bluuebunny/crossref_metadata_2025 huggingface dataset:
Vpu-induced degradation of CD4: requirement for specific amino acid residues in the cytoplasmic domain of CD4
Authors: Lenburg M E , Landau N R
Published: January 2020

Two functions have been attributed to the product of the human immunodeficiency virus type 1 vpu open reading frame: it increases virion release from infected cells and induces rapid degradation of CD4 shortly after its synthesis. In the absence of Vpu, newly synthesized gp160 and CD4 associate in the endoplasmic reticulum (ER), forming a complex whose further maturation is blocked and which is eventually degraded. In studies using NL4-3-based expression vectors, it has been previously shown that Vpu induces the release of gp160 from the complex that it forms with CD4 in the ER. This release, which appears to be due to the rapid degradation of CD4 induced by Vpu, allows gp160 to transit to the Golgi, where it matures further. We investigated which regions of CD4 are important for its susceptibility to Vpu-induced degradation by transfecting HeLa cells with isogenic vpu-positive and vpu-negative proviruses and vectors expressing various truncated or mutated CD4 molecules. The results suggested that the cytoplasmic domain of CD4 contains a determinant lying within amino acids 418 to 425 that is critical for susceptibility to Vpu-induced degradation. Neither the phosphorylation sites in the cytoplasmic domain nor the Lck interaction region was required for the effect. Vpu-induced degradation was specific for CD4, since CD8, even when retained in the ER, was not degraded. In addition, under conditions of high-level Vpu expression, CD4 degradation could be observed in the absence of gp160 or other means of retaining CD4 in the ER.
Replication of lymphocytic choriomeningitis virus is restricted in terminally differentiated neurons
Authors: de la Torre J C , Rall G , Oldstone C , Sanna P P , Borrow P , Oldstone M B
Published: January 2020

We have investigated the replication of lymphocytic choriomeningitis virus (LCMV) before and after the nerve growth factor (NGF)-induced transdifferentiation of PC12 cells from the chromaffin to the neuron-like phenotype. Untreated and NGF-treated cells were equally susceptible to LCMV infection; however, the viral yield was found to be 1,000-fold lower in NGF-differentiated PC12 cells. The reduced viral yield correlated with restricted LCMV replication and transcription within the infected cell, which was not caused by the lack of cell proliferation in the NGF-treated cells but rather was related to the induction or changes in expression levels of specific gene product(s) associated with the cell commitment to a neuronal phenotype. The return to the chromaffin phenotype after withdrawal of NGF restored normal LCMV yields as well as levels of viral replication and transcription. The finding of reduced viral replication in terminally differentiated neuronal cells has important implications for understanding the mechanism by which neurotropic viruses, such as LCMV, are able to establish a long-term persistent infection in the central nervous system in the absence of severe pathological changes.
Epstein-Barr virus recombinants with specifically mutated BCRF1 genes
Authors: Swaminathan S , Hesselton R , Sullivan J , Kieff E
Published: January 2020

Epstein-Barr virus (EBV) recombinants with specifically mutated BCRF1 genes were constructed and compared with wild-type BCRF1 recombinants derived in parallel for the ability to initiate and maintain latent infection and growth transformation in primary human B lymphocytes. A stop codon insertion after codon 116 of the 170-codon BCRF1 open reading frame or deletion of the entire gene had no effect on latent infection, B-lymphocyte proliferation into long-term lymphoblastoid cell lines (LCLs), or virus replication. LCLs infected with the stop codon recombinant were indistinguishable from wild-type recombinant-infected LCLs in tumorigenicity in SCID mice. However, mutant BCRF1 recombinant-infected cells differed from wild-type recombinant-infected cells in their inability to block gamma interferon release in cultures of permissively infected LCLs incubated with autologous human peripheral blood mononuclear cells. This is the first functional assay for BCRF1 expression from the EBV genome. BCRF1 probably plays a key role in modulating the specific and nonspecific host responses to EBV infection.
HLA A2.1-restricted cytotoxic T cells recognizing a range of Epstein-Barr virus isolates through a defined epitope in latent membrane protein LMP2
Authors: Lee S P , Thomas W A , Murray R J , Khanim F , Kaur S , Young L S , Rowe M , Kurilla M , Rickinson A B
Published: January 2020

Cytotoxic T-lymphocyte (CTL) responses induced by persistent Epstein-Barr virus (EBV) infection in normal B-lymphoid tissues could potentially be directed against EBV-positive malignancies if expression of the relevant viral target proteins is maintained in tumor cells. For malignancies such as nasopharyngeal carcinoma and Hodgkin's disease, this will require CTL targeting against the nuclear antigen EBNA1 or the latent membrane proteins LMP1 and LMP2. Here we analyze in detail a B95.8 EBV-reactivated CTL response which is specific for LMP2 and restricted through a common HLA allele, A2.1. We found that in vitro-reactivated CTL preparations from several A2.1-positive virus-immune donors contained detectable reactivity against A2.1-bearing target cells expressing either LMP2A or the smaller LMP2B protein from recombinant vaccinia virus vectors. Peptide sensitization experiments then mapped the A2.1-restricted response to a single epitope, the nonamer CLGGLLTMV (LMP2A residues 426 to 434), whose sequence accords well with the proposed peptide binding motif for A2.1. Most Caucasian and African virus isolates (whether of type 1 or type 2) were identical in sequence to B95.8 across this LMP2 epitope region, although 2 of 12 such isolates encoded a Leu-->Ile change at epitope position 6. In contrast, most Southeast Asian and New Guinean isolates (whether of type 1 or type 2) constituted a different virus group with a Cys-->Ser mutation at epitope position 1. CTLs raised against the B95.8-encoded epitope were nevertheless able to recognize these variant epitope sequences in the context of A2.1 whether they were provided exogenously as synthetic peptides or generated endogenously in B cells transformed with the variant viruses. A CTL response of this kind could have therapeutic potential in that it is directed against a protein expressed in many EBV-positive malignancies, is reactive across a range of virus isolates, and is restricted through a relatively common HLA allele.
In vivo footprinting analysis of constitutive and inducible protein-DNA interactions at the long terminal repeat of human immunodeficiency virus type 1
Authors: Demarchi F , D'Agaro P , Falaschi A , Giacca M
Published: January 2020

The regulation of the rate of transcription of human immunodeficiency virus type 1 is mainly exerted through the long terminal repeat (LTR) at the 5' end of the provirus. A large number of cis-acting regulatory elements have been identified in the LTR by in vitro binding studies; the biological role of these sites within living infected cells, however, is still not clear. We have studied the interactions of nuclear proteins with the LTR in the U1 monocytic cell line by in vivo dimethylsulfate footprinting, using the ligation-mediated polymerase chain reaction technique. In this cell line, transcription of the virus, which is very low under basal conditions, is highly inducible by treatment with phorbol esters; therefore, this system is likely to represent a suitable cellular model to study viral latency. Independently of the level of viral transcription, major in vivo footprints appear at the two Sp1 sites adjacent to the enhancer, the downstream-positioned enhancer repeat, the NFAT binding site, and one of the purine-rich sites of the negative regulatory element. Upon transcriptional activation by phorbol myristate acetate, the only perturbation in the footprinting pattern is a dramatic increase in dimethylsulfate sensitivity of guanine at position -92 in the downstream enhancer repeat. This modification is correlated with the transient induction of two enhancer-binding activities, as determined by gel retardation assays. While the transcriptional rate is still increasing and the in vivo footprinting pattern is unchanged at up to 24 h postactivation, these enhancer-binding factors are considerably reduced at this time. Therefore, further levels of regulation have to be considered to explain the maintenance of the induced state.
Role of spike protein conformational changes in fusion of Semliki Forest virus
Authors: Justman J , Klimjack M R , Kielian M
Published: January 2020

The alphavirus Semliki Forest virus (SFV) and a number of other enveloped animal viruses infect cells via a membrane fusion reaction triggered by the low pH within endocytic vesicles. In addition to having a low pH requirement, SFV fusion and infection are also strictly dependent on the presence of cholesterol in the host cell membrane. A number of conformational changes in the SFV spike protein occur following low-pH treatment, including dissociation of the E1-E2 dimer, conformational changes in the E1 and E2 subunits, and oligomerization of E1 to a homotrimer. To allow the ordering of these events, we have compared the kinetics of these conformational changes with those of fusion, using pH treatment near the fusion threshold and low-temperature incubation to slow the fusion reaction. Dimer dissociation, the E1 conformational change, and E1 trimerization all occur prior to the mixing of virus and cell membranes. Studies of cells incubated at 20 degrees C showed that as with virus fusion, E1 trimerization occurred in the endosome before transport to lysosomes. However, unlike the strictly cholesterol-dependent membrane fusion reaction, the E1 homotrimer was produced in vivo during virus uptake by cholesterol-depleted cells or in vitro by low-pH treatment of virus in the presence of artificial liposomes with or without cholesterol. Purified, lipid-free spike protein rosettes were assayed to determine the requirement for virus membrane cholesterol in E1 homotrimer formation. Spike protein rosettes were found to undergo E1 oligomerization upon exposure to low pH and target liposomes and showed an enhancement of oligomerization with cholesterol-containing membranes. The E1 homotrimer may represent a perfusion complex that requires cholesterol to carry out the final coalescence of the viral and target membranes.
F0-containing noninfectious Sendai virus can initiate replication in mouse lungs but requires a relatively long incubation period
Authors: Kiyotani K , Sakaguchi T , Fujii Y , Yoshida T
Published: January 2020

The replication of LLC-MK2-grown noninfectious Sendai virus, containing exclusively fusion (F) glycoprotein precursors, was examined in the mouse lung to study the accessibility of virus inoculated intranasally to the virus activator present in the lung. When mice were intranasally inoculated with various doses of the virus after in vitro activation with trypsin, the 50% mouse infectious dose (MID50) was determined to be 0.7 cell-infectious units (CIU) per mouse, indicating that one infectious unit of Sendai virus is enough to initiate replication in the mouse lung and that the present experimental system is highly sensitive. On the other hand, in mice inoculated with virus not treated with trypsin, virus replication in the lung was recognized even in mice inoculated with samples containing no infectious virus, and the MID50 was determined to be 67.5 CIU per mouse (here, CIU were assayed after in vitro trypsin treatment). When mice were infected with 20 MID50 of trypsin-treated infectious and untreated noninfectious viruses (an approximately 100-fold greater amount of noninfectious virus than of infectious virus was used), the noninfectious virus was found to require 2 more days of incubation than the infectious virus, and many of the F proteins synthesized in the lungs of mice infected with the F0-containing virus were present in the cleaved form. In addition, the infection of mice with noninfectious virus was strongly suppressed by aprotinin, a serine protease inhibitor. These results indicate that Sendai virus can initiate replication in the mouse lung even with the F0-containing noninfectious virus and strongly suggest that this infection process is mediated by cleavage activation of the F0 proteins of inoculated viruses by a serine protease(s) present in the lumen of the mouse respiratory tract but that activation of the noninfectious virus is an inefficient process.
Isoprenylation mediates direct protein-protein interactions between hepatitis large delta antigen and hepatitis B virus surface antigen
Authors: Hwang S B , Lai M M
Published: January 2020

Hepatitis delta antigen (HDAg) consists of two protein species of 195 and 214 amino acids, respectively, which are identical in sequence except that the large HDAg has additional 19 amino acids at its C terminus and is prenylated. Previous studies have shown that the large HDAg and the surface antigen of hepatitis B virus (HBsAg) together can form empty hepatitis delta virus (HDV) particles. To understand the molecular mechanism of HDV virion morphogenesis, we investigated the possible direct protein-protein interaction between HDAg and HBsAg. We constructed recombinant baculoviruses expressing the major form of HBsAg and various mutant HDAgs and used these proteins for far-Western protein binding assays. We demonstrated that HBsAg interacted specifically with the large HDAg but not with the small HDAg. Using mutant HDAgs which have defective or aberrant prenylation, we showed that this interaction required isoprenylates on the cysteine residue of the C terminus of the large HDAg. Isoprenylation alone, without the remainder of the C-terminal amino acids of the large HDAg, was insufficient to mediate interaction with HBsAg. This study demonstrates a novel role of prenylates in HDV virion assembly.
Mechanism of interferon action: evidence for intermolecular autophosphorylation and autoactivation of the interferon-induced, RNA-dependent protein kinase PKR
Authors: Thomis D C , Samuel C E
Published: January 2020

The interferon-induced RNA-dependent protein kinase (PKR) is postulated to have an important regulatory role in the synthesis of viral and cellular proteins. Activation of the enzyme requires the presence of a suitable activator RNA and is accompanied by an autophosphorylation of PKR. Active PKR phosphorylates the alpha subunit of protein synthesis eukaryotic initiation factor 2, resulting in an inhibition of translation initiation. The mechanism of autophosphorylation is not well understood. Here we present evidence that the autophosphorylation of human PKR can involve intermolecular phosphorylation events, i.e., one PKR protein molecule phosphorylating a second PKR molecule. Both wild-type PKR and the point mutant PKR(K296R) synthesized in vitro were phosphorylated, even though PKR(K296R) was deficient in kinase catalytic activity. Phosphorylation of both wild-type PKR and PKR(K296R) was inhibited in the presence of 2-aminopurine. Furthermore, purified human recombinant PKR(K296R) was a substrate for the purified wild-type human PKR kinase. This intermolecular phosphorylation of mutant PKR(K296R) by wild-type PKR was dependent on double-stranded RNA and was inhibited by 2-aminopurine. Finally, PKR mRNA was capable of mediating an autoactivation of wild-type PKR kinase autophosphorylation in vitro.
NF-IL6 represses early gene expression of human papillomavirus type 16 through binding to the noncoding region
Authors: Kyo S , Inoue M , Nishio Y , Nakanishi K , Akira S , Inoue H , Yutsudo M , Tanizawa O , Hakura A
Published: January 2020

The expression of human papillomavirus type 16 (HPV16) early genes, including E6 and E7 transforming genes, is regulated by several cellular factors binding to the noncoding region (NCR), such as the glucocorticoid receptor, NF-I, and AP1, all of which are positive regulators. We demonstrated that the nuclear factor for interleukin 6 expression (NF-IL6) specifically binds to the HPV16 NCR ranging from nucleotides 7007 to 7766 and represses the early gene expression of HPV16. The responsive element in HPV16 NCR was determined within the region ranging from nucleotides 7454 to 7766. In this region, many binding sites for other cellular transactivators, such as NF-I and AP1, have been detected. Interestingly, three of seven binding sites for NF-I and two of two binding sites for AP1 in this region overlap with the putative NF-IL6 binding sites identified by computer analysis. Competition experiments with the oligonucleotides containing such NF-I or AP1 sites indicated that NF-IL6 certainly binds to them. Furthermore, in a chloramphenicol acetyltransferase assay using mutant NF-IL6 expression vectors, the DNA binding domain of NF-IL6 was shown to be necessary for repression, whereas the functional domain was not. These findings indicate that repression may be caused by competition with other transcriptional activators, such as NF-I and AP1. Thus, NF-IL6 may play a significant role in the regulation of viral transcription as a part of the host's resistance to viral infection.
Measles virus-induced changes in leukocyte function antigen 1 expression and leukocyte aggregation: possible role in measles virus pathogenesis
Authors: Attibele N , Wyde P R , Trial J , Smole S C , Smith C W , Rossen R D
Published: January 2020

Measles virus (MV) infection of U937 cell or peripheral blood leukocyte cultures was shown to induce changes in the expression of leukocyte function antigen 1 (LFA-1) and cause marked aggregation of these cells. Addition of selected monoclonal antibodies specific for LFA-1 epitopes that did not neutralize MV in standard neutralization assays were found to block both virus-induced leukocyte aggregation and virus dissemination. These data suggest that MV modulation of LFA-1 expression on leukocytes may be an important step in MV pathogenesis.
Introduction of human genomic sequences renders CHO-K1 cells susceptible to infection by amphotropic retroviruses
Authors: Eglitis M A , Kadan M J , Wonilowicz E , Gould L
Published: January 2020

To learn more about the nature of the block to infection by amphotropic retroviruses exhibited by Chinese hamster cells (CHO-K1), CHO-K1 cells were made susceptible to amphotropic retrovirus infection by introducing genomic DNA from infectable human cells. A clone, designated CHO18, was obtained and shown to be infected as efficiently as NIH 3T3 fibroblasts. Susceptibility of CHO18 cells to infection was specific to retroviruses and vectors bearing an amphotropic envelope. By comparison to CHO-K1 cells, CHO18 cells may provide a useful model for analysis of the molecular events involved in the retrovirus-receptor interaction.
Human immunodeficiency virus type 1 DNA integration: fine structure target analysis using synthetic oligonucleotides
Authors: Hong T , Murphy E , Groarke J , Drlica K
Published: January 2020

The target specificity of DNA strand transfer mediated by human immunodeficiency virus type 1 integrase was examined in vitro with synthetic oligonucleotides. Although insertion occurred at most locations in the target, some sites were preferred over others by at least 15-fold. Changing the nucleotide sequence of the target changed the distribution of preferred sites in complex ways, some of which included changes in target preference distant from the sequence alteration. Alignment of target sequences revealed that adenosine is preferred adjacent to the insertion site. Strand transfer occurred to within 2 nucleotides of the 3' end and to within 3 nucleotides of the 5' end of the target. This suggests that only 2 or 3 nucleotides flanking the target site are required for integration; such restricted contact with target DNA would allow integrase to insert the two ends of viral DNA into two closely spaced sites in host DNA, consistent with the concerted in vivo integration reaction that generates a 5-bp target duplication.
Effect of rearrangements and duplications of the Cys-His motifs of Rous sarcoma virus nucleocapsid protein
Authors: Bowles N E , Damay P , Spahr P F
Published: January 2020

It has been widely documented that the nucleocapsid protein p12 (NC) of Rous sarcoma virus (RSV) has a role in the encapsidation and maturation of the virus genomic RNA during particle formation, and particularly important appear to be the Cys-His motifs of this protein. Since some retroviruses only have one such motif, we have investigated the significance of the two distinct Cys-His motifs of RSV NC. The analysis of the phenotype of virus NC mutants with precise rearrangements or duplications of the motifs highlights the following features. (i) The two motifs are not functionally equivalent. (ii) The order and number of Cys-His motifs are less important for RSV NC than the presence of two distinct motifs for both the encapsidation of virus genomic RNA and maintenance of the integrity of the RNA after particle formation. (iii) The proximal motif has a distinct function in the virus replication cycle other than RNA encapsidation and dimerization. (iv) The presence of three Cys-His motifs reduces virus infectivity and leads to high-frequency deletion events (of one of the motifs) after infection: the resulting RNA species encode a wild type-like NC protein restoring full infectivity to the progeny virus particles. Additionally, the data suggest that this occurs only after infection. The deletion probably arises by intramolecular displacement of the replication complex between repeat sequences.
Sendai virus assembly: M protein binds to viral glycoproteins in transit through the secretory pathway
Authors: Sanderson C M , McQueen N L , Nayak D P
Published: January 2020

We have examined the relative ability of Sendai virus M (matrix) protein to associate with membranes containing viral glycoproteins at three distinct stages of the exocytic pathway prior to cell surface appearance. By the use of selective low-temperature incubations or the ionophore monensin, the transport of newly synthesized viral glycoproteins was restricted to either the pre-Golgi intermediate compartment (by incubation at 15 degrees C), the medial Golgi (in the presence of monensin), or the trans-Golgi network (by incubation at 20 degrees C). All three of these treatments resulted in a marked accumulation of the M protein on perinuclear Golgi-like membranes which in each case directly reflected the distribution of the viral F protein. Subsequent redistribution of the F protein to the plasma membrane by removal of the low-temperature (20 degrees C) block resulted in a concomitant redistribution of the M protein, thus implying association of the two components during intracellular transit. The extent of M protein-glycoprotein association was further examined by cell fractionation studies performed under each of the three restrictive conditions. Following equilibrium sedimentation of membranes derived from monensin-treated cells, approximately 40% of the recovered M protein was found to cofractionate with membranes containing the viral glycoproteins. Also, by flotation analyses, a comparable subpopulation of M protein was found to be membrane associated whether viral glycoproteins were restricted to the trans-Golgi network, the medial Golgi, or the pre-Golgi intermediate compartment. Additionally, transient expression of M protein alone from cloned cDNA showed that neither membrane association nor Golgi localization occurs in the absence of Sendai virus glycoproteins.
B epitopes and selection pressures in feline immunodeficiency virus envelope glycoproteins
Authors: Pancino G , Chappey C , Saurin W , Sonigo P
Published: January 2020

In order to map linear B epitopes in feline immunodeficiency virus (FIV) envelope glycoproteins (Env), a random library of FIV Env polypeptides fused to beta-galactosidase and expressed in Escherichia coli was screened by using sera from experimentally FIV-infected cats. We mapped five antibody-binding domains in the surface envelope glycoprotein (SU1 to SU5) and four in the transmembrane envelope glycoprotein (TM1 to TM4). Immunological analysis with 48 serum samples from naturally or experimentally infected cats of diverse origins revealed a broad group reactivity for epitopes SU2, TM2, and TM3, whereas SU3 appeared as strictly type specific. To study selection pressures acting on the identified immunogenic domains, we analyzed structural constraints and distribution of synonymous and nonsynonymous mutations (amino acids unchanged or changed). Two linear B epitopes (SU3 and TM4) appeared to be submitted to positive selection for change, a pattern of evolution predicting their possible involvement in antiviral protection. These experiments provide a pertinent choice of oligopeptides for further analysis of the protective response against FIV envelope glycoproteins, as a model to understand the role of antibody escape in lentiviral persistence and to design feline AIDS vaccines.
Coupled translation and replication of poliovirus RNA in vitro: synthesis of functional 3D polymerase and infectious virus
Authors: Barton D J , Flanegan J B
Published: January 2020

Poliovirus RNA polymerase and infectious virus particles were synthesized by translation of virion RNA in vitro in HeLa S10 extracts. The in vitro translation reactions were optimized for the synthesis of the viral proteins found in infected cells and in particular the synthesis of the viral polymerase 3Dpol. There was a linear increase in the amount of labeled protein synthesized during the first 6 h of the reaction. The appearance of 3Dpol in the translation products was delayed because of the additional time required for the proteolytic processing of precursor proteins. 3Dpol was first observed at 1 h in polyacrylamide gels, with significant amounts being detected at 6 h and later. Initial attempts to assay for polymerase activity directly in the translation reaction were not successful. Polymerase activity, however, was easily detected by adding a small amount (3 microliters) of translation products to a standard polymerase assay containing poliovirion RNA. Full-length minus-strand RNA was synthesized in the presence of an oligo(U) primer. In the absence of oligo(U), product RNA about twice the size of virion RNA was synthesized in these reactions. RNA stability studies and plaque assays indicated that a significant fraction of the input virion RNA in the translation reactions was very stable and remained intact for 20 h or more. Plaque assays indicated that infectious virus was synthesized in the in vitro translation reactions. Under optimal conditions, the titer of infectious virus produced in the in vitro translation reactions was greater than 100,000 PFU/ml. Virus was first detected at 6 h and increased to maximum levels by 12 h. Overall, the kinetics of poliovirus replication (protein synthesis, polymerase activity, and virus production) observed in the HeLa S10-initiation factor in vitro translation reactions were similar to those observed in infected cells.
An antibody- and synthetic peptide-defined rubella virus E1 glycoprotein neutralization domain
Authors: Wolinsky J S , Sukholutsky E , Moore W T , Lovett A , McCarthy M , Adame B
Published: January 2020

We previously described a monoclonal antibody (MAb) library generated by infecting BALB/c mice with rubella virus (RV) and selected by an enzyme-linked immunosorbent assay (ELISA) using purified virion targets. Plasmid pARV02-01, which expresses the fusion protein RecA1-35-GIGDLGSP-E1(202)-E1(283)-GDP-LacZ9-1015 in Escherichia coli, was shown to be a ligand for MAbs E1-18 and E1-20 (J. S. Wolinsky, M. McCarthy, O. Allen-Cannady, W. T. Moore, R. Jin, S. N. Cao, A. Lovett, and D. Simmons, J. Virol. 65:3986-3994, 1991). Both of these MAbs neutralize RV infectivity. A series of five overlapping synthetic peptides was made to further explore the requirements of this MAb binding domain. One of these peptides (SP15; E1(208) to E1(239)) proved an effective ligand for both MAbs in the ELISA. Stepwise synthesis of SP15 defined the minimal amino-terminal requirement for binding MAb E1-18 as E1(221) and that of MAb E1-20 as E1(223); the minimal carboxyl-terminal requirement is uncertain but does not exceed E1(239). Immunization of mice and rabbits with SP15 induced polyvalent antibody reactive with SP15, with other overlapped and related but not unrelated synthetic peptides, and with RV. The rabbit anti-SP15 antibody showed neutralization activity to RV similar to that of MAbs E1-18 and E1-20 but lacked hemagglutination inhibition activity. These data define a neutralization domain on E1 and suggest that the RV epitopes conserved by SP15 may be critical for protective host humoral immune responses.
Identification of a DNA-binding domain in the amino terminus of adeno-associated virus Rep proteins
Authors: Owens R A , Weitzman M D , Kyöstiö S R , Carter B J
Published: January 2020

The Rep78 and Rep68 proteins of adeno-associated virus (AAV) bind to the AAV terminal repeat hairpin DNA and are required for viral replication. We have expressed a series of mutant rep genes from the human immunodeficiency virus type 1 long terminal repeat promoter in human 293 cells and in an in vitro transcription-translation system. Mutant proteins were analyzed for AAV hairpin DNA binding and AAV terminal resolution functions. Deletion of amino acid residues 523 through 621 of Rep 78 had no effect on these functions. Amber mutant Rep proteins truncated at either amino acid 237 or amino acid 243 showed no detectable hairpin DNA binding or terminal resolution activity. A frameshift mutant Rep protein which contained Rep78 amino acids 1 through 241 lacked terminal resolution functions but bound specifically to the AAV hairpin DNA. The carboxyl-terminal missense sequence in this mutant appeared to have complemented an AAV-specific DNA-binding domain within the amino terminus of the Rep protein. mutant Rep protein in which methionine 225 of Rep78 was deleted (M225dl) was reduced threefold in AAV hairpin binding and had no terminal resolution functions. A mutant Rep protein in which a glycine was substituted at position 225 (M225G) was fully functional in these assays. When M225dl extract was mixed with wild-type Rep78 extract, AAV terminal resolution by Rep78 was inhibited. These results suggest that the amino-terminal portion of Rep78 and Rep68 contains a domain which can direct binding to AAV terminal hairpin DNA and that elements within the central region of the protein stabilize binding.
Integration is essential for efficient gene expression of human immunodeficiency virus type 1
Authors: Sakai H , Kawamura M , Sakuragi J , Sakuragi S , Shibata R , Ishimoto A , Ono N , Ueda S , Adachi A
Published: January 2020

A mutant of human immunodeficiency virus type 1 which carries a frameshift insertion in the integrase/endonuclease region of pol gene was constructed in vitro. Upon transfection into cells, although this mutant exhibited a normal phenotype with respect to expression of gag, pol, and env genes and to generation of progeny virions, no replication-competent virus in CD4-positive cells emerged. An assay for the single-step replication of a defective viral genome dependent on trans complementation by rev protein was established and used to monitor the early phase of viral infection process. Viral clones with a mutation in the vif, vpr, or vpu gene displayed no abnormality in the early phase. In contrast, the integrase mutant did not direct a marker gene expression after infection. Together with an observation that the mutant lacked the ability to integrate, these results indicated that the integration was required for efficient viral gene expression and productive infection of human immunodeficiency virus type 1.
Simian immunodeficiency viruses from central and western Africa: evidence for a new species-specific lentivirus in tantalus monkeys
Authors: Müller M C , Saksena N K , Nerrienet E , Chappey C , Hervé V M , Durand J P , Legal-Campodonico P , Lang M C , Digoutte J P , Georges A J
Published: January 2020

Although up to 50% of African green monkeys (AGMs) are infected by simian immunodeficiency viruses (SIV) in their natural habitat, they remain asymptomatic carriers of these lentiviruses. They provide an attractive model to study not only the origin but also the link among genetic variation, host-virus adaptation, and pathogenicity of primate lentiviruses. SIVagm have been isolated from three species of AGM: the vervet (Cercopithecus pygerythrus), the grivet (Cercopithecus aethiops), and the sabaeus (Cercopithecus sabaeus) monkey. We studied four new SIVagm isolates from a fourth AGM species, the tantalus monkey (Cercopithecus tantalus), caught in the Central African Republic, and four new isolates from feral sabaeus monkeys from Senegal. Antigenic properties and partial env sequences were used to evaluate the diversity among these isolates. Alignment of env sequences in SIVagm isolated from tantalus and sabaeus monkeys permitted detailed mapping of the variable and conserved domains in the external glycoprotein. Genetic distances indicated that SIVagm isolates from tantalus monkeys are the most divergent among SIVagm in feral AGMs in Africa. The fact that AGMs are infected by four distinct lentiviruses, each specific for a single AGM species, supports the hypothesis of a coevolution of these viruses and their natural hosts and suggests that SIV transmission is a rare event among separated AGM species in the wild.
Episomal and integrated copies of Epstein-Barr virus coexist in Burkitt lymphoma cell lines
Authors: Delecluse H J , Bartnizke S , Hammerschmidt W , Bullerdiek J , Bornkamm G W
Published: January 2020

The Epstein-Barr virus genome is present in more than 95% of the African cases of Burkitt lymphoma. In this tumor, the viral genome is usually maintained in multiple episomal copies. Viral integration has been described only for Namalwa, a cell line lacking episomes. In this study, we have addressed the question of whether integrated and episomal copies can coexist in Burkitt lymphoma cells. Gel electrophoresis was used to demonstrate the presence of episomal as well as free linear DNA in three Burkitt lymphoma cell lines. The numbers of episomal copies per cell were estimated to be 5 to 10 in BL36 and BL137 cells and below 1 in BL60 cells, indicating that BL60 does not represent a homogeneous cell population. Fluorescence in situ hybridization was combined with chromosomal banding to study the association of the viral DNA with metaphase chromosomes. A symmetrical pattern of signals at both chromatids located at the same chromosomal sites in many if not all metaphases was taken as evidence for viral integration. In each of the three cell lines, one site of integration was identified: at chromosome 11p15 in BL36 cells, at chromosome 1p34 in BL137 cells, and at the site of a reciprocal t(11;19) translocation in BL60 cells. Integrated, episomal and linear copies of Epstein-Barr virus DNA thus coexist in Burkitt lymphoma cells. The biological significance of viral integration in Burkitt lymphoma cells remains to be elucidated.
Erythropoietin receptor (EpoR)-dependent mitogenicity of spleen focus-forming virus correlates with viral pathogenicity and processing of env protein but not with formation of gp52-EpoR complexes in the endoplasmic reticulum
Authors: Wang Y , Kayman S C , Li J P , Pinter A
Published: January 2020

Recent evidence suggests that interactions between spleen focus-forming virus (SFFV) env products and the erythropoietin receptor (EpoR) are responsible for viral pathogenicity. Infection of factor-dependent cell lines expressing epoR (the cloned gene for EpoR) with SFFVP is mitogenic, generating cell lines that are no longer dependent on added growth factor, and an immunoprecipitable complex between EpoR and immature env protein in the endoplasmic reticulum has been identified. The dependence of these in vitro activities on env protein processing and their relationship to pathogenicity of SFFV were explored by using glycosylation site mutants of SFFV env. Mutants carrying Asn-->Asp mutations at each of the two consensus signals for N-linked glycosylation in the N-terminal domain of SFFVAP-L env (gs1 and gs2), the gs1-2- double mutant, and the gs0 quadruple mutant (mutated at all four signals utilized for N-linked glycosylation in SFFVAP-L env) were made. The primary translation products (gp52) of single-site mutant envs were processed into more highly glycosylated forms, and the corresponding viruses induced splenomegaly in susceptible mice, whereas the gs1-2- and gs0 proteins were not processed, and these viruses were not pathogenic. Unprocessed env proteins of both pathogenic and nonpathogenic mutants coprecipitated with EpoR. In the BaF3 cell assay for epoR-dependent mitogenicity, the pathogenic single mutants induced factor-independent growth efficiently whereas the nonpathogenic gs1-2- and gs0 mutants did not. These data demonstrate that the ability of gp52 to form complexes with EpoR in the endoplasmic reticulum is not sufficient for either mitogenicity in cell culture or induction of splenomegaly in mice while supporting the hypothesis that pathogenicity and mitogenicity of SFFV both result from an interaction between EpoR and SFFV env protein.
Transcriptional activity and mutational analysis of recombinant vesicular stomatitis virus RNA polymerase
Authors: Sleat D E , Banerjee A K
Published: January 2020

The 241-kDa large (L) protein of vesicular stomatitis virus (VSV) is the multifunctional catalytic component of the viral RNA polymerase. A protocol has been developed for the synthesis of recombinant L protein that will support viral mRNA synthesis in vitro. COS cells were transfected with a transient expression vector (pSV-VSL1 [M. Schubert, G. G. Harmison, C. D. Richardson, and E. Meier, Proc. Natl. Acad. Sci. USA 82:7984-7988, 1985]) which contains the simian virus 40 late promoter for the transcription of a cDNA copy of the L protein of the Indiana serotype of VSV. Cytoplasmic extracts of these cells efficiently transcribed VSV mRNAs in vitro in conjunction with N protein-RNA template purified from virus and recombinant phosphoprotein synthesized in Escherichia coli. mRNA synthesis was completely dependent upon addition of both bacterial phosphoprotein and extracts from cells transfected with the L gene. Extracts from mock-transfected cells or from cells transfected with the expression vector alone did not support VSV RNA synthesis. RNA synthesis was proportional to the concentration of cell extract used, with an optimum of 0.2 mg/ml. Rhabdoviruses and paramyxoviruses contain a highly conserved GDNQ motif which was mutated in the transfected L gene. All constructs with mutations within the core GDN abrogated transcriptional activity except for the mutant containing GDD, which retained 25% activity. Conserved amino acid changes outside of the core GDN and changes corresponding to other paromyxovirus and rhabdovirus L proteins retained variable transcriptional activity. These findings provide experimental evidence that the GDN of negative-strand, nonsegmented RNA viruses is a variant of the GDD motif of plus-strand RNA viruses and of the XDD motif of DNA viruses and reverse transcriptases.
Mutations in herpes simplex virus type 1 genes encoding VP5 and VP23 abrogate capsid formation and cleavage of replicated DNA
Authors: Desai P , DeLuca N A , Glorioso J C , Person S
Published: January 2020

The herpes simplex virus type 1 capsid is composed of seven capsid proteins which are termed VP5, VP19c, VP21, VP22a, VP23, VP24, and VP26. Major capsid protein VP5 is encoded by the gene UL19. UL18, whose transcript is 3' coterminal with that of VP5, specifies capsid protein VP23. Vero cell lines have been isolated that are transformed with either the BglII N (UL19) or EcoRI G (UL16 to UL21) fragment of KOS. These cell lines, selected for the ability to support the replication of a temperature-sensitive VP5 mutant, were used to isolate VP5 and VP23 null mutants. The mutations in VP5 (K5 delta Z) and VP23 (K23Z) were generated by insertion of the lacZ gene at the beginning of the coding sequences of the genes. Both mutants failed to form plaques on the nonpermissive cell line, and therefore, VP23, like VP5, is an essential gene product for virus replication. Both mutants expressed wild-type levels of infected-cell proteins upon infection of permissive and nonpermissive cell lines. However, the VP5 (150-kDa) and VP23 (33-kDa) polypeptides were absent in lysates prepared from K5 delta Z- and K23Z-infected Vero cells, respectively. No capsid structures were observed by electron microscopic analysis of thin sections of K5 delta Z- and K23Z-infected Vero cells. Following sedimentation of lysates from cells infected by the mutants, capsid proteins were not observed in the fractions where capsids normally sediment. The amounts of DNA replicated in the VP5 and VP23 mutant and in KOS-infected Vero cells were the same as in permissive cells. However, genomic ends were not evident in Vero cells infected with the mutants, suggesting that the DNA remains in concatemers and is not processed into unit length genomes.
Low-pH conformational changes of rabies virus glycoprotein and their role in membrane fusion
Authors: Gaudin Y , Ruigrok R W , Knossow M , Flamand A
Published: January 2020

Fusion of rabies virus with membranes occurs at acidic pH and is mediated by the viral spike glycoprotein (G). In this paper, we provide the basis for a description of structural transitions associated with exposure to low pH and of their role in membrane fusion. First, we have extended previous studies of fusion kinetics and we have shown that low-pH inhibition of fusion is detectable at 0.5 pH units higher than fusion. Second, low-pH-induced conformational changes were analyzed by using electron microscopy and monoclonal antibody binding assays. The existence of a pH-dependent equilibrium between the native and a low-pH inactive conformation was demonstrated. Third, besides these two conformations, we, using the fluorescent probe ANS (8-anilino-1-naphthalenesulfonic acid), provide evidence for the existence of a transient third state which appears to be more hydrophobic than the native state. Our results suggest that this transient state is responsible for viral aggregation at low pH and could play a role in the first steps of the fusion mechanism.
Characterization of functional Sp1 transcription factor binding sites in the hepatitis B virus nucleocapsid promoter
Authors: Zhang P , Raney A K , McLachlan A
Published: January 2020

The hepatitis B virus nucleocapsid minimal promoter contains sequence elements which are similar to the Sp1 transcription factor binding site consensus sequence. The interaction of these regulatory elements with Sp1 was examined by DNase I footprinting with purified Sp1 protein and DNase I footprinting and gel retardation analysis with nuclear extracts from human cell lines and was examined functionally with transient transfection assays in human hepatoma and Drosophila melanogaster Schneider line-2 cells. DNase I footprinting identified two regions of the nucleocapsid promoter, representing three recognition elements, that bound purified Sp1. Gel retardation analysis with Huh7 nuclear extracts demonstrated that each of the three recognition elements bound the same or similar transcription factor(s) as that recognized by the Sp1 consensus sequence recognition element. The function of the nucleocapsid promoter elements was examined by transient transfection assays in D. melanogaster Schneider line-2 cells by using these binding sites cloned into a minimal promoter element. Each of these regulatory regions transactivated transcription from the minimal promoter element in response to exogenously expressed Sp1. In addition, the second Sp1 site was shown to be an essential element of the nucleocapsid promoter in human hepatoma cells. This demonstrates that the hepatitis B virus nucleocapsid promoter contains three functional Sp1 binding sites which may contribute to the level of transcription from this promoter during viral infection.
Cell fusion by the envelope glycoproteins of persistent measles viruses which caused lethal human brain disease
Authors: Cattaneo R , Rose J K
Published: January 2020

Measles virus (MV) rarely induces lethal diseases of the human central nervous system characterized by reduced expression of the viral envelope proteins and by lack of viral budding. The MV envelope contains two integral membrane proteins, termed fusion (F) protein and hemagglutinin (H) protein, and a membrane-associated matrix (M) protein. Previously, analysis of MV genes from autopsy material indicated that the M protein and the F protein intracellular domain are often drastically altered by mutations. Here, we present evidence that truncation of the F protein intracellular domain does not impair fusion function, and we suggest that this alteration interferes with viral budding. Unexpectedly, certain combinations of functional F and H proteins were unable to induce syncytium formation, an observation suggesting that specific F-H protein interactions are required for cell fusion. We also found that three of four H proteins of persistent MVs are defective in intracellular transport, oligosaccharide modification, dimerization, and fusion helper function. Thus, MVs replicating in the brain at the terminal stage of infection are typically defective in M protein and in the two integral membrane proteins. Whereas the M protein appears dispensable altogether, partial preservation of F-protein function and H-protein function seems to be required, presumably to allow local cell fusion. Certain subtle alterations of the F and H proteins may be instrumental for disease development.
Deletion analysis of the capsid protein of Sindbis virus: identification of the RNA binding region
Authors: Geigenmüller-Gnirke U , Nitschko H , Schlesinger S
Published: January 2020

The capsid protein of Sindbis virus has multiple functions in the life cycle of the virus. One essential function is to interact with the genomic RNA of the virus to form the nucleocapsid. The experiments described in this article define a region of the protein that is required for binding to Sindbis virus RNA. The assay we used measured the binding of in vitro-translated proteins to RNA on the basis of their migration with the RNA during electrophoresis in an agarose gel. Binding to RNA showed specificity; more protein bound to an RNA containing the previously defined packaging signal in Sindbis virus RNAs than to a similar RNA lacking this sequence. We were able to produce a variety of deleted forms of the capsid protein by constructing cDNAs with in-frame deletions throughout the coding region of the capsid protein gene. These cDNAs were then transcribed into mRNAs and translated in vitro. C-terminal deletions in the capsid protein were obtained by preparing transcripts from cDNAs linearized at sites within the coding region. Our studies identified a 32-amino-acid region that is essential for the specificity in RNA binding, and they defined a 68-amino-acid minimal sequence which displays almost the complete specific RNA binding activity of the intact Sindbis virus capsid protein containing 264 amino acids.