV core protein in the liver of HBV transgenic mice is transiently reduced during massive liver regeneration following partial hepatectomy while the steady-state content of viral RNA is unchanged. This antiviral effect is triggered by interferon and tumor necrosis factor that are induced in the liver following hepatectomy and either prevent the formation or accelerate the degradation of viral nucleocapsids in the cytoplasm of the hepatocyte. Despite massive hepatocellular turnover, this effect is independent of liver cell division, indicating that HBV replicates efficiently in resting and dividing hepatocytes.
Efficient pseudotyping of murine leukemia virus particles with chimeric human foamy virus envelope proteins
Authors: Lindemann D , Bock M , Schweizer M , Rethwilm A
Published: January 2020

Incorporation of human foamy virus (HFV) envelope proteins into murine leukemia virus (MuLV) particles was studied in a transient transfection packaging cell system. We report here that wild-type HFV envelope protein can pseudotype MuLV particles, albeit at low efficiency. Complete or partial removal of the HFV cytoplasmic tail resulted in an abolishment or reduction of HFV-mediated infectivity, implicating a role of the HFV envelope cytoplasmic tail in the pseudotyping of MuLV particles. Mutation of the endoplasmic reticulum retention signal present in the HFV envelope cytoplasmic tail did not result in a higher relative infectivity of pseudotyped retroviral vectors. However, a chimeric envelope protein, containing an unprocessed MuLV envelope cytoplasmic domain fused to a truncated HFV envelope protein, showed an enhanced HFV specific infectivity as a result of an increased incorporation of chimeric envelope proteins into MuLV particles.
Multiple extracellular domains of CCR-5 contribute to human immunodeficiency virus type 1 entry and fusion
Authors: Picard L , Simmons G , Power C A , Meyer A , Weiss R A , Clapham P R
Published: January 2020

Human immunodeficiency virus type 1 (HIV-1) entry is governed by the interaction of the viral envelope glycoprotein (Env) with its receptor. The HIV-1 receptor is composed of two molecules, the CD4 binding receptor and a coreceptor. The seven-membrane-spanning chemokine receptor CCR-5 is one of the coreceptors used by primary isolates of HIV-1. We demonstrate that the mouse homolog of CCR-5 (mCCR-5) does not function as an HIV-1 coreceptor. A set of chimeras of human CCR-5 and mCCR-5 was studied for Env-induced cell fusion and HIV-1 infection. Using the HIV-1ADA envelope glycoprotein in a syncytium formation assay, we show that replacement of any fragment containing extracellular domains of mCCR-5 by its human counterparts is sufficient to allow Env-induced fusion. Conversely, replacement of any fragment containing human extracellular domains by its murine counterpart did not lead to coreceptor function loss. These results show that several domains of CCR-5 participate in coreceptor function. In addition, using a panel of primary nonsyncytium-inducing and syncytium-inducing isolates that use CCR-5 or both CXCR-4 and CCR-5 as coreceptors, we show that the latter dual-tropic isolates are less tolerant to changes in CCR-5 than strains with a more restricted coreceptor use. Thus, different strains are likely to have different ways of interacting with the CCR-5 coreceptor.
Tissue culture adaptation of foot-and-mouth disease virus selects viruses that bind to heparin and are attenuated in cattle
Authors: Sa-Carvalho D , Rieder E , Baxt B , Rodarte R , Tanuri A , Mason P W
Published: January 2020

Isolates of foot-and-mouth disease virus (FMDV) exist as complex mixtures of variants. Two different serotype O1 Campos preparations that we examined contained two variants with distinct plaque morphologies on BHK cells: a small, clear-plaque virus that replicates in BHK and CHO cells, and a large, turbid-plaque virus that only grows in BHK cells. cDNAs encoding the capsids of these two variants were inserted into a genome-length FMDV type A12 infectious cDNA and used to produce chimeric viruses that exhibited the phenotype of the original variants. Analyses of these viruses, and hybrids created by exchanging portions of the capsid gene, identified codon 56 in VP3 (3056) as the critical determinant of both cell tropism and plaque phenotype. Specifically, the CHO growth/clear-plaque phenotype is dependent on the presence of the highly charged Arg residue at 3056, and viruses with this phenotype and genotype were selected during propagation in tissue culture. The genetically engineered Arg 3056 virus was highly attenuated in bovines, but viruses recovered from animals inoculated with high doses of this virus had lost the ability to grow in CHO cells and contained either an uncharged residue at 3056 or a negatively charged Glu substituted for a Lys at a spatially and antigenically related position on VP2 (2134). Comparison of these animal-derived viruses to other natural and engineered viruses demonstrated that positively charged residues are required at both 2134 and 3056 for binding to heparin. Taken together, these results indicate that in vitro cultivation of FMDV type O selects viruses that bind to heparin and that viruses with the heparin-binding phenotype are attenuated in the natural host.
Identification and analysis of three myristylated vaccinia virus late proteins
Authors: Martin K H , Grosenbach D W , Franke C A , Hruby D E
Published: January 2020

Previous studies have shown that at least three vaccinia virus (VV) late proteins (with apparent molecular asses of 37, 35, and 25 kDa) label with myristic acid. Time course labeling of VV-infected cells with [3H]myristic acid reveals at least three additional putative myristylproteins, with apparent molecular masses of 92, 17, and 14 kDa. The 25-kDa protein has previously been identified as that encoded by the L1R open reading frame, leaving the identities of the remaining proteins to be determined. Sequence analysis led to the preliminary identification of the 37-, 35-, and 17-kDa proteins as G9R, A16L, and E7R, respectively. Using synthetic oligonucleotides and PCR techniques, each of these open reading frames was amplified by using VV DNA as a template and then cloned individually into expression vectors behind T7 promoters. These plasmid constructs were then transcribed in vitro, and the resulting mRNAs were translated in wheat germ extracts and radiolabeled with either [35S]methionine or [3H]myristic acid. Each wild-type polypeptide was labeled with [35S]methionine or [3H]myristic acid in the translation reactions, while mutants containing an alanine in place of glycine at the N terminus were labeled only with [35S]methionine, not with myristic acid. This result provided strong evidence that the open reading frames had been correctly identified and that each protein is myristylated on a glycine residue adjacent to the initiating methionine. Subcellular fractionations of VV-infected cells suggested that A16L and E7R are soluble, in contrast to L1R, which is a membrane-associated protein.
Characterization of humoral and CD4+ cellular responses after genetic immunization with retroviral vectors expressing different forms of the hepatitis B virus core and e antigens
Authors: Sällberg M , Townsend K , Chen M , O'Dea J , Banks T , Jolly D J , Chang S M , Lee W T , Milich D R
Published: January 2020

The humoral and CD4+ cellular immune responses in mice following genetic immunization with three retroviral vectors encoding different forms of hepatitis B virus core antigen (HBcAg) and e antigen (HBeAg) were analyzed. The retroviral vectors induced expression of intracellular HBcAg (HBc[3A4]), secreted HBeAg (HBe[5A2]), or an intracellular HBcAg-neomycin phosphoryltransferase fusion protein (HBc-NEO[6A3]). Specific antibody levels and immunoglobulin G isotype restriction were highly dependent on both the host major histocompatibility complex and the transferred gene. Humoral and CD4+ cellular HBcAg and/or HBeAg (HBc/eAg)-specific immune responses following retroviral vector immunization were of a lower magnitude but followed the same characteristics compared with those after immunization with HBc/eAg in adjuvant. Two factors influenced the humoral responses. First, in vivo depletion of CD8+ cells in HBc-NEO[6A3]-immunized H-2k mice abrogated both HBcAg-specific antibodies and in vitro-detectable cytotoxic T lymphocytes. Second, priming of H-2b mice with an HBc/eAg-derived T-helper (Th) peptide in adjuvant prior to retroviral vector immunization greatly enhanced the HBc/eAg-specific humoral responses to all three vectors, suggesting that insufficient HBc/eAg-specific CD4+ Th-cell priming limits the humoral responses. In conclusion, direct injection of retroviral vectors seems to be effective in priming HBc/eAg-specific CD8+ but comparatively inefficient in priming CD4+ Th cells and subsequently specific antibodies. However, the limited HBc/eAg-specific CD4+ cell priming can effectively be circumvented by prior administration of a recombinant or synthetic form of HBc/eAg in adjuvant.
The NS1 protein of the autonomous parvovirus minute virus of mice blocks cellular DNA replication: a consequence of lesions to the chromatin?
Authors: Op De Beeck A , Caillet-Fauquet P
Published: January 2020

The nonstructural protein NS1 of the autonomous parvovirus minute virus of mice interferes with cell division and can cause cell death, depending on the cell transformation state. Upon infection, the synthesis of NS1 protein is massively initiated during S phase. In this article, we show that minute virus of mice-infected cells accumulate in this phase. To investigate the link between NS1 accumulation and S-phase arrest, we have used stably transfected cells in which NS1 expression is under the control of a glucocorticoid-inducible promoter (the long terminal repeat of mouse mammary tumor virus). NS1 expression interferes with cell DNA replication, and consequently, the cell cycle stops in S phase. NS1 expression also induces nicks in the cell chromatin, as detected by an in situ nick translation assay. The nicks are observed several hours before any cell cycle perturbation. As cell cycle arrest is a common consequence of DNA damage, we propose that NS1 exerts its cytostatic activity by inducing lesions in cell chromatin.
Insertions within epsilon affect synthesis of minus-strand DNA before the template switch for duck hepatitis B virus
Authors: Jiang H , Loeb D D
Published: January 2020

Duck hepatitis B virus (DHBV) is a DNA virus that replicates via reverse transcription of a pregenomic RNA (pgRNA). Synthesis of the first strand of DNA (minus-strand DNA) for DHBV can be divided into two steps: (i) synthesis of the first four nucleotides of minus-strand DNA, which is primed by the viral polymerase (P) protein and copied from the sequence 5'-UUAC-3' within the phylogenetically conserved bulge in the encapsidation signal (epsilon) near the 5' end of pgRNA; and (ii) a template switch of the four-nucleotide minus-strand DNA from epsilon to an acceptor site near the 3' end of pgRNA and synthesis of a complete minus-strand DNA. To understand why only four nucleotides of minus-strand DNA were synthesized before the template switch, we introduced small insertions immediately 5' to the UUAC sequence in epsilon and determined whether these epsilon variants were competent for protein priming and whether minus strands longer than four nucleotides were synthesized. Then we determined, in cell culture, whether the longer minus-strand DNAs were competent to undergo a template switch. Also, we analyzed the structure of the epsilon variants, in solution. We found that the epsilon variants were functional for protein priming and RNA encapsidation and that the insertions were copied into minus-strand DNA. However, two mutant viruses that contained two different three-nucleotide insertions failed to synthesize minus-strand DNA efficiently from the acceptor site, even though seven nucleotides of the donor and acceptor sites were identical. These results suggest that the length and/or sequence of the minus-strand DNA copied from epsilon can be important for an efficient template switch. The RNA structural analysis of the epsilon variants indicated alteration in the position and size of the bulge. Overall, these results are consistent with the notion that the template within epsilon is limited to four nucleotides because the remaining two nucleotides located within the bulge are inaccessible for polymerization.
Theiler's virus and Mengo virus induce cross-reactive cytotoxic T lymphocytes restricted to the same immunodominant VP2 epitope in C57BL/6 mice
Authors: Dethlefs S , Escriou N , Brahic M , van der Werf S , Larsson-Sciard E L
Published: January 2020

C57BL/6 mice develop a virus-specific cytotoxic T-lymphocyte (CTL) response after intraperitoneal inoculation with either the DA strain of Theiler's virus or Mengo virus, two members of the Cardiovirus genus. These CTLs contribute to viral clearance in the case of Theiler's virus but do not protect the mice from the fatal encephalomyelitis caused by Mengo virus. In this study we show that DA and Mengo virus-induced CTLs are cross-reactive. The cross-reactivity is due to a conserved, H-2Db-restricted epitope located between amino acid residues 122 and 130 of the VP2 capsid protein (VP2(122-130)). This epitope is immunodominant in C57BL/6 mice infected with Theiler's virus. The VP2(122-130) epitope, initially identified for Mengo virus, is the first CTL epitope described for Theiler's virus.
High level of transgene expression in cell cultures and in the mouse by replication-incompetent adenoviruses harboring modified VAI genes
Authors: Eloit M , Adam M , Gallais I , Fournier A
Published: January 2020

Replication-incompetent adenoviruses are currently used in gene therapy trials. Most of the work designed to increase the expression from these vectors concerns the modification of cis sequences of the foreign transcription unit, so as to improve the transcription level or the stability of the mRNA. In this report, we show that an alternative strategy based on the coexpression of modified VAI genes can efficiently increase gene expression both in cell cultures and in animals. The VAI RNA is synthesized mainly during the late phase of the adenovirus cycle and increases the translation of late adenovirus gene products by counteracting the effect of an interferon-induced kinase, the PKR. We have constructed several modified VAI genes in which the central domain was deleted or substituted by exogenous sequences. These modified VAI genes, or the native VAI gene, were cloned into the left part of adenovirus type 5 genomes harboring their own endogenous VAI gene. One of the resulting viruses (Ad-VAr) increased 12.5- to 502-fold the expression level of reporter genes, either expressed as a constitutive cell line from an extrachromosomal DNA or introduced into cells by coinfection with another adenovirus vector. This effect was independent of the promoter, the coding sequence, and the 5' untranslated mRNA sequence and was obvious in the two non-E1-complementing cell lines tested (HeLa and Vero). Coinfection of Ad-VAr with adenoviruses expressing the luciferase gene from the major late promoter or Rous sarcoma virus (RSV) promoter by the intravenous route in mice increased by more than 33 (MLP)- to 128 (RSV)- and 4,700 (MLP)- to 30,000 (RSV)-fold the expression level of the reporter gene in the lungs and liver, respectively. The intramuscular coinoculation of Ad-VAr and Ad-MLP-gD (a recombinant adenovirus vaccine expressing gD from the pseudorabies herpes virus) led to a 10-fold decrease in the protective dose of Ad-gD in mice. Ad-VAfull, a similar adenovirus in which the native VAI gene was cloned at the left part of the genome, showed no evidence of efficacy in cell culture and in mice. These results suggest that the use of modified VAI genes expressed at the early phase of the cycle can be helpful in the design of potent adenovirus vectors.
Comprehensive quantification of herpes simplex virus latency at the single-cell level
Authors: Sawtell N M
Published: January 2020

To date, characterization of latently infected tissue with respect to the number of cells in the tissue harboring the viral genome and the number of viral genomes contained within individual latently infected cells has not been possible. This level of cellular quantification is a critical step in determining (i) viral or host cell factors which function in the establishment and maintenance of latency, (ii) the relationship between latency burden and reactivation, and (iii) the effectiveness of vaccines or antivirals in reducing or preventing the establishment of latent infections. Presented here is a novel approach for the quantitative analysis of nucleic acids within the individual cells comprising complex solid tissues. One unique feature is that the analysis reflects the nucleic acids within the individual cells as they were in the context of the intact tissue-hence the name CXA, for contextual analysis. Trigeminal ganglia latently infected with herpes simplex virus (HSV) were analyzed by CXA of viral DNA. Both the type and the number of cells harboring the viral genome as well as the number of viral genomes within the individual latently infected cells were determined. Here it is demonstrated that (i) the long-term repository of HSV-1 DNA in the ganglion is the neuron, (ii) the viral-genome copy number within individual latently infected neurons is variable, ranging over 3 orders of magnitude from <10 to >1,000, (iii) there is a direct correlation between increasing viral input titer and the number of neurons in which latency is established in the ganglion, (iv) increasing viral input titer results in more neurons with greater numbers of viral-genome copies, (v) treatment with acyclovir (ACV) during acute infection reduces the number of latently infected ganglionic neurons 20-fold, and (vi) ACV treatment results in uniformly low (<10)-copy-number latency. This report represents the first comprehensive quantification of HSV latency at the level of single cells. Beyond viral latency, CXA has the potential to advance many studies in which rare cellular events occur in the background of a complex solid tissue mass, including microbial pathogenesis, tumorigenesis, and analysis of gene transfer.
Sendai virus efficiently infects cells via the asialoglycoprotein receptor and requires the presence of cleaved F0 precursor proteins for this alternative route of cell entry
Authors: Bitzer M , Lauer U , Baumann C , Spiegel M , Gregor M , Neubert W J
Published: January 2020

Biochemical evidence suggests that the asialoglycoprotein receptor (ASGP-R) can be used as an alternative receptor for a temperature-sensitive Sendai virus (SV) mutant. We now have investigated this possible alternative route of infection for SV wild-type (SV-wt) strain Fushimi by using a pair of cell lines which differ only with regard to ASGP-R expression. Infection studies after enzymatic destruction of conventional sialic acid-containing SV receptors (SA-R) revealed that only ASGP-R-expressing cells could be infected by SV-wt. This alternative route of cell entry could be completely blocked by incubation of cells with ASGP-R-specific antibodies prior to infection. Furthermore, cleavage of SV-F0 precursor protein into the subunits F1 and F2 was necessary to establish infection via ASGP-R, suggesting a fusion-mediated cell entry after binding of SV-wt to the ASGP-R on host cells. Interestingly, infection via ASGP-R was found to be nearly as efficient as infection via conventional sialic acid-containing SV receptors. A possible physiological role of the ASGP-R-mediated route of SV infection is discussed.
Formation of intracellular particles by hepatitis B virus large surface protein
Authors: Xu Z , Bruss V , Yen T S
Published: January 2020

Hepatitis B virus small surface protein is synthesized as a transmembrane protein of the rough endoplasmic reticulum (RER) and then buds into the lumen in the form of subviral particles that are secreted. The closely related large surface protein is also targeted to the RER but is retained in a pre-Golgi compartment and cannot be secreted. It has been assumed that the large surface protein remains as a transmembrane RER protein and hence cannot form particles, possibly because of binding to a host factor on the cytosolic face of the RER membranes. We have reexamined this question and found the following results. (i) The retained large surface protein is associated not with RER but, rather, with a more distal compartment. (ii) Electron microscopy reveals intravesicular 20-nm particles, similar to those formed by the small surface protein. (iii) The large surface protein colocalizes with and binds to calnexin, an ER chaperone protein. Therefore, our results indicate that the large surface protein is capable of budding and forming particles, and hence its intracellular retention cannot be attributed to a cytosolic factor. We interpret the data as evidence that the large surface protein is retained by virtue of interacting with calnexin, a component of what is considered the quality control mechanism of the ER.
The kappaB sites in the human immunodeficiency virus type 1 long terminal repeat enhance virus replication yet are not absolutely required for viral growth
Authors: Chen B K , Feinberg M B , Baltimore D
Published: January 2020

The dependence of human immunodeficiency virus type 1 (HIV-1) on its NF-kappaB binding sites (kappaB sites) for replication in transformed and primary T-cell targets was examined by infecting cells with HIV-1 reporter viruses containing kappaB site enhancer mutations. Viral transcription was measured either with luciferase-expressing HIV-1 that infects for a single round or by flow cytometric analyses with HIV-1 expressing placental alkaline phosphatase (PLAP) or green-fluorescent protein (GFP). Both PLAP- and GFP-expressing viruses spread from cell to cell and allowed analysis of viral gene expression patterns in single cells. Infection of a panel of T-cell lines with different basal levels of NF-kappaB demonstrated a direct correlation between the amount of constitutive nuclear NF-kappaB and the degree to which a wild-type virus outperformed kappaB site mutants. One T-cell line with a constitutively high level of nuclear NF-kappaB, PM1, showed a 20-fold decrease in transcription when its kappaB sites were mutated. In contrast, in a T-cell line with a low basal level of NF-kappaB, SupT1, mutation of the kappaB site in the enhancer had no effect on viral transcription or growth rate. Phytohemagglutinin-activated peripheral blood mononuclear cells showed a large dependence on the kappaB sites for optimal virus growth. Viruses without marker genes corroborated the finding that mutations to the kappaB sites impair virus production in cells with a high basal level of NF-kappaB. These data show that in T cells, HIV-1 can use NF-kappaB to enhance its growth but the virus is clearly able to grow in its absence.
Cloning of a new murine endogenous retrovirus, MuERV-L, with strong similarity to the human HERV-L element and with a gag coding sequence closely related to the Fv1 restriction gene
Authors: Bénit L , De Parseval N , Casella J F , Callebaut I , Cordonnier A , Heidmann T
Published: January 2020

We had previously identified a new family of human endogenous retrovirus-like elements, the HERV-L elements (human endogenous retrovirus with leucine tRNA primer), whose pol gene was most closely related to that of the foamy viruses. HERV-L pol-related sequences were also detected in other mammalian species. The recent cloning of the mouse Fv1 gene (S. Best, P. Le Tissier, G. Towers, and J. P. Stoye, Nature 382:826-829, 1996) has shed light on another HERV-L domain--identified as a gag gene based on its location within the provirus--which was found to be 60% identical, at the nucleotide level, to the Fv1 open reading frame (ORF). We have now cloned the murine homolog of HERV-L which, in contrast to HERV-L, displays fully open reading frames in the gag and pol genes. Its predicted Gag protein shares 43% identity with the Fv1 ORF product. Moreover, the characteristic major homology region of the capsid subdomain can be identified within both proteins, thus strongly emphasizing the gag-like origin of Fv1.
Subunit-specific mutagenesis of the cysteine 280 residue of the reverse transcriptase of human immunodeficiency virus type 1: effects on sensitivity to a specific inhibitor of the RNase H activity
Authors: Loya S , Gao H Q , Avidan O , Boyer P L , Hughes S H , Hizi A
Published: January 2020

Treatment of human immunodeficiency virus type 1 reverse transcriptase (HIV-1 RT) with N-ethylmaleimide (NEM) selectively inhibits the RNase H activity. The cysteine residue at position 280 (C280) is the target for NEM; HIV-1 RT carrying the mutation C280S is resistant to NEM. Since HIV-1 RT is composed of two related subunits (p66 and p51) that play distinct roles, we asked whether the C280 in p51 or the C280 in p66 is responsible for the sensitivity of the enzyme to NEM. HIV-1 RT versions were prepared that had one mutant and one wild-type subunit. When these chimeric enzymes were tested, both the p51 and p66 subunits were found to contribute to the sensitivity of the enzyme to NEM. The implications of these results are discussed in the context of the structure of the enzyme.
Two distinct oncornaviruses harbor an intracytoplasmic tyrosine-based basolateral targeting signal in their viral envelope glycoprotein
Authors: Lodge R , Delamarre L , Lalonde J P , Alvarado J , Sanders D A , Dokhélar M C , Cohen E A , Lemay G
Published: January 2020

It has been clearly established that the budding of the human immunodeficiency virus (HIV-1), a lentivirus, occurs specifically through the basolateral membrane in polarized epithelial cells. More recently, the signal was assigned to a tyrosine-based motif located in the intracytoplasmic domain of the envelope glycoprotein, as previously observed on various other viral and cellular basolateral proteins. In the present study, expression of human T-cell leukemia virus type 1 (HTLV-1) or Moloney murine leukemia virus envelope glycoproteins was used for trans-complementation of an envelope-negative HIV-1. This demonstrated the potential of oncornaviral retrovirus envelope glycoproteins to confer polarized basolateral budding in epithelial Madin-Darby canine kidney cells (MDCK cells). Site-directed mutagenesis confirmed the importance of a common motif encompassing at least one crucial membrane-proximal intracytoplasmic tyrosine residue. The conservation of a similar basolateral maturation signal in different retroviruses further supports its importance in the biology of this group of viruses.
Human immunodeficiency virus type 1 (HIV-1) protein Vif inhibits the activity of HIV-1 protease in bacteria and in vitro
Authors: Kotler M , Simm M , Zhao Y S , Sova P , Chao W , Ohnona S F , Roller R , Krachmarov C , Potash M J , Volsky D J
Published: January 2020

Human immunodeficiency virus type 1 (HIV-1) Vif is required for productive infection of T lymphocytes and macrophages. Virions produced in the absence of Vif have abnormal core morphology and those produced in primary T cells carry immature core proteins and low levels of mature capsid (M. Simm, M. Shahabuddin, W. Chao, J. S. Allan, and D. J. Volsky, J. Virol. 69:4582-4586, 1995). To investigate whether Vif influences the activity of HIV-1 protease (PR), the viral enzyme which is responsible for processing Gag and Gag-Pol precursor polyproteins into mature virion components, we transformed bacteria to inducibly express truncated Gag-Pol fusion proteins and Vif. We examined the cleavage of polyproteins consisting of matrix to PR (Gag-PR), capsid to PR (CA-PR), and p6Pol to PR (p6Pol-PR) and evaluated HIV-1 protein processing at specific sites by Western blotting using antibodies against matrix, capsid, and PR proteins. We found that Vif modulates HIV-1 PR activity in bacteria mainly by preventing the release of mature MA and CA from Gag-PR, CA from CA-PR, and p6Pol from p6Pol-PR, with other cleavages being less affected. Using subconstructs of Vif, we mapped this activity to the N-terminal half of the molecule, thus identifying a new functional domain of Vif. Kinetic study of p6Pol-PR autocatalysis in the presence or absence of Vif revealed that Vif and N'Vif reduce the rate of PR-mediated proteolysis of this substrate. In an assay of in vitro proteolysis of a synthetic peptide substrate by purified recombinant PR we found that recombinant Vif and the N-terminal half of the molecule specifically inhibit PR activity at a molar ratio of the N-terminal half of Vif to PR of about 1. These results suggest a mechanism and site of action of Vif in HIV-1 replication and demonstrate novel regulation of a lentivirus PR by an autologous viral protein acting in trans.